InteractiveFly: GeneBrief

Lkb1 kinase: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

Gene name - Lkb1 kinase

Synonyms - PAR-4

Cytological map position - 87F7--9

Function - signaling

Keywords - cell polarity, oogenesis, JNK pathway, apoptosis, oncogene

Symbol - lkb1

FlyBase ID: FBgn0038167

Genetic map position - 3R

Classification - serine/threonine kinase

Cellular location - cytoplasmic

NCBI links: Precomputed BLAST | EntrezGene
Recent literature
Gailite, I., Aerne, B. L. and Tapon, N. (2015). Differential control of Yorkie activity by LKB1/AMPK and the Hippo/Warts cascade in the central nervous system. Proc Natl Acad Sci [Epub ahead of print] PubMed ID: 26324895
The Hippo (Hpo) pathway is a highly conserved tumor suppressor network that restricts developmental tissue growth and regulates stem cell proliferation and differentiation. At the heart of the Hpo pathway is the progrowth transcriptional coactivator Yorkie. Yki activity is restricted through phosphorylation by the Hpo/Warts core kinase cascade, but increasing evidence indicates that core kinase-independent modes of regulation also play an important role. This study examined Yki regulation in the Drosophila larval CNS and uncovered a Hpo/Warts-independent function for the tumor suppressor kinase LKB1 and its downstream effector, the energy sensor AMP-activated protein kinase (AMPK), in repressing Yki activity in the central brain. Thus Yki is inhibited by the nutrient-sensing LKB1/AMPK cascade independent of Hpo/ Warts in a population of neural progenitors in the developing Drosophila larval brain. Although the Hpo/Warts core cascade restrains Yki in the optic lobe, it is dispensable for Yki target gene repression in the late larval central brain. Thus, this study demonstrates a dramatically different wiring of Hpo signaling in neighboring cell populations of distinct developmental origins in the central nervous system.

Raja, E., Tzavlaki, K., Vuilleumier, R., Edlund, K., Kahata, K., Zieba, A., Moren, A., Watanabe, Y., Voytyuk, I., Botling, J., Soderberg, O., Micke, P., Pyrowolakis, G., Heldin, C. H. and Moustakas, A. (2015). The protein kinase LKB1 negatively regulates bone morphogenetic protein receptor signaling. Oncotarget [Epub ahead of print]. PubMed ID: 26701726

The protein kinase LKB1 regulates cell metabolism and growth and is implicated in intestinal and lung cancer. Bone morphogenetic protein (BMP) signaling regulates cell differentiation during development and tissue homeostasis. This study demonstrates that LKB1 physically interacts with BMP type I receptors and requires Smad7 to promote downregulation of the receptor. Accordingly, LKB1 suppresses BMP-induced osteoblast differentiation and affects BMP signaling in Drosophila wing longitudinal vein morphogenesis. LKB1 protein expression and Smad1 phosphorylation analysis in a cohort of non-small cell lung cancer patients demonstrated a negative correlation predominantly in a subset enriched in adenocarcinomas. Lung cancer patient data analysis indicated strong correlation between LKB1 loss-of-function mutations and high BMP2 expression, and these two events further correlated with expression of a gene subset functionally linked to apoptosis and migration. This new mechanism of BMP receptor regulation by LKB1 has ramifications in physiological organogenesis and disease (Raja, 2015).

Dogliotti, G., Kullmann, L., Dhumale, P., Thiele, C., Panichkina, O., Mendl, G., Houben, R., Haferkamp, S., Puschel, A. W. and Krahn, M. P. (2017). Membrane-binding and activation of LKB1 by phosphatidic acid is essential for development and tumour suppression. Nat Commun 8: 15747. PubMed ID: 28649994
The serine/threonine kinase LKB1 regulates various cellular processes such as cell proliferation, energy homeostasis and cell polarity and is frequently downregulated in various tumours. Many downstream pathways controlled by LKB1 have been described but little is known about the upstream regulatory mechanisms. This study shows that targeting of the kinase to the membrane by a direct binding of LKB1 to phosphatidic acid is essential to fully activate its kinase activity. Consequently, LKB1 mutants that are deficient for membrane binding fail to activate the downstream target AMPK to control mTOR signalling. Furthermore, the in vivo function of LKB1 during development of Drosophila depends on its capacity to associate with membranes. Strikingly, LKB1 found to be downregulated in malignant melanoma, which exhibit aberrant activation of Akt and overexpress phosphatidic acid generating Phospholipase D. These results provide evidence for a fundamental mechanism of LKB1 activation and its implication in vivo and during carcinogenesis.
Couderc, J. L., Richard, G., Vachias, C. and Mirouse, V. (2017). Drosophila LKB1 is required for the assembly of the polarized actin structure that allows spermatid individualization. PLoS One 12(8): e0182279. PubMed ID: 28767695
In mammals, a testis-specific isoform of the protein kinase LKB1 is required for spermiogenesis, but its exact function and specificity are not known. Human LKB1 rescues the functions of Drosophila Lkb1 essential for viability, but these males are sterile, revealing a new function for this genes in fly. A testis-specific transcript was identified, generated by an alternative promoter; it only differs by a longer 5'UTR. dLKB1 is required in the germline for the formation of the actin cone, the polarized structure that allows spermatid individualization and cytoplasm excess extrusion during spermiogenesis. Three of the nine LKB1 classical targets in the Drosophila genome (AMPK, NUAK and KP78b) are required for proper spermiogenesis, but later than dLKB1. dLkb1 mutant phenotype is reminiscent of that of myosin V mutants, and both proteins show a dynamic localization profile before actin cone formation. Together, these data highlight a new dLKB1 function and suggest that dLKB1 posttranscriptional regulation in testis and involvement in spermatid morphogenesis are evolutionarily conserved features.

The PAR-4 and PAR-1 kinases are necessary for the formation of the anterior-posterior (A-P) axis in Caenorhabditis elegans (Kemphues, 1988; Watts, 2000; Guo, 1995). PAR-1 is also required for A-P axis determination in Drosophila. The Drosophila par-4 homologue, lkb1, is required for the early A-P polarity of the oocyte, and for the repolarization of the oocyte cytoskeleton that defines the embryonic A-P axis. LKB1 is phosphorylated by PAR-1 in vitro, and overexpression of LKB1 partially rescues the par-1 phenotype. These two kinases therefore function in a conserved pathway for axis formation in flies and worms. lkb1 mutant clones also disrupt apical-basal epithelial polarity, suggesting a general role in cell polarization. The human homologue, LKB1, is mutated in Peutz-Jeghers syndrome (Hemminki, 1998; Jenne, 1998) and is regulated by prenylation and by phosphorylation by protein kinase A (Collins, 2000; Sapkota, 2001). Protein kinase A phosphorylates Drosophila LKB1 on a conserved site that is important for its activity. Thus, Drosophila and human LKB1 may be functional homologues, suggesting that loss of cell polarity may contribute to tumour formation in individuals with Peutz-Jeghers syndrome (Martin, 2003).

The A-P axis of Drosophila is specified during oogenesis when a signal from the posterior follicle cells induces the formation of a polarized oocyte microtubule cytoskeleton, in which most minus ends are nucleated from the anterior cortex, with the plus ends extending towards the posterior pole. These polarized microtubules direct both the localization of bicoid messenger RNA to the anterior of the oocyte to specify where the head and thorax will develop, and the transport of oskar mRNA to the posterior, where it induces the formation of polar granules that contain the abdominal and germline determinants (Martin, 2003).

To identify other genes required for formation of the A-P axis, a genetic screen was carried out in germline clones for mutants that disrupt the localization of Staufen tagged with green fluorescent protein (GFP), which colocalizes with bicoid and oskar mRNAs. Mutants in one lethal complementation group of two alleles abolish the posterior localization of both Staufen and oskar mRNA in 80%-90% of oocytes. In over 40% of mutant oocytes, both bicoid and K10 mRNAs are detected around the whole oocyte cortex, rather than only at the anterior pole. In contrast to wild-type oocytes, which have an anterior-to-posterior gradient of microtubules, mutant oocytes have a high density of microtubules all around the cortex, with the lowest concentration in the centre. Kinesin-ß-galactosidase, a microtubule plus-end marker, also fails to concentrate at the posterior of the oocyte and accumulates instead in the centre. Thus, these mutants disrupt bicoid and oskar mRNA localization by preventing the A-P polarization of the microtubules (Martin, 2003).

Hypomorphic mutants in Drosophila par-1 show defects in the polarization of the oocyte microtubule cytoskeleton and in the localization of bicoid and oskar mRNA that are very similar to the defects of lkb1 mutants. The PAR-1 kinase is also required much earlier in oogenesis for the determination of the oocyte. The oocyte is selected from a cyst of 16 germline cells in the germarium and forms a microtubule-organizing centre, which directs the microtubule-dependent localization of oocyte-specific factors, such as ORB, to this cell. The microtubule-organizing centre then moves from the anterior to the posterior of the oocyte in region 3, the most posterior region of the germarium, and oocyte-specific factors accumulate posteriorly. This anterior-to-posterior switch is disrupted in par-1 null mutants, and the oocyte exits meiosis and becomes a nurse cell (Martin, 2003).

To study the role of lkb1 in this process, mutant germline clones marked by the loss of GFP were induced. ORB still accumulates in the presumptive oocyte in lkb1 cysts but often fails to move to the posterior in region 3; instead, it disperses throughout the cyst as the oocyte exits meiosis and adopts the nurse cell fate. Thus, lkb1 and par-1 share very similar phenotypes in both oocyte determination and polarization, suggesting that they function together. Because the penetrance of the early lkb1 phenotype increases with age (67% of mutant clones in 5-day-old females, rising to 85% after 12 d), wild-type LKB1 activity seems to perdure for several days after the clones are induced, and this presumably accounts for the escapers that show the later oocyte polarity phenotype (Martin, 2003).

Since PAR-1 and LKB1 seem to act in a common pathway, whether either kinase could phosphorylate the other was tested. Although recombinant PAR-1 is not a substrate for LKB1, immunoprecipitated GFP-PAR-1 phosphorylates recombinant LKB1 in vitro. It was found that the phosphorylation site or sites are located in the amino-terminal half of the protein, although it has not been possible to map them precisely. Also a strong genetic interaction is observed between the two genes. The oskar mRNA localization defects in hypomorphic combinations of par-1 alleles cause loss of abdominal segments in the embryo, which can be strongly enhanced by removing one copy of lkb1. In addition, overexpression of GFP-LKB1 partially rescues the A-P polarity phenotype of par-16323/par-1W3, the strongest allelic combination that produces late-stage oocytes. Whereas Staufen localizes to the posterior normally in only 12% of these oocytes, 80% have wild-type amounts of Staufen at the posterior when LKB1 is overexpressed. This phenotype is not rescued by a kinase-dead form of LKB1, indicating that this suppression requires kinase activity. By contrast, overexpression of PAR-1 does not rescue the phenotype of lkb1 mutant germline clones. These results are consistent with a model in which LKB1 is a direct target of PAR-1 regulation in vivo and functions as a downstream effector in the polarization of the oocyte microtubule cytoskeleton (Martin, 2003).

A GFP-LKB1 fusion construct under the control of the endogenous promoter rescues both the lethality and oogenesis phenotypes of lkb1 mutants and is expressed in very low amounts in both the germline and somatic follicle cells of the ovary. The highest expression was observed in the germarium, where GFP-LKB1 colocalizes with PAR-1 on the fusome, a branched membranous organelle that connects the germ cells in a cyst. This localization presumably reflects their common function in early oocyte polarity and determination, because the cell that inherits most fusome is selected to become the oocyte. During the rest of oogenesis, GFP-LKB1 shows a uniform cortical localization in both the germline and the follicle cells. It is enriched in the oocyte from stage 7, when the A-P axis is polarized, and colocalizes with cortical actin, but not with pole plasm components (Martin, 2003).

Follicle cell clones mutant for lkb1 also show a defect in polarity. In severely affected clones, the follicular monolayer is disorganized, with mutant cells rounding up and sorting out of the epithelium. Morphologically wild-type clones show defects in the apical localization of atypical protein kinase C (aPKC) and Armadillo, which become either diffuse or ectopically localized along lateral membranes. In less severely affected cells, the apical localization is discontinuous. These phenotypes are penetrant in large stem-cell clones but not in small clones, indicating that LKB1 activity perdures. Expression of the wild-type or S535E transgenes with arm-GAL4 in lkb1 mutants rescues these follicle cell phenotypes, whereas expression of S535A rescues lethality but gives rise to a completely disorganized follicular epithelium, in which most cells appear unpolarized. Thus, LKB1 is required for cell polarity in the germ line and the follicle cells, and is probably regulated by phosphorylation on the conserved C-terminal serine in both processes (Martin, 2003).

In Drosophila, par-1 and lkb1, the homologue of C. elegans par-4, show very similar phenotypes. In addition, LKB1 is an in vitro substrate for PAR-1 and can suppress the polarity phenotype of par-1 mutants when overexpressed. These results suggest that LKB1 functions downstream of PAR-1. This conclusion is consistent with genetic data in C. elegans that show that par-4 mutants display only a subset of the par-1 A-P polarity phenotypes. Notably, mutants in par-4 and par-1, but not in other par genes, show a disappearance of P granules in the one-cell zygote (Kemphues, 1988). Thus, LKB1 and PAR-1 function in a conserved pathway that is required for the polarization of the A-P axis in both worms and flies (Martin, 2003).

Drosophila LKB1 is closely related to human LKB1, and conserved prenylation and PKA phosphorylation sites are essential for the in vivo function of both proteins, indicating that the two are likely to be functional homologues. Mutants in LKB1 cause Peutz-Jeghers syndrome (Hemminki, 1998; Jenne, 1998), which is characterized by the formation of intestinal polyps and a high incidence of adeno-carcinomas (tumours of epithelial origin) (Hemminki, 1999). In addition, mutations in lkb1 have been identified in several sporadic epithelial cancers (Sanchdz-Cdspedes, 2002). The role of LKB1 as a tumour suppressor is not well understood, however, and LKB1 has been proposed to regulate apoptosis, the cell cycle or angiogenesis (Yoo, 2002; Bardeesy, 2002). In addition, LKB1 seems to function in a context-dependent manner that is different from classical tumour-suppressor genes such as ras or p53. Given that Drosophila lkb1 is required to polarize the epithelial follicle cells, an alternative model is proposed, that loss of LKB1 leads to polyp and tumour formation by disrupting epithelial polarity (Martin, 2003).

Feeding and fasting signals converge on the LKB1-SIK3 pathway to regulate lipid metabolism in Drosophila

LKB1 plays important roles in governing energy homeostasis by regulating AMP-activated protein kinase (AMPK) and other AMPK-related kinases, including the salt-inducible kinases (SIKs). However, the roles and regulation of LKB1 in lipid metabolism are poorly understood. This study shows that Drosophila LKB1 mutants display decreased lipid storage and increased gene expression of brummer, the Drosophila homolog of adipose triglyceride lipase (ATGL). These phenotypes were consistent with those of Salt-inducible kinase 3 (SIK3) mutants and were rescued by expression of constitutively active SIK3 in the fat body, suggesting that SIK3 is a key downstream kinase of LKB1. Using genetic and biochemical analyses, HDAC4, a class IIa histone deacetylase, was identified as a lipolytic target of the LKB1-SIK3 pathway. Interestingly, it was found that the LKB1-SIK3-HDAC4 signaling axis was modulated by dietary conditions. In short-term fasting, the adipokinetic hormone (AKH) pathway, related to the mammalian glucagon pathway, inhibited the kinase activity of LKB1 as shown by decreased SIK3 Thr196 phosphorylation, and consequently induced HDAC4 nuclear localization and brummer gene expression. However, under prolonged fasting conditions, AKH-independent signaling decreased the activity of the LKB1-SIK3 pathway to induce lipolytic responses. It was also identified that the Drosophila insulin-like peptides (DILPs) pathway, related to mammalian insulin pathway, regulated SIK3 activity in feeding conditions independently of increasing LKB1 kinase activity. Overall, these data suggest that fasting stimuli specifically control the kinase activity of LKB1 and establish the LKB1-SIK3 pathway as a converging point between feeding and fasting signals to control lipid homeostasis in Drosophila (Choi, 2015).

Perturbation of energy homeostasis either directly or indirectly causes human health problems such as obesity and type II diabetes. Lipid stores are the major energy reserves in animals and are dynamically regulated by alternating between the lipogenesis and lipolysis cycles in response to food availability. Dissecting the regulatory mechanisms of lipid homeostasis is therefore essential to understanding of how energy metabolism is maintained (Choi, 2015).

Drosophila has emerged as a useful genetic model organism for studying lipid homeostasis and energy metabolism. Drosophila lipid reserves are mainly stored as triacylglycerol (TAG) in the fat body, the insect equivalent of mammalian adipose tissue. In addition, lipolytic factors are evolutionarily conserved between insects and mammals. Brummer (Bmm) is the Drosophila homolog of ATGL, a key regulator of lipolysis. bmm mutant flies are obese and display partial defects in lipid mobilization. Furthermore, hormonal regulation of lipid metabolism is also highly conserved in Drosophila. Under starvation conditions, the primary role of AKH, the functional analogue of glucagon and β-adrenergic signaling in mammals, is to stimulate lipid mobilization by activating Adipokinetic hormone receptor (AKHR) and consequently inducing cAMP/PKA signaling in the fat body. A report demonstrated that AKH acts in parallel with Bmm to regulate lipolysis and that AKHR mutation leads to obesity phenotypes and defects in fat mobilization. However, bmm expression is hyperstimulated in starved AKHR mutants, implying the existence of an unknown regulatory mechanism between Bmm and AKHR in Drosophila (Choi, 2015).

LKB1 (liver kinase B1, also known as STK11) is a serine/threonine kinase that was first identified as a tumor suppressor gene associated with Peutz-Jeghers syndrome. LKB1 phosphorylates and activates AMP-activated protein kinase (AMPK) in response to cellular energy status, thus controlling cell metabolism, cell structures, apoptosis, etc. Moreover, LKB1 is the master upstream protein kinase for 12 AMPK-related kinases, including salt-inducible kinases (SIKs), suggesting that it plays diverse roles. Although the metabolic functions of AMPK have been highly studied, the in vivo functions of LKB1 and AMPK-related kinases in metabolism, including lipid homeostasis, are still largely unknown. Recent reports showed that LKB1 is required for the growth and differentiation of white adipose tissue and that SIK3 maintains lipid storage size in adipose tissues. In addition, Drosophila SIK family kinases regulate lipid levels and starvation responses (Choi, 2011; Wang, 2011). However, to further understand the roles and mechanisms of LKB1 signaling in lipid metabolism, proper genetic animal models are urgently required (Choi, 2015).

This study has demonstrated the role of LKB1 and its downstream SIK3 in the regulation of lipid homeostasis using Drosophila as an in vivo model system. LKB1-activated SIK3 regulates the nucleocytoplasmic localization of HDAC4 to control lipolytic gene expression. This study also identified DILPs modulate SIK3 activity via Akt-dependent phosphorylation and that the AKH pathway regulates LKB1 activity in phosphorylating SIK3 to control its lipolytic responses upon short-term fasting. Furthermore, AKH-independent signaling modulates the LKB1-SIK3-HDAC4 pathway upon prolonged fasting. Altogether, these studies showed that the LKB1-SIK3 signaling pathway plays a crucial regulatory role in maintaining lipid homeostasis in Drosophila (Choi, 2015).

This study provides evidence that LKB1 is necessary for maintaining Drosophila lipid storage via the regulation of lipolysis through the activation of SIK3. Consistent with thtes results in Drosophila, adipose tissue-specific LKB1 knockout mice showed decreased serum triglycerides, and the basal lipogenesis activity of adipocytes was significantly lower in LKB1 hypomorphic mice. Recently, SIK3 null mice were also found to display a malnourished phenotype with lipodystrophy and were resistant to high-fat diets. Thus, the LKB1-SIK3 pathway is indeed an evolutionally conserved regulatory mechanism for lipid homeostasis (Choi, 2015).

LKB1 is ubiquitously expressed and constitutively active in mammalian cells, which raises the question of how dietary conditions change the activity of LKB1 and SIK3 to control lipid homeostasis. The curreny findings suggested that fasting and the AKH pathway inhibit LKB1 activity to regulate SIK3 Thr196 phosphorylation. It is possible that fasting- and AKH-induced inhibition of LKB1 activity can be achieved by altered subcellular localization, protein conformation, stability, and/or protein-protein interactions of LKB1 and its associated proteins. Interestingly, in HEK-293 cells, fasting triggers autophosphorylation of human LKB1 at Thr336 that corresponds to Thr460 in Drosophila LKB1. This phosphorylation promotes the protein-protein interaction between LKB1 and 14-3-3 proteins and inhibits the ability of LKB1 for suppressing cell growth (Choi, 2015).

In addition, the AKH pathway activates cAMP/PKA signaling in Drosophila. Mammalian PKA inhibits SIK activity by phosphorylating a conserved serine residue that corresponds to Ser563 in Drosophila SIK3], suggesting that the AKH pathway also controls SIK3 activity via PKA-dependent phosphorylation. On the other hand, the Drosophila insulin-like peptides (DILPs) did not increase SIK3 Thr196 phosphorylation, but induced Akt-mediated SIK3 phosphorylation, suggesting that DILPs directly regulate SIK3 activity independently of affecting LKB1 activity. Interestingly, these Drosophila signaling circuits are highly similar to mammalian insulin and glucagon pathways in controlling lipid metabolism and storage, raising questions regarding whether the LKB1-SIK3-HDAC4 signaling pathway is also conserved in mammalian systems as a converging point between feeding and fasting signals to control lipid homeostasis (Choi, 2015).

Is SIK3 also involved in the modulation of other LKB1 functions, such as the regulation of cell polarity and mitosis? SIK3 null mutants showed normal epithelial polarity and mitosis. Additionally, transgenic expression of constitutively active SIK3 (SIK3 T196E) failed to suppress the cell polarity and mitosis defects of LKB1 mutants, suggesting that SIK3 does not participate in the regulation of cell polarity and mitosis by LKB1. In addition, both fat body-specific expression of LKB1 and ablation of HDAC4 failed to rescue the lethality of LKB1 null mutants, indicating that LKB1 has SIK3/HDAC4-independent roles and additional targets in other tissues and developmental processes (Choi, 2015).

In summary, this study has demonstrated that the LKB1-SIK3 pathway is important for maintaining lipid homeostasis in Drosophila. As alterations in lipolysis are closely associated with human obesity, future studies will be required to unravel the relationship between LKB1-SIK3-HDAC4 signaling and obesity-related metabolic diseases (Choi, 2015).

Sgt1 acts via an LKB1/AMPK pathway to establish cortical polarity in larval neuroblasts

Drosophila neuroblasts are a model system for studying stem cell self-renewal and the establishment of cortical polarity. Larval neuroblasts generate a large apical self-renewing neuroblast, and a small basal cell that differentiates. A genetic screen was performed to identify regulators of neuroblast self-renewal, and a mutation was identified in sgt1 (suppressor-of-G2-allele-of-skp1) that had fewer neuroblasts. sgt1 neuroblasts have two polarity phenotypes: failure to establish apical cortical polarity at prophase, and lack of cortical Scribble localization throughout the cell cycle. Apical cortical polarity was partially restored at metaphase by a microtubule-induced cortical polarity pathway. Double mutants lacking Sgt1 and Pins (a microtubule-induced polarity pathway component) resulted in neuroblasts without detectable cortical polarity and formation of 'neuroblast tumors.' Mutants in hsp83 (encoding the predicted Sgt1-binding protein Hsp90), LKB1 (PAR-4), or AMPKα all show similar prophase apical cortical polarity defects (but no Scribble phenotype), and activated AMPKα rescued the sgt1 mutant phenotype. It is proposed that an Sgt1/Hsp90-LKB1-AMPK pathway acts redundantly with a microtubule-induced polarity pathway to generate neuroblast cortical polarity, and the absence of neuroblast cortical polarity can produce neuroblast tumors (Anderson, 2012).

This study presents evidence that the evolutionary-conserved protein Sgt1 acts with Hsp90, LKB1 and AMPK to promote apical localization of the Par and Pins complexes in prophase neuroblasts. It is proposed that Sgt1/Hsp90 proteins function together based on multiple lines of evidence: (1) they show conserved binding from plants to humans; (2) the sgt1s2383 mutant results in a five amino acid deletion within the CS domain, which is the Hsp90 binding domain; (3) sgt1 and hsp83 have similar cell cycle phenotypes; and (4) sgt1 and hsp83 have similar neuroblast polarity phenotypes. The Sgt1/Hsp90 complex either stabilizes or activates client proteins (Zuehlke, 2010); it is suggested that Sgt1 activates LKB1, rather than stabilizing it, because it was not possible to rescue the sgt1 mutant phenotype by simply overexpressing wild type LKB1 protein. No tests were performed for direct interactions between Sgt1 and LKB1 proteins, and thus the mechanism by which Sgt1 activates LKB1 remains unknown (Anderson, 2012).

LKB1 is a 'master kinase' that activates at least 13 kinases in the AMPK family. It is suggested that LKB1 activates AMPK to promote neuroblast polarity because overexpression of phosphomimetic, activated AMPKα can rescue the lkb1 and sgt1 mutant phenotype. It remains unclear how AMPK activity promotes apical protein localization. An antibody to activated AMPKα (anti-phosphoT385-AMPKα shows spindle and cytoplasmic staining that is absent in ampkα mutants, and centrosomal staining that persists in AMPKα null mutants, but no sign of asymmetric localization in neuroblasts. AMPK activity is thought to directly or indirectly activate myosin regulatory light chain to promote epithelial polarity. AMPK is activated by a rise in AMP/ATP levels that occur under energy stress or high metabolism; AMP binds to the γ regulatory subunit of the heterotrimeric complex and results in allosteric activation of the α subunit. ampkα mutants grown under energy stress have defects in apical/basal epithelial cell polarity in follicle cells within the ovary. In contrast, AMPKα mutants grown on nutrient rich food still show defects in embryonic epithelial polarity, neuroblast apical polarity, and visceral muscle contractio. Larval neuroblasts, embryonic ectoderm, and visceral muscle may have a high metabolic rate, require low basal AMPK activity, or use a different mechanism to activate AMPK than epithelial cells. What are the targets of AMPK signaling for establishing apical cortical polarity in larval neuroblasts? AMPK could directly phosphorylate Baz to destabilize the entire pool of apical proteins, but currently there is no evidence supporting such a direct model. AMPK may act via regulating cortical myosin activity: clear defects have been seen in cortical motility, ectopic patchy activated myosin at the cortex, and failure of cytokinesis in sgt1, lkb1, and ampkα mutants. This strongly suggests defects in the regulation of myosin activity, but how or if gain/loss/mispositioning of myosin activity leads to failure to establish apical cortical polarity remains unknown. Lastly, the defects in apical cell polarity seen at prophase could be due to the prometaphase cell cycle delays (Anderson, 2012).

What activates the Sgt1-LKB1-AMPK pathway to promote cell polarity during prophase? In budding yeast, Sgt1 requires phosphorylation on Serine 361 (which is conserved in Drosophila Sgt1) for dimerization and function (Bansal, 2009); this residue is conserved in Drosophila Sgt1 but its functional significance is unknown (Anderson, 2012).

Sgt1/Hsp90/LKB1/AMPK are all required for apical Par/Pins complex localization, but Sgt1 must act via a different pathway to promote Dlg/Scrib cortical localization, because only the sgt1 mutant affects Dlg/Scrib localization, and overexpression of activated AMPKα is unable to restore cortical Scrib in sgt1 mutants. The mechanism by which Sgt1 promotes Dlg/Scrib cortical localization is unknown (Anderson, 2012).

This study has shown that sgt1 mutants lack Par/Pins apical polarity in prophase neuroblasts, but these proteins are fairly well polarized in metaphase neuroblasts. The rescue of cortical polarity is microtubule dependent, probably occurring via the previously described microtubule-dependent cortical polarity pathway containing Pins, Dlg and Khc-73. The weak polarity defects still observed in sgt1 metaphase neuroblasts may be due to the poor spindle morphology. The lack of microtubule-induced polarity at prophase, despite a robust microtubule array in prophase neuroblasts, suggests that the microtubule-induced cortical polarity pathway is activated at metaphase. Activation of the pathway could be via expression of the microtubule-binding protein Khc-73; via phosphorylation of Pins, Dlg or Khc-73 by a mitotic kinase like Aurora A; or via a yet unknown pathway (Anderson, 2012).


Protein Interactions

Drosophila LKB1 also has a conserved RKLS consensus phosphorylation site near its C terminus. In mammalian LKB1, this site is phosphorylated in vitro and in vivo by protein kinase A (PKA) and is required for its ability to suppress cell growth in culture (Collins, 2000; Sapkota, 2001). Like its vertebrate counterparts, Drosophila LKB1 is phosphorylated in a PKA-dependent manner. Coexpression of wild-type LKB1 and PKA in S2 cells induces a phosphatase-sensitive mobility shift of LKB1 on Western blots, which is abolished when serine 535 is mutated to alanine. To assay the significance of phosphorylation of this conserved site, transgenes were generated in which the serine was mutated to either alanine (S535A) to prevent phosphorylation, or to glutamic acid (S535E) to mimic the presence of a charged phosphate group. Expression of the transgenes with arm-GAL4 allows the recovery of lkb1 mutant flies expressing low amounts of either wild-type or mutant proteins in the germ line. GFP-LKB1S535A does not rescue the localization of Staufen to the posterior of the oocyte, whereas GFP-LKB1S535E rescues even more efficiently than the wild-type control, strongly suggesting that LKB1 is positively regulated by phosphorylation at this site. Because S535 is phosphorylated by PKA in vivo and PKA is required in the germ line to polarize the oocyte, it is speculate that PKA regulates Drosophila LKB1 in the germ line. Additional signals must regulate LKB1, however, because the lack of phosphorylation on S535 does not abolish LKB1 activity completely. Tenfold overexpression of S535A in lkb1 germline clones partially rescues the localization of Staufen to the posterior of the oocyte, albeit less efficiently than the wild-type transgene, whereas a kinase-dead version (K174M) shows no rescuing activity on overexpression. Thus, LKB1 may be regulated by both PAR-1 and PKA, and may function to integrate the two signalling pathways during the polarization of the oocyte (Martin, 2003).

Energy-dependent regulation of cell structure by AMP-activated protein kinase

AMP-activated protein kinase (AMPK, also known as SNF1A: see Drosophila Alicorn) has been primarily studied as a metabolic regulator that is activated in response to energy deprivation. Although there is relatively ample information on the biochemical characteristics of AMPK, not enough data exist on the in vivo function of the kinase. Using the Drosophila model system, animals with no AMPK activity were genrated and physiological functions of the kinase investigated. Surprisingly, AMPK-null mutants are lethal with severe abnormalities in cell polarity and mitosis, similar to those of lkb1-null mutants. Constitutive activation of AMPK restores many of the phenotypes of lkb1-null mutants, suggesting that AMPK mediates the polarity- and mitosis-controlling functions of the LKB1 serine/threonine kinase. Interestingly, the regulatory site of non-muscle myosin regulatory light chain (MRLC, Spaghetti-squash; also known as MLC2 was directly phosphorylated by AMPK. Moreover, the phosphomimetic mutant of MRLC rescued the AMPK-null defects in cell polarity and mitosis, suggesting MRLC is a critical downstream target of AMPK. Furthermore, the activation of AMPK by energy deprivation was sufficient to cause dramatic changes in cell shape, inducing complete polarization and brush border formation in the human LS174T cell line, through the phosphorylation of MRLC. Taken together, these results demonstrate that AMPK has highly conserved roles across metazoan species not only in the control of metabolism, but also in the regulation of cellular structures (Lee, 2007).

The catalytic subunit of Drosophila AMPK is a single orthologue of its human and yeast counterparts, and is activated by LKB1 on energy deprivation. By imprecise excision of the EP-element (enhancer- and promoter-containing P-element) from the AMPKG505 line, AMPK-null mutant lines, AMPKD1 and AMPKD2 were generated. Interestingly, all AMPK-null mutant flies are lethal before the mid-pupal stage and fail to enter adulthood, even in the presence of sufficient nutrients. Although transgenic expression of wild-type AMPK (AMPKWT) allowed AMPK-null mutants to successfully develop into adults, the expression of kinase-dead AMPK (AMPKKR) failed to rescue the lethality, demonstrating that the phosphotransferase activity of AMPK is crucial for its function. In summary, AMPK was found to be essential for normal development of Drosophila (Lee, 2007).

The developmental role of AMPK was further investigated by generating AMPK-null germ-line clone (AMPK-GLC) embryos, which are completely deprived of both the maternal and zygotic AMPK proteins. Surprisingly, AMPK-GLC embryos never developed into larvae, showing the requirement of AMPK during embryogenesis. In AMPK-GLC embryos, cuticle structures were severely deformed, and ventral denticle belts were missing. Furthermore, the surface of AMPK-GLC embryos was roughened and the columnar structure of the epidermis was disorganized, implicating defects in underlying epithelial structures (Lee, 2007).

To examine the embryonic epithelial structures, AMPK-GLC epithelia were examined with various polarity markers. Bazooka (Baz, apical complex marker) and β-catenin (Arm, adherens junction marker) lost their apical localization and were found in various locations around the basolateral cell surfaces. The Discs-large (Dlg, basolateral marker was also irregularly distributed throughout the epithelium in AMPK-GLC embryos. Moreover, actin staining demonstrated that the AMPK-GLC epithelium contains many unpolarized round cells that had lost contact with the underlying tissue. This disorganization of epithelial structures was not a result of cell death, because it could not be restored by overexpression of apoptosis inhibitor p35. In addition, wing discs of AMPK-null mutants also showed defective epithelial organization with ectopic actin structures in the basolateral region. These results indicate that AMPK is indispensable for epithelial integrity (Lee, 2007).

In addition, abnormally enlarged nuclei were found in some cells of AMPK-GLC embryos. Mitotic chromosome staining with anti-phospho-histone H3 (PH3) antibody demonstrated that AMPK-GLC embryos frequently contained defective mitotic cells with lagging or polyploid chromosomes. Consistently, aceto-orcein staining of squashed AMPK-null larval brains revealed polyploidy in ~30% of mitotic cells, and anti-PH3 staining showed a highly increased amount of chromosome content in some of the neuroblasts. These results indicate that AMPK is also required for the maintenance of genomic integrity (Lee, 2007).

Recently, it has been proposed that LKB1, a kinase upstream of AMPK, is involved in the regulation of epithelial polarity and mitotic cell division. Indeed, the abnormal polarity and mitosis phenotypes of lkb1-null mutants were highly similar to those of AMPK-null mutants. To test whether AMPK mediates the polarity- and mitosis-controlling functions of LKB1, constitutively active AMPK (AMPKTD), which is catalytically active even without phosphorylation by LKB1, was expressed in lkb1-null mutants. Remarkably, AMPKTD suppresses the epithelial polarity defects and the genomic instability of lkb1-null mutants, suggesting that AMPK is a critical downstream mediator of LKB1, controlling mitosis and cell polarity (Lee, 2007).

To understand the molecular mechanism underlying the AMPK-dependent control of mitosis and cell polarity, attempts were made to identify the downstream targets of AMPK. Intriguingly, MRLC, a critical molecule for the execution of mitosis and cell polarity establishment, contains a peptide sequence that can be phosphorylated by AMPK. Therefore, various experiments were performed to evaluate the ability of AMPK to phosphorylate MRLC. AMPK holoenzyme purified from rat liver strongly phosphorylated full-length MRLC, which was further enhanced by the addition of AMP. The phosphorylation of MRLC was more efficient than that of acetyl-CoA carboxylase 2 (ACC2), a representative substrate of AMPK, indicating that MRLC is a good in vitro substrate of AMPK. It was deduced that this phosphorylation is specifically performed by AMPK because Compound C, a specific inhibitor of AMPK, inhibited the phosphorylation, whereas ML-7, an inhibitor of another MRLC-phosphorylating kinase (MLCK), did not. A mutant form of MRLC, whose regulatory phosphorylation site (corresponding to Thr 21/Ser 22 in Drosophila and Thr 18/Ser 19 in human) was mutated into non-phosphorylatable alanines, was not phosphorylated by AMPK, suggesting that MRLC is exclusively phosphorylated at the regulatory phosphorylation site. Both the human and Drosophila forms of AMPK were able to phosphorylate MRLC from each of the respective species, which further demonstrates that the AMPK phosphorylation of MRLC is highly conserved between species (Lee, 2007).

Moreover, it was found that MRLC phosphorylation is indeed regulated by AMPK in vivo. The phosphorylation of MRLC was dramatically reduced in AMPK- and lkb1-GLC epithelia when compared with the wild-type epithelia, although the protein level of MRLC was unaffected. The reduced phosphorylation of MRLC in the AMPK-GLC epithelia was completely restored by transgenic expression of AMPK but not by overexpression of LKB1. Furthermore, in Drosophila S2 cells, energy deprivation induced by 2-deoxyglucose (2DG) enhanced MRLC phosphorylation, which was suppressed by double-strand-RNA-mediated silencing of lkb1 or AMPK. Collectively, these data strongly suggest that MRLC is specifically phosphorylated by AMPK both in vitro and in vivo (Lee, 2007).

To find out whether the phosphorylation of MRLC is critical for the physiological functions of AMPK, an active form of MRLC (MRLCEE), whose regulatory phosphorylation site was mutated into phosphomimetic glutamates, was expressed in AMPK-GLC embryos. Strikingly, MRLCEE rescued the epithelial polarity defects caused by the loss of AMPK, and increased the percentage of cuticle-forming embryos from ~10% to ~30. MRLCEE also restored the epithelial polarity defects of lkb1-null wing imaginal discs. Furthermore, the genomic polyploidy of AMPK- and lkb1-null larval brain neuroblasts was suppressed by the expression of MRLCEE. Therefore, it is concluded that MRLC is a critical downstream target of AMPK controlling cell polarity and mitosis (Lee, 2007).

Notably, the larval brains of MRLC loss-of-function mutants (spaghetti-squash1) showed extensive polyploidy (~40% of mitotic neuroblasts), and their imaginal discs showed severe disorganization in epithelial structure, similar to those of lkb1- and AMPK-null mutants. These phenotypic similarities further support the conclusion that MRLC is an important functional mediator of LKB1 and AMPK (Lee, 2007).

Finally, it was asked whether AMPK is critical for directing cell polarity in mammalian cells as well. To assess this, it was asked whether the activation of AMPK by 2DG treatment could induce polarization of unpolarized epithelial cells such as LS174T, which can be polarized by the activation of LKB1, in a cell-autonomous manner. Surprisingly, on 2DG treatment, LS174T cells undergo a dramatic change in cell shape to have polarized actin cytoskeleton with a brush-border-like structure. Moreover, although brush border marker villin, apical marker CD66/CEA, and basal marker CD71/transferrin were distributed throughout untreated cells, they became dramatically polarized on 2DG treatment, supporting that the activation of AMPK by energy deprivation is sufficient to induce complete polarization of LS174T cells (Lee, 2007).

It was also found that the phosphorylation of MRLC by AMPK is involved in the energy-dependent polarization of LS174T cells. Phosphorylated MRLC was colocalized with the 2DG-induced polarized actin structures, and this phosphorylation, as well as the actin polarization, was suppressed by the AMPK-specific inhibitor Compound C. Overexpression of dominant-negative AMPK (AMPKDN) and short interfering (si)RNA-mediated inhibition of MRLC (siMRLC) also blocked the polarization, although inhibition of Par-1, another downstream kinase of LKB1, by Par-1 siRNA (siPar-1) or overexpression of dominant-negative Par-1 (Par-1DN) failed to cause a block. More strikingly, human MRLCEE itself was sufficient to polarize LS174T cells, even without energy deprivation, showing that phosphorylation of MRLC is critical for the AMPK-dependent polarization (Lee, 2007).

Until now, the importance of AMPK has been limited to its role as a regulator of metabolism. However, by generating the first animal model with no AMPK activity, additional functions of AMPK were characterized: AMPK regulates mitotic cell division and epithelial polarity downstream of LKB1 by controlling the activity of MRLC through direct phosphorylation. The findings revealed a link between energy status and cell structures, providing a new perspective to the diverse molecular function of AMPK. Further studies are needed on the cell-structure-controlling function of AMPK with respect to the various metabolic and physiological contexts, which may also help to understand AMPK-related diseases such as cancer and diabetes (Lee, 2007).


EGFR signaling promotes the identity of follicle stem cells via maintenance of partial cell polarity

Epithelial stem cells divide asymmetrically, such that one daughter replenishes the stem cell pool and the other differentiates. This study found that in the epithelial follicle stem cell (FSC) lineage of the Drosophila ovary, EGFR signaling functions specifically in the FSCs to promote the unique partially polarized state of the FSC, establish apical-basal polarity throughout the lineage, and promote FSC maintenance in the niche. In addition, a novel connection was identified between EGFR signaling and the cell polarity regulator, LKB1, which indicates that EGFR signals through both the Ras-Raf-MEK-Erk pathway and through the LKB1-AMPK pathway to suppress apical identity. The development of apical-basal polarity is the earliest visible difference between FSCs and their daughters, and these findings demonstrate that the EGFR-mediated regulation of apical-basal polarity is essential for the segregation of stem cell and daughter cell fates (Castanieto, 2014: PubMed).


Although recent progress has unveiled the diverse in vivo functions of LKB1, detailed molecular mechanisms governing these processes still remain enigmatic. This study shows that Drosophila LKB1 negatively regulates organ growth by caspase-dependent apoptosis, without affecting cell size and cell cycle progression. Through genetic screening for LKB1 modifiers, the JNK pathway was discovered as a novel component of LKB1 signaling. The JNK pathway is activated by LKB1 and mediates the LKB1-dependent apoptosis. Consistently, the LKB1-null mutant is defective in embryonic apoptosis and displayed a drastic hyperplasia in the central nervous system: these phenotypes are fully rescued by ectopic JNK activation as well as wild-type LKB1 expression. Furthermore, inhibition of LKB1 results in epithelial morphogenesis failure, which is associated with a decrease in JNK activity. Collectively, these studies unprecedentedly elucidate JNK as the downstream mediator of the LKB1-dependent apoptosis, and provide a new paradigm for understanding the diverse LKB1 functions in vivo (Lee, 2005).

Tumor suppressor LKB1 is a serine/threonine kinase that is heterozygotically mutated in the germ line of Peutz-Jegher syndrome (PJS) patients. PJS patients have a highly increased risk of malignant tumors, especially in the gastrointestinal tract, breast, uterine cervix, and ovary. Many of these tumors acquire somatic mutations in the remaining wild-type allele of LKB1. Moreover, various sporadic cancers are also associated with the loss of LKB1, implicating the general role of LKB1 in tumor suppression. Since point mutations of LKB1 found in PJS mostly reside in the highly conserved kinase domain, it is very likely that the kinase activity of LKB1 is essential for its tumor suppressive function (Lee, 2005 and references therein).

Since the discovery of LKB1 in 1998, various functions of LKB1 molecule have been suggested to be responsible for its tumor-suppressing activity, such as inhibition of cell cycle progression, cell growth retardation, apoptotic cell death, and cell polarity control. The LKB1 protein was originally characterized as a cell cycle inhibitor, and its putative downstream targets such as Brg1 and p21 were suggested to mediate LKB1-dependent cell cycle arrest. It has been recently proposed that LKB1 also regulates cellular growth by controlling another tumor suppressor, tuberous sclerosis complex (TSC), via the AMP kinase (AMPK)-dependent pathway. In addition, studies showing the absence of apoptosis in PJS polyps reveal that LKB1 is an inducer of apoptosis in vivo. Another tumor suppressor, p53, the first identified in vitro substrate of LKB1, was proposed as a mediator of this apoptosis. Finally, LKB1 was shown to be necessary for the polarization of intestinal epithelial cells and Drosophila ovary cells, demonstrating another important function of LKB1 in regulating cell structure (Lee, 2005).

Although considerable progress has been made to characterize the in vivo function of LKB1, only a limited amount of information concerning its molecular mechanisms has been found in detail; the major biological pathway responsible for the tumor suppressive function of LKB1 remains to be clarified (Lee, 2005).

The c-Jun N-terminal kinase (JNK) is a member of the mitogen-activated protein kinase (MAPK) family that mediates various cellular responses including programmed cell death, epithelial sheet movement, and planar polarity. The activity of JNK is tightly regulated by reversible phosphorylation, which is stimulated by a sequential cascade of protein kinases and inhibited by JNK-specific phosphatases. Activation of the JNK pathway is required for the release of cytochrome c from the mitochondria and the subsequent activation of the caspase cascade. Therefore, abrogation of the JNK signaling pathway causes various defects in developmental or stress-induced apoptosis. In addition to controlling apoptosis, regulation of epithelial morphogenesis is another well-known function of the JNK signaling pathway. Depletion of JNK activity results in various epithelial defects, including those of dorsal closure and planar polarity in Drosophila, and of the optic and neuronal system in mice (Lee, 2005 and references therein).

Since JNK is required for apoptosis and epithelial organization, disruption of the JNK signaling pathway is thought to be a prerequisite of tumorigenesis by enabling tumor cells to evade programmed cell death and to acquire a metastatic potential. Indeed, JNK1 and JNK2 are both required for the suppression of oncogenic transformation and tumorigenesis. Loss of JNK3, which is selectively expressed in neuronal cells, is also closely associated with human brain tumors. Furthermore, the Mkk4 gene, which encodes an upstream kinase of JNK, has been identified as a tumor suppressor gene and a metastasis suppressor gene (Lee, 2005 and references therein).

This paper shows a genetic connection between tumor suppressor LKB1 and the JNK signaling pathway. The results indicate that LKB1 activates the JNK pathway in vivo and that the JNK pathway mediates the LKB1-dependent apoptotic cell death (Lee, 2005 )

Using various Gal4 drivers, a high-level expression was obtained of both wild-type and kinase-dead LKB1 in the specific region of various tissues: the posterior region of the eye imaginal disc by the glass multiple reporter-Gal4 driver (gmr-Gal4), the dorsal region of the wing disc by the apterous-Gal4 driver (ap-Gal4), and the central pouch region of the wing disc by the ms1096-Gal4 driver). The expression levels of the LKB1 protein were determined by immunostaining using Drosophila LKB1-specific antisera. Eye-specific expression of wild-type LKB1 by the gmr-Gal4 driver induced a slight reduction in overall eye size, and this reduction became severer when the LKB1 expression level was elevated. However, the kinase-dead LKB1 overexpression did not induce this specific phenotype, showing that the kinase activity of LKB1 is important in inducing tissue-size reduction. The dorsal region-specific expression of wild-type and kinase-dead LKB1 by the ap-Gal4 driver displayed more dramatic phenotypes. Since Drosophila wing discs are composed of two layers of tissues, dorsal and ventral, alterations in size of only the dorsal layer give rise to bent-up or bent-down wing phenotypes. Although the dorsal tissue-specific expression of kinase-dead LKB1 did not induce any alterations, wild-type LKB1 induced a dramatic bent-up wing phenotype in a dose-dependent manner. Consistently, the ms1096-Gal4-driven expression of wild-type LKB1 dose-dependently reduced the overall wing size, although kinase-dead LKB1 did not. Therefore, it is concluded that Drosophila LKB1 negatively regulates the tissue and organ size in a kinase activity-dependent manner (Lee, 2005).

To examine the effect of LKB1 in individual cells, clonal analyses was performed by inducing LKB1 overexpression in a specific subset of cells within wing imaginal discs. Compared to the control clones expressing only GFP, the LKB1-expressing clones expanded very poorly. Similarly, overexpression of the TSC complex, another well-known tumor suppressor, also decreased the clone size, although with a milder severity than the overexpression of LKB1 (Lee, 2005).

Since the LKB1-dependent reduction of tissue size appropriately reflected the tumor-suppressing activity of LKB1, whether this tissue-size reduction is caused by cell cycle arrest or apoptotic cell death was assessed. Both these phenomena have been proposed as major cellular processes underlying the tumor-suppressing function of LKB1. To check the G1-S cell cycle progression of LKB1-expressing tissues, the bromodeoxyuridine (BrdU) incorporation assay, which specifically labels S-phase cells, was performed. Although LKB1 is highly expressed in these tissues, no significant alterations in the S-phase cell number were detected with respect to the control tissues. Similarly, there was no change in the antiphosphospecific histone 3 (PH3) antibody staining pattern between the wild-type and the LKB1-expressing tissues, demonstrating that G2-M cell cycle transition was also unaffected. However, when the equivalent tissues were subjected to acridine orange (AO) staining, which specifically labels the dying cells with disrupted membrane integrity, they displayed prominent death signals, specifically in the region of LKB1 overexpression. These results strongly implied that the LKB1-induced organ size reduction is caused by apoptotic cell death, not by cell cycle arrest. Furthermore, TUNEL staining of the eye and wing discs also confirmed the existence of extensive apoptosis in the LKB1-expressing cells. Therefore, it was concluded that LKB1 is a potent apoptosis instigator in Drosophila (Lee, 2005).

Tumor suppressor LKB1 is a unique serine/threonine kinase with no other close relatives in the mammalian genome. Likewise, only a single LKB1 orthologue exists in the Drosophila genome, implying well-conserved functional characteristics between the mammalian and Drosophila LKB1. This study has discovered a novel functional connection between LKB1 and the JNK pathway: the JNK pathway is present downstream of the LKB1-dependent signaling pathway and controls apoptosis and organ size in response to LKB1 activities (Lee, 2005).

Previous studies on classical tumor suppressors in the Drosophila model system showed that hyperactivation of each tumor suppressor negatively regulates organ size by cell size reduction, cell cycle inhibition, or cell death promotion. LKB1 negatively regulates organ size in Drosophila in a kinase activity-dependent manner. Recent reports on the TSC tumor suppressor suggested that LKB1 suppresses TOR activity via the TSC complex, ultimately resulting in inhibition of cell growth and proliferation. Likewise, it was observed that TSC overexpression dramatically reduces cell size in Drosophila clonal overexpression analyses. However, unexpectedly, LKB1 overexpression did not induce any cell size reduction, in stark contrast to the case of TSC. Moreover, LKB1 overexpression in the clones of endoreplicating tissues altered neither cell size nor DNA content, confirming that the tumor-suppressing activity of LKB1 is not related to the negative regulation of cell growth. Therefore, it is concluded there is no physiological connection between LKB1 and TSC in the context of cell growth control in Drosophila (Lee, 2005).

However, histological analyses clearly demonstrate that the LKB1-overexpressing tissues suffer caspase-dependent programmed cell death, without apparent alteration in G1/S and G2/M cell cycle transition. The cell death induced by LKB1 overexpression displays the most typical phenotypes of apoptosis, such as membrane integrity disruption, DNA fragmentation, and caspase activation. Furthermore, endogenous LKB1 was physiologically required for normal embryonic apoptosis, and also essential for the size control of the central nervous system. Consistent with the results, LKB1 is reported to be highly expressed in apoptotic cells of the small intestine in humans, and PJS polyps that lack the functional LKB1 protein possessed fewer apoptotic cells than in adjacent normal tissues. Collectively, these results strongly suggested that instigation of apoptosis is a critical function of tumor suppressor LKB1 (Lee, 2005).

Surprisingly, in contradiction to previous research done with the mammalian system, the LKB1-induced apoptosis in Drosophila is a p53-independent event; it is not blocked by the expression of a dominant-negative version of p53. Moreover, apoptosis induced by DNA damage or p53 overexpression is also unaffected by downregulation of LKB1 activity. Furthermore, diagnostic symptoms and major tumor types of PJS patients are highly distinct from those of Li-Fraumeni syndrome patients Therefore, it is very likely that LKB1 and p53 are indirectly related since they both have caspase activation as a common downstream signaling event (Lee, 2005).

Genetic screening using the Drosophila model system enabled discovery or the JNK pathway as a downstream effector of the LKB1-dependent signaling pathway. Through simple genetic screening, more than 500 genes for interaction with LKB were found; this study covered various signal transduction pathways including the Ras/ERK, JNK, PI3K, TOR, Wnt, TGF-beta, and NF-kappaB pathways. Among them, some components of the JNK signaling pathway showed outstanding genetic interactions with LKB1, strongly enhancing the tissue-size reduction phenotype of LKB1. This result implied the specific involvement of the JNK pathway in LKB1-dependent signal transduction, prompting additional studies carried out concerning the relationship between LKB1 and the JNK signaling pathway (Lee, 2005).

These biochemical and histological analyses clearly demonstrated that JNK can be activated by LKB1 overexpression. Since the kinase-dead mutant of LKB1 did not induce this JNK activation, this phenomenon seems to be a direct consequence of LKB1 phosphotransferase activity. Moreover, blockage of the JNK signaling pathway strongly suppresses the LKB1-induced apoptosis, as well as the tissue-size reduction. Furthermore, the activation of JNK signaling is shown to be involved in the physiological apoptosis induced by endogenous LKB1. Therefore, it is concluded that the JNK pathway acts as a downstream effector of LKB1 by mediating the LKB1-induced apoptosis. Accordingly, any signals upregulating the kinase activity of LKB1 would also activate the JNK pathway, ultimately leading to apoptosis in the cell. In fact, some microtubule-disrupting agents that induce the LKB1-dependent cell death also activate the JNK pathway to promote apoptosis (Lee, 2005).

Another interesting point of this research is that inhibition of LKB1 results in defective epithelial morphogenesis, which is associated with low JNK activity. Several previous studies already demonstrated the requirement of LKB1 and JNK for epithelial integrity and polarity, both in the mammalian and Drosophila systems. Consistently, it was observed that inhibition of LKB1 activity during development causes various morphogenesis defects including malformation of adult thoraxes and failure in embryonic dorsal closure. Furthermore, LKB1 is shown to be required for maintaining adequate JNK activity during embryonic and larval development. Since loss of tissue integrity is a common feature of PJS polyps and cancer, the role of LKB1 in epithelial organization seems to be also important for mediating its tumor-suppressing activity (Lee, 2005).

This study has demonstrated that the JNK pathway is involved in the LKB1-dependent apoptosis and epithelial morphogenesis in Drosophila. Since deregulation of both LKB1 and the JNK pathways is highly implicated in cancer development, the novel connection between LKB1 and the JNK signaling pathway provides a new direction for future studies in better understanding the complex functions of tumor suppressor LKB1 (Lee, 2005).


Function of Par-4, a homolog of LKB1, in C. elegans

Eight strict maternal effect mutations identifying four genes, par-1, par-2, par-3, and par-4, required for cytoplasmic localization in early embryos of the nematode C. elegans have been isolated and analyzed. Mutations in these genes lead to defects in cleavage patterns, timing of cleavages, and localization of germ line-specific P granules. Four mutations in par-1 and par-4 are fully expressed maternal effect lethal mutations; all embryos from mothers homozygous for these mutations arrest as amorphous masses of differentiated cells but are specifically lacking intestinal cells. Four mutations in par-2, par-3, and par-4 are incompletely expressed maternal effect lethal mutations and are also grandchildless; some embryos from homozygous mothers survive and grow to become infertile adults due to the absence of functional germ cells. It is proposed that all of these defects result from the failure of a maternally encoded system for intracellular localization in early embryos (Kemphues, 1988).

During the first cell cycle of C. elegans embryogenesis, asymmetries are established that are essential for determining the subsequent developmental fates of the daughter cells. The maternally expressed par genes are required for establishing this polarity. The products of several of the par genes have been found to be themselves asymmetrically distributed in the first cell cycle. The par-4 gene of C. elegans encodes a putative serine-threonine kinase with similarity to a human kinase associated with Peutz-Jeghers Syndrome, LKB1, and a Xenopus egg and embryo kinase, XEEK1. Several strong par-4 mutant alleles are missense mutations that alter conserved residues within the kinase domain, suggesting that kinase activity is essential for PAR-4 function. The PAR-4 protein is present in the gonads, oocytes and early embryos of C. elegans, and is both cytoplasmically and cortically distributed. The cortical distribution begins at the late 1-cell stage, is more pronounced at the 2- and 4-cell stages and is reduced at late stages of embryonic development. No asymmetry is found in the distribution of PAR-4 protein in C. elegans embryos. The distribution of PAR-4 protein in early embryos is unaffected by mutations in the other par genes (Watts, 2000).

The KH domain protein MEX-3 is central to the temporal and spatial control of PAL-1 expression in the C. elegans early embryo. PAL-1 is a Caudal-like homeodomain protein that is required to specify the fate of posterior blastomeres. While pal-1 mRNA is present throughout the oocyte and early embryo, PAL-1 protein is expressed only in posterior blastomeres, starting at the four-cell stage. To better understand how PAL-1 expression is regulated temporally and spatially, MEX-3 interacting proteins (MIPs) have been identified and two that are required for the patterning of PAL-1 expression are described in detail. RNA interference of MEX-6, a CCCH zinc-finger protein, or SPN-4, an RNA recognition motif protein, causes PAL-1 to be expressed in all four blastomeres starting at the four-cell stage. Genetic analysis of the interactions between these mip genes and the par genes, which provide polarity information in the early embryo, defines convergent genetic pathways that regulate MEX-3 stability and activity to control the spatial pattern of PAL-1 expression. These experiments suggest that par-1 and par-4 affect distinct processes. par-1 is required for many aspects of embryonic polarity, including the restriction of MEX-3 and MEX-6 activity to the anterior blastomeres. PAL-1 is not expressed in par-1 mutants, because MEX-3 and MEX-6 remain active in the posterior blastomeres. The role of par-4 is less well understood. This analysis suggests that par-4 is required to inactivate MEX-3 at the four-cell stage. Thus, PAL-1 is not expressed in par-4 mutants because MEX-3 remains active in all blastomeres. It is proposed that MEX-6 and SPN-4 act with MEX-3 to translate the temporal and spatial information provided by the early acting par genes into the asymmetric expression of the cell fate determinant PAL-1 (Huang, 2002).

In C. elegans, reduced insulin-like signalling induces developmental quiescence, reproductive delay and lifespan extension. The C. elegans orthologues of LKB1 and AMPK cooperate during conditions of reduced insulin-like signalling to establish cell cycle quiescence in the germline stem cell population, in addition to prolonging lifespan. The inactivation of either protein causes aberrant germline proliferation during diapause-like 'dauer' development, whereas the loss of AMPK uncouples developmental arrest from lifespan extension. Reduced TGF-beta activity also triggers developmental quiescence independent of the insulin-like pathway. These data suggest that these two signalling pathways converge on the C. elegans PTEN orthologue to coordinate germline proliferation with somatic development during dauer formation, via the regulation of AMPK and its upstream activator LKB1, rather than through the canonical insulin-like signalling cascade. In humans, germline mutations in TGF-beta family members, PTEN or LKB1 result in related tumour-predisposing syndromes. These findings establish a developmental relationship that may underscore their shared, characteristic aetiology (Narbonne, 2006).

Anteroposterior polarity in early C. elegans embryos is required for the specification of somatic and germline lineages, and is initiated by a sperm-induced reorganization of the cortical cytoskeleton and PAR polarity proteins. Through mechanisms that are not understood, the kinases PAR-1 and PAR-4, and other PAR proteins cause the cytoplasmic zinc finger protein MEX-5 to accumulate asymmetrically in the anterior half of the one-cell embryo. MEX-5 asymmetry requires neither vectorial transport to the anterior, nor protein degradation in the posterior. MEX-5 has a restricted mobility before fertilization and in the anterior of one-cell embryos. However, MEX-5 mobility in the posterior increases as asymmetry develops, presumably allowing accumulation in the anterior. The MEX-5 zinc fingers and a small, C-terminal domain are essential for asymmetry; the zinc fingers restrict MEX-5 mobility, and the C-terminal domain is required for the increase in posterior mobility. A crucial residue in the C-terminus, Ser 458, is phosphorylated in vivo. PAR-1 and PAR-4 kinase activities are required for the phosphorylation of S458, providing a link between PAR polarity proteins and the cytoplasmic asymmetry of MEX-5 (Tenlen, 2008).

Activation of LKB1 by the induced expression of STRAD polarizes intestinal epithelial cells in a cell-autonomous fashion

The LKB1 gene encodes a serine/threonine kinase that is mutated in the Peutz-Jeghers cancer syndrome. An LKB1-specific adaptor protein, STRAD, activates LKB1 and translocates it from nucleus to cytoplasm. Intestinal epithelial cell lines have been constructed in which inducible STRAD activates LKB1. Upon LKB1 activation, single cells rapidly remodel their actin cytoskeleton to form an apical brush border. The junctional proteins ZO-1 and p120 redistribute in a dotted circle peripheral to the brush border, in the absence of cell-cell contacts. Apical and basolateral markers sort to their respective membrane domains. It is concluded that LKB1 can induce complete polarity in intestinal epithelial cells. In contrast to current thinking on polarization of simple epithelia, these cells can fully polarize in the absence of junctional cell-cell contacts (Baas, 2004).

Thus, activation of LKB1 by the induced expression of STRAD polarizes intestinal epithelial cells in a cell-autonomous fashion. Activated LKB1 induces three major aspects of epithelial polarity, i.e., the formation of an apical brush border, the positioning of junctional proteins surrounding this brush border, and the correct sorting of apical and basolateral plasma membrane markers. From these observations, it appears that LKB1 is a bona fide mammalian polarity gene and is thus a genuine ortholog of C. elegans Par-4 and Drosophila dLKB1. These data further suggest that orthologs of the STE20-like pseudokinase STRAD could play comparable activator roles in these two model organisms. Indeed, the Drosophila genome contains a pseudokinase with high homology to STRAD (Ste20-like kinase), which harbors very similar kinase-inactivating amino acid substitutions (Baas, 2004).

The control of asymmetry of individual cells in multicellular organisms is only beginning to be understood. Cellular polarity involves many independent features, such as an asymmetry of the actin cytoskeleton; the localized distribution of specific mRNAs; the asymmetric localization of the mitotic spindle, of cell junctions, and of intracellular secretory granules, and the sorting of surface molecules to polarized membrane domains. Genetic studies on asymmetric events during early C. elegans development have led to the identification of six Par genes (Par-1 through Par-6) that appear to function as master regulators of polarity. Loss of these Par proteins results in loss of multiple aspects of polarity. Most of the Par proteins in C. elegans are themselves distributed in an asymmetric fashion in the early embryo with the notable exception of Par-4, which is diffusely expressed in the cytoplasm and the cellular cortex. The individual phenotypes of the Par genes are overlapping yet unique (Baas, 2004).

The absence of gain-of-function PAR phenotypes has hampered epistatic analysis. As a consequence, the connections between the Par proteins have remained largely unknown. It has been proposed that Par-4/LKB1 and Par-1 constitute a kinase cascade, because (1) the two phenotypes are similar; (2) overexpression of Par-4/LKB1 in Drosophila partially rescues a Par-1 mutation; and (3) Drosophila Par-4/LKB1 is phosphorylated by Par-1 in vitro (Martin, 2003). The phenotype of Drosophila LKB1 is of particular interest to the current study. It not only affects the anterior-posterior axis of embryos, but it also disrupts the polarity of the follicular epithelium in mutant clones, a simple epithelium which, like the intestinal epithelium of mammals, normally forms apical adherent junctions (Baas, 2004).

The study of polarity of simple epithelia in mammals has largely been restricted to long-term cultures of epithelial cell lines. These cell lines slowly acquire polarity upon reaching confluency. Cell culture systems in which the induction of epithelial polarity occurs rapidly and in a tightly controlled, cell-autonomous fashion are not available. Therefore, the cellular model described in this study may be a valuable complement to the genetic loss-of-function studies in model organisms. The gain-of-function effects induced by STRAD in the cell lines used in this study can be combined with siRNA technology and other approaches to resolve molecular details of the various aspects of polarization of simple epithelia (Baas, 2004).

One unexpected finding of the current study is the observation that individual epithelial cells can be induced to polarize in the absence of cell-cell contacts, which contradicts current thinking. Previous studies on model cell lines such as MDCK and Caco-2 have always been restricted to the spontaneous appearance of polarity after the cells are allowed to form cell junctions (Baas, 2004).

The dynamics of the actin reorganization in the cells used in this study suggest that the brush border formation is a direct consequence of LKB1 activation and may be required for the additional polarity phenomena to occur. It appears very likely that the actin reorganization is mediated by Rho family small GTPases. Cdc42 has been found to colocalize with Par-3/-6 and represents a good candidate effector of LKB1-induced polarity. The brush border, once formed, may serve as a landmark to accumulate junctional proteins around its periphery, prior to the assembly of junctional complexes between adjacent cells. The third aspect of epithelial polarity, the definition of apical versus basolateral membrane domains may also directly result from the establishment of the brush border. Possibly, brush border components such as ezrin may specifically anchor and retain apical surface proteins (Baas, 2004).

It is widely held that tight junctions are crucial to the definition of apical versus basolateral domains, since they serve as an absolute barrier for lateral diffusion of transmembrane proteins. In this light, it is interesting that the cells used in this study do not form cell-cell contacts or junctional complexes (in LS174T), yet are capable of sorting proteins to specific membrane domains (Baas, 2004).

As has been reported for Par-4 previously (Watts, 2000), LKB1 is never observed to be distributed in a polarized fashion. Yet, the actin cap that precedes the formation of a brush border consistently appears directly above the center of the cell. This implies that the cell has spatial cues prior to the induction of STRAD, since it knows where polarized structures should be created. It has been proposed that cells in simple epithelia and in cell culture dishes derive such spatial cues from integrin signaling occurring at the membrane domain that touches the basal lamina in vivo, or the laminin-coated culture dish in vitro. Of note, the surrogate basolateral domain that is defined by transferrin receptor expression upon LKB1 activation does not coincide with the membrane domain that touches the laminin coat. The former encompasses the entire cellular membrane outside the brush border (Baas, 2004).

It is currently unknown what controls Par-4/LKB1 activity in the various model systems. cAMP-dependent kinase has been demonstrated to phosphorylate Par-4/LKB1 in Drosophila (Martin, 2003) and man (Collins, 2000 and Sapkota, 2001). The current data suggest that the regulated expression of STRAD or the regulated LKB1-STRAD interaction may be a key control point of an LKB1/Par-4 polarity-signaling pathway (Baas, 2004).

LKB1 has been identified originally as a tumor suppressor. It has since been shown to affect a wide variety of cell growth characteristics. Of note, the cell lines used in this study do undergo growth arrest upon induction of STRAD. This is first observed after 24 hr, long after the cells express the polarized phenotype. When the same cells are induced to undergo a G1 arrest by blocking the Wnt cascade or by inducible expression of cell-cycle inhibitor p21, they do not polarize. It is believed that many of the phenomena that have been attributed to LKB1 may be the indirect consequence of changes in cellular polarity. As suggested previously (Martin, 2003), loss of the tumor suppressor LKB1 may result in cellular transformation as a consequence of disrupted epithelial polarity (Baas, 2004).

The human Lkb1 kinase, encoded by the ortholog of the invertebrate Par4 polarity gene, is mutated in Peutz-Jeghers cancer syndrome. Lkb1 activity requires complex formation with the pseudokinase Strad and the adaptor protein Mo25. The complex can induce complete polarization in a single isolated intestinal epithelial cell. This study describes an interaction between Mo25alpha and a human serine/threonine kinase termed Mst4. A homologous interaction occurs in the yeast Schizosaccharomyces pombe in the control of polar tip growth. Human Mst4 translocates from the Golgi to the subapical membrane compartment upon activation of Lkb1. Inhibition of Mst4 activity inhibits Lkb1-induced brush border formation, whereas other aspects of polarity such as the formation of lateral junctions remain unaffected. As an essential event in brush border formation, Mst4 phosphorylates the regulatory T567 residue of Ezrin. These data define a brush border induction pathway downstream of the Lkb1/Strad/Mo25 polarization complex, yet separate from other polarity events (ten Klooster, 2009).

Differential requirements for STRAD in LKB1-dependent functions in C. elegans

The protein kinase LKB1 is a crucial regulator of cell growth/proliferation and cell polarity and is the causative gene in the cancer-predisposing disease Peutz-Jeghers syndrome (PJS). The activity of LKB1 is greatly enhanced following its association with the Ste20-like adapter protein STRAD. Unlike LKB1 however, mutations in STRAD have not been identified in PJS patients and thus, the key tumour suppressive role(s) of LKB1 might be STRAD independent. This study reports that Caenorhabditis elegans strd-1/STRAD mutants recapitulate many phenotypes typical of par-4/LKB1 loss of function, showing defects during early embryonic and dauer development. Interestingly, although the growth/proliferation defects in severe par-4 and strd-1 mutant dauers are comparable, strd-1 mutant embryos do not share the polarity defects of par-4 embryos. Most of par-4-dependent regulation of germline stem cell (GSC) quiescence occurs through AMPK, whereby PAR-4 requires STRD-1 to phosphorylate and activate AMPK. Consistent with this, even though AMPK plays a major role in the regulation of cell proliferation, like strd-1 it does not affect embryonic polarity. Instead, it was found that the PAR-4-mediated phosphorylation of polarity regulators such as PAR-1 and MEX-5 in the early embryo occurs in the absence of STRD-1. Thus, PAR-4 requires STRD-1 to phosphorylate AMPK to regulate cell growth/proliferation under reduced insulin signalling conditions, whereas PAR-4 can promote phosphorylation of key proteins, including PAR-1 and MEX-5, to specify early embryonic polarity independently of STRD-1. These results therefore identify a key strd-1/STRAD-independent function of par-4/LKB1 in polarity establishment that is likely to be important for tumour suppression in humans (Narbonne, 2010).

PAR-4/LKB1 mobilizes nonmuscle myosin through anillin to regulate C. elegans embryonic polarization and cytokinesis

The serine/threonine kinase LKB1 regulates cell growth and polarity in metazoans, and loss of LKB1 function is implicated in the development of some epithelial cancers. Despite its fundamental role, the mechanism by which LKB1 regulates polarity establishment and/or maintenance is unclear. This study used C. elegans to investigate the role of the LKB1 ortholog PAR-4 in actomyosin contractility, a cellular process essential for polarity establishment and cell division in the early embryo. Using high-resolution time-lapse imaging of GFP-tagged nonmuscle myosin II (NMY-2), it was found that par-4 mutations reduce actomyosin contractility during polarity establishment, leading to the mispositioning of anterior PAR proteins and to defects in contractile ring ingression during cytokinesis. Fluorescence recovery after photobleaching analysis revealed that the mobility of a cortical population of NMY-2 was reduced in par-4 mutants. Interestingly, the contractility defects of par-4 mutants depend on the reciprocal activity of ANI-1 and ANI-2, two C. elegans homologs of the actin cytoskeletal scaffold protein anillin. Because loss of PAR-4 promoted inappropriate accumulation of ANI-2 at the cell cortex, it is proposed that PAR-4 controls C. elegans embryonic polarity by regulating the activity of anillin family scaffold proteins, thus enabling turnover of cortical myosin and efficient actomyosin contractility. This work provides the first description of a cellular mechanism by which PAR-4/LKB1 mediates cell polarization (Chartier, 2011).

The N-terminal part of Drosophila or human anillin contains formin-, myosin-, and actin-binding domains; the C-terminal part contains an anillin homology domain (AHD), which interacts with actin regulator RhoA and the small GTPase regulator MgcRacGAP, and a PH domain that can interact with septins. In C. elegans, the canonical anillin protein ANI-1 is predicted to have all four domains; it organizes cortical contractility during polarization and the asymmetric closure of the cytokinetic furrow. ANI-2 is a shorter isoform of anillin that lacks the N-terminal domains predicted to bind myosin and actin. By competing with ANI-1 for the binding of C-terminal domain partners, this shorter isoform could function in a dominant-negative manner and thus negatively regulate actomyosin contractility. To test this, ANI-1 was depleted from wild-type and par-4 embryos, and the kinetics of contractile ring closure was measured, as well as the position of PAR-6::GFP domain at the end of polarization. It was found that although ANI-1 depletion had no effect on the position of cortical PAR-6::GFP, it caused an increase in the duration of cytokinetic ring ingression in par-4 mutant embryos. This is consistent with previous work showing that ANI-1 is dispensable for proper polarization, whereas it is required for asymmetric ingression during cytokinesis. Importantly, simultaneous depletion of ANI-2 and ANI-1 failed to suppress both phenotypic defects displayed by par-4 embryos. These results indicate that the suppression of par-4 phenotypes by ANI-2 depletion requires the presence of the canonical anillin ANI-1 and support a model in which the contractility defects observed in par-4 mutants are due to the perturbation of ANI-1 function by ANI-2 (Chartier, 2011).

Surprisingly, in FRAP experiments, depleting ANI-1 phenocopied ANI-2 depletion and resulted in a restoration of myosin mobility at the cortex of par-4 mutant embryos. However, the actomyosin cortex is severely disorganized in ani-1(RNAi) embryos: NMY-2 fails to coalesce into robust patches. ANI-1-depleted embryos have normal polarity and can complete cytokinesis, indicating that ANI-1 does not regulate contractility per se, but rather regulates the organization of the actomyosin cortex. It is concluded that the gain of ANI-2 function observed in par-4 embryos only affects cortical myosin turnover in NMY-2 patches and that disrupting the formation of these patches abrogates the negative regulation by ANI-2. This is consistent with previous reports indicating that the degree of organization of the actomyosin network inversely correlates with the turnover of its components (Chartier, 2011).

PAR-4/LKB1 regulates DNA replication during asynchronous division of the early C. elegans embryo

Regulation of cell cycle duration is critical during development, yet the underlying molecular mechanisms are still poorly understood. The two-cell stage Caenorhabditis elegans embryo divides asynchronously and thus provides a powerful context in which to study regulation of cell cycle timing during development. Using genetic analysis and high-resolution imaging, this study found that deoxyribonucleic acid (DNA) replication is asymmetrically regulated in the two-cell stage embryo and that the PAR-4 (Drosophila Lkb1) and PAR-1 polarity proteins dampen DNA replication dynamics specifically in the posterior blastomere, independently of regulators previously implicated in the control of cell cycle timing. These results demonstrate that accurate control of DNA replication is crucial during C. elegans early embryonic development and further provide a novel mechanism by which PAR proteins control cell cycle progression during asynchronous cell division (Benkemoun, 2014).

Signaling upstream of LKB1

Peutz-Jeghers syndrome (PJS) is an autosomal dominant disease characterized by melanocytic macules, hamartomatous polyps and an increased risk for numerous cancers. The human LKB1 (hLKB1) gene encodes a serine/threonine protein kinase that is deficient in the majority of patients with PJS. The murine LKB1 (mLKB1) cDNA was isolated, sequenced and shown to produce a 2.4-kb transcript encoding a 436 amino acid protein with 90% identity with hLKB1. RNA blot and RNase-protection analysis revealed that mLKB1 mRNA is expressed in all tissues and cell lines examined. The widespread expression of LKB1 transcripts is consistent with the elevated risk of multiple cancer types in PJS patients. The predicted LKB1 protein sequence terminates with a conserved prenylation motif [Cys(433)-Lys-Gln-Gln(436)] directly downstream from a consensus cAMP-dependent protein kinase (PKA) phosphorylation site [Arg(428)-Arg-Leu-Ser(431)]. The expression of enhanced green fluorescent protein (EGFP)-mLKB1 chimaeras demonstrated that LKB1 possesses a functional prenylation motif that is capable of targeting EGFP to cellular membranes. Mutation of Cys(433) to an alanine residue, but not phosphorylation by PKA, blocks membrane localization. These findings suggest that PKA does phosphorylate LKB1, although this phosphorylation does not alter the cellular localization of LKB1 (Collins, 2000).

The mechanism by which LKB1 is regulated in cells is not known. Stimulation of Rat-2 or embryonic stem cells with activators of ERK1/2 or of cAMP-dependent protein kinase induces phosphorylation of endogenously expressed LKB1 at Ser(431). Pharmacological and genetic evidence is presented that p90(RSK) mediates this phosphorylation in response to agonists that activate ERK1/2, and cAMP-dependent protein kinase mediates this phosphorylation in response to agonists that activate adenylate cyclase. Ser(431) of LKB1 lies adjacent to a putative prenylation motif, and full-length LKB1 expressed in 293 cells is prenylated by addition of a farnesyl group to Cys(433). These data suggest that phosphorylation of LKB1 at Ser(431) does not affect farnesylation and that farnesylation does not affect phosphorylation at Ser(431). Phosphorylation of LKB1 at Ser(431) does not alter the activity of LKB1 to phosphorylate itself or the tumor suppressor protein p53 or alter the amount of LKB1 associated with cell membranes. The reintroduction of wild-type LKB1 into a cancer cell line that lacks LKB1 suppressed growth, but mutants of LKB1 in which Ser(431) was mutated to Ala to prevent phosphorylation of LKB1 were ineffective in inhibiting growth. In contrast, a mutant of LKB1 that cannot be prenylated is still able to suppress the growth of cells (Sapkota, 2001).

LKB1 functions upstream of AMP-activated protein kinase (AMPK)

Somatic mutations of STK11/LKB11, the gene responsible for Peutz-Jeghers syndrome (PJS), are found in a small proportion of sporadic pancreatic adenocarcinomas, intraductal papillary mucinous neoplasms (IPMNs), and biliary adenocarcinomas. This study characterizes the expression of Stk11, the protein product of the STK11 gene, in a larger series of pancreatic and biliary neoplasms. Initially, the specificity of the Stk11 antibody was established in 23 neoplasms (22 IPMNs and 1 biliary adenocarcinoma) with known STK11 gene status. Complete absence of labeling was seen in the neoplastic cells of 3 of the 3 cases with previously documented biallelic inactivation of the STK11 gene, whereas 16 of the 20 IPMNs, presumably with at least one wild-type STK11 gene, retained Stk11 expression in the neoplastic cells. The marked decrease or absence of Stk11 expression in four neoplasms with wild-type STK11 suggests that additional mechanisms may account for the lack of Stk11 expression. Subsequently, in order to further evaluate Stk11 expression in pancreatic and biliary neoplasms, tissue microarrays (TMAs) were constructed from a series of nearly 100 ductal adenocarcinomas and biliary neoplasms. Stk11 expression was lost in 4 of the 56 pancreatic adenocarcinomas and 1 of the 38 biliary cancers by immunohistochemistry; the absence of labeling was confirmed by repeated immunohistochemical labeling of complete tissue sections for the same cases. Thus, Stk11 expression is abrogated in a small proportion of pancreatic and biliary neoplasms. The inactivation of Stk11 in 27% of IPMNs versus 7% of pancreatic adenocarcinomas suggests genetic disparities in the pathogenesis of these closely related neoplasms. Immunohistochemical analysis for Stk11 expression may be a valid surrogate for genetic analysis of STK11 gene mutations in cancers (Sahin, 2003).

The LKB1 tumour suppressor kinase phosphorylates and activates a number of protein kinases belonging to the AMP-activated protein kinase (AMPK) subfamily. A modified tandem affinity purification strategy was used to identify proteins that interact with AMPKalpha, as well as the twelve AMPK-related kinases that are activated by LKB1. The AMPKbeta and AMPKgamma regulatory subunits were associated with AMPKalpha, but not with any of the AMPK-related kinases, explaining why AMP does not influence the activity of these enzymes. In addition, novel binding partners were identified that interacted with one or more of the AMPK subfamily enzymes, including fat facets/ubiquitin specific protease-9 (USP9), AAA-ATPase-p97, adenine nucleotide translocase, protein phosphatase 2A holoenzyme and isoforms of the phospho-protein binding adaptor 14-3-3. Interestingly, the 14-3-3 isoforms binds directly to the T-loop Thr residue of QSK and SIK, after these are phosphorylated by LKB1. Consistent with this, the 14-3-3 isoforms fail to interact with non-phosphorylated QSK and SIK, in LKB1 knockout muscle or in HeLa cells in which LKB1 is not expressed. Moreover, mutation of the T-loop Thr phosphorylated by LKB1, prevents QSK and SIK from interacting with 14-3-3 in vitro. Binding of 14-3-3 to QSK and SIK, enhanced catalytic activity towards the TORC2 protein and the AMARA peptide, and is required for the cytoplasmic localization of SIK and for localization of QSK to punctate structures within the cytoplasm. This study provides the first example of 14-3-3 binding directly to the T-loop of a protein kinase and influencing its catalytic activity and cellular localization (Al-Hakim, 2005).

The LKB1 tumour suppressor protein kinase is the major 'upstream' activator of the energy sensor AMP-activated protein kinase (AMPK). Mice were examined in which LKB1 is expressed at only approximately 10% of the normal levels in muscle and most other tissues, or that lack LKB1 entirely in skeletal muscle. Muscle expressing only 10% of the normal level of LKB1 has significantly reduced phosphorylation and activation of AMPKalpha2. In LKB1-lacking muscle, the basal activity of the AMPKalpha2 isoform is greatly reduced and is not increased by the AMP-mimetic agent, 5-aminoimidazole-4-carboxamide riboside (AICAR), by the antidiabetic drug phenformin, or by muscle contraction. Moreover, phosphorylation of acetyl CoA carboxylase-2, a downstream target of AMPK, is profoundly reduced. Glucose uptake stimulated by AICAR or muscle contraction, but not by insulin, is inhibited in the absence of LKB1. Contraction increases the AMP:ATP ratio to a greater extent in LKB1-deficient muscles than in LKB1-expressing muscles. These studies establish the importance of LKB1 in regulating AMPK activity and cellular energy levels in response to contraction and phenformin (Sakamoto, 2005).

The Snf1/AMP-activated protein kinase (AMPK) family is important for metabolic regulation in response to stress. In the yeast Saccharomyces cerevisiae, the Snf1 kinase cascade comprises three Snf1-activating kinases, Pak1, Tos3, and Elm1. The only established mammalian AMPK kinase is LKB1. This study shows that LKB1 functions heterologously in yeast. In pak1Delta tos3Delta elm1Delta cells, LKB1 activates Snf1 catalytic activity and confers a Snf(+) growth phenotype. Coexpression of STRADalpha and MO25alpha, which form a complex with LKB1, enhances LKB1 function. Thus, the Snf1/AMPK kinase cascade is functionally conserved between yeast and mammals. Ca(2+)/calmodulin-dependent kinase kinase (CaMKK) shows more sequence similarity to Pak1, Tos3, and Elm1 than does LKB1. When expressed in pak1Delta tos3Delta elm1Delta cells, CaMKKalpha activated Snf1 catalytic activity, restored the Snf(+) phenotype, and also phosphorylates the activation loop threonine of Snf1 in vitro. These findings indicate that CaMKKalpha is a functional member of the Snf1/AMPK kinase family and support CaMKKalpha as a likely candidate for an AMPK kinase in mammalian cells. Analysis of the function of these heterologous kinases in yeast provides insight into the regulation of Snf1. When activated by LKB1 or CaMKKalpha, Snf1 activity is significantly inhibited by glucose, suggesting that a mechanism independent of the activating kinases can mediate glucose signaling in yeast. Finally, this analysis provided evidence that Pak1 functions in another capacity, besides activating Snf1, to regulate the nuclear enrichment of Snf1 protein kinase in response to carbon stress (Hong, 2005).

Germline mutations of the LKB1 gene are responsible for the cancer-prone Peutz-Jeghers syndrome (PJS). LKB1 encodes a serine-threonine kinase that acts as a regulator of cell cycle, metabolism and cell polarity. The majority of PJS missense mutations abolish LKB1 enzymatic activity and thereby impair all functions assigned to LKB1. The functional consequences have been investigated of recurrent missense mutations identified in PJS and in sporadic tumors which map in the LKB1 C-terminal non-catalytic region. These C-terminal mutations neither disrupt LKB1 kinase activity nor interfere with LKB1-induced growth arrest. However, these naturally occuring mutations lessened LKB1-mediated activation of the AMP-activated protein kinase (AMPK) and impaired downstream signaling. Furthermore, C-terminal mutations compromise LKB1 ability to establish and maintain polarity of both intestinal epithelial cells and migrating astrocytes. Consistent with these findings, mutational analysis reveals that the LKB1 tail exerts an essential function in the control of cell polarity. Overall, these results ascribe a crucial regulatory role to the LKB1 C-terminal region. These findings further indicate that LKB1 tumor suppressor activity is likely to depend on the regulation of AMPK signaling and cell polarization (Forcet, 2005).

Recent work indicates that the LKB1 tumour suppressor protein kinase, which is mutated in Peutz-Jeghers cancer syndrome, phosphorylates and activates a group of protein kinases that are related to AMPK (AMP-activated protein kinase). Ten of the 14 AMPK-related protein kinases activated by LKB1, including SIK (salt-induced kinase), MARK (microtubule-affinity-regulating kinase) and BRSK (brain-specific kinase) isoforms, possess a ubiquitin-associated (UBA) domain immediately C-terminal to the kinase catalytic domain. These are the only protein kinases in the human genome known to possess a UBA domain, but their roles in regulating AMPK-related kinases are unknown. This study investigated the roles that the UBA domain may play in regulating these enzymes. Limited proteolysis of MARK2 revealed that the kinase and UBA domains were contained within a fragment that is resistant to trypsin proteolysis. SAXS (small-angle X-ray scattering) analysis of inactive and active LKB1-phosphorylated MARK2 revealed that activation of MARK2 is accompanied by a significant conformational change that alters the orientation of the UBA domain with respect to the catalytic domain. The results indicate that none of the UBA domains found in AMPK-related kinases interact with polyubiquitin or other ubiquitin-like molecules. Instead, the UBA domains appear to play an essential conformational role and are required for the LKB1-mediated phosphorylation and activation of AMPK-related kinases. This is based on the findings that mutation or removal of the UBA domains of several AMPK-related kinases, including isoforms of MARK, SIK and BRSK, markedly impair the catalytic activity and LKB1-mediated phosphorylation of these enzymes. Evidence is provided that the UBA domains do not function as LKB1-STRAD (STE20-related adaptor)-MO25 (mouse protein 25) docking/interacting sites and that mutations in the UBA domain of SIK suppresses the ability of SIK to localize within punctate regions of the nucleus. Taken together, these findings suggest that the UBA domains of AMPK-related kinases play an important role in regulating the conformation, activation and localization of these enzymes (Jaleel, 2006).

The phosphoinositide 3-kinase-dependent activation of the 5'-AMP-activated kinase (AMPK) by peroxynitrite (ONOO-) and hypoxia-reoxygenation occurs in cultured endothelial cells. Molecular mechanism of activation of this pathway is described. Exposure of bovine aortic endothelial cells to ONOO- significantly increases the phosphorylation of both Thr172 of AMPK and Ser1179 of endothelial nitric-oxide synthase, a known downstream enzyme of AMPK. In addition, activation of AMPK by ONOO- is accompanied by increased phosphorylation of protein kinase Czeta (PKCzeta) (Thr410/403) and translocation of cytosolic PKCzeta into the membrane. Further, inhibition of PKCzeta abrogates ONOO- -induced AMPK-Thr172 phosphorylation as that of endothelial nitric-oxide synthase. Furthermore, overexpression of a constitutively active PKCzeta mutant enhances the phosphorylation of AMPK-Thr172, suggesting that PKCzeta is upstream of AMPK activation. In contrast, ONOO- activates PKCzeta in LKB1-deficient HeLa-S3 but affects neither AMPK-Thr172 nor AMPK activity. These data suggest that LKB1 is required for PKCzeta-enhanced AMPK activation. In vitro, recombinant PKCzeta phosphorylates LKB1 at Ser428, resulting in phosphorylation of AMPK at Thr172. Further, direct mutation of Ser428 of LKB1 into alanine, like the kinase-inactive LKB1 mutant, abolishes ONOO- -induced AMPK activation. In several cell types originating from human, rat, and mouse, inhibition of PKCzeta significantly attenuates the phosphorylation of both LKB1-Ser428 and AMPK-Thr172 that are enhanced by ONOO-. Taken together, it is concluded that PKCzeta can regulate AMPK activity by increasing the Ser428 phosphorylation of LKB1, resulting in association of LKB1 with AMPK and consequent AMPK Thr172 phosphorylation by LKB1 (Xie, 2006).

AMP-activated protein kinase (AMPK) is a highly conserved sensor of cellular energy status found in all eukaryotic cells. AMPK is activated by stimuli that increase the cellular AMP/ATP ratio. Essential to activation of AMPK is its phosphorylation at Thr-172 by an upstream kinase, AMPKK, whose identity in mammalian cells has remained elusive. Biochemical and genetic evidence indicates that the LKB1 serine/threonine kinase, the gene inactivated in the Peutz-Jeghers familial cancer syndrome, is the dominant regulator of AMPK activation in several mammalian cell types. LKB1 directly phosphorylates Thr-172 of AMPKalpha in vitro and activates its kinase activity. LKB1-deficient murine embryonic fibroblasts show nearly complete loss of Thr-172 phosphorylation and downstream AMPK signaling in response to a variety of stimuli that activate AMPK. Reintroduction of WT, but not kinase-dead, LKB1 into these cells restores AMPK activity. Furthermore, LKB1 plays a biologically significant role in this pathway, because LKB1-deficient cells are hypersensitive to apoptosis induced by energy stress. On the basis of these results, a model is proposed to explain the apparent paradox that LKB1 is a tumor suppressor, yet cells lacking LKB1 are resistant to cell transformation by conventional oncogenes and are sensitive to killing in response to agents that elevate AMP. The role of LKB1/AMPK in the survival of a subset of genetically defined tumor cells may provide opportunities for cancer therapeutics (Shaw, 2004a).

Regulation of the TSC pathway by LKB1

Tuberous sclerosis complex (TSC) and Peutz-Jeghers syndrome (PJS) are dominantly inherited benign tumor syndromes that share striking histopathological similarities. LKB1, the gene mutated in PJS, acts as a tumor suppressor by activating TSC2, the gene mutated in TSC. Like TSC2, LKB1 inhibits the phosphorylation of the key translational regulators S6K and 4EBP1. Furthermore, LKB1 activates TSC2 through the AMP-dependent protein kinase (AMPK: Drosophila homolog SNF1A/AMP-activated protein kinase), indicating that LKB1 plays a role in cell growth regulation in response to cellular energy levels. These results suggest that PJS and other benign tumor syndromes could be caused by dysregulation of the TSC2/mTOR pathway (Corradetti, 2004).

Peutz-Jeghers syndrome (PJS), a dominantly inherited genetic disorder, is characterized by the formation of gastrointestinal hamartomas that are histologically similar to those observed in TSC patients. PJS is associated with mutations in the lkb1 tumor suppressor gene, which codes for a serine/threonine kinase. Although extensive work has been performed on the molecular pathogenesis of TSC, the molecular mechanism of LKB1 as a tumor suppressor has remained elusive (Corradetti, 2004 and references therein).

Recent studies from several laboratories have demonstrated that LKB1 phosphorylates and activates AMPK, representing the first convincing physiological target of LKB1. AMPK is a multimeric protein, and its kinase activity is enhanced by both phosphorylation and high intracellular AMP levels. The amount of AMP in the cell is inversely proportional to the amount of ATP, and high levels of AMP are present under low energy conditions. Under such conditions, AMPK is activated and phosphorylates numerous substrates to suppress anabolism and enhance catabolism, thereby regulating cellular energy homeostasis. LKB1 potentiates the effect of AMP on AMPK by phosphorylating AMPK on Thr 172, a residue found in the AMPK activation loop (Corradetti, 2004 and references therein).

Activated AMPK phosphorylates and activates the TSC2 tumor suppressor protein. This AMPK-dependent regulation of TSC2 is especially important for cellular energy response because cells expressing TSC2 mutants that cannot be phosphorylated by AMPK undergo apoptosis under energy starvation conditions. TSC2 displays GTPase activating protein (GAP) activity toward the small G-protein Rheb (Ras homolog enriched in brain), and both biochemical and genetic studies have established that TSC2 acts through Rheb to inhibit mTOR function. Recent studies have also found that mTOR activation is important for the secretion of vascular endothelial growth factor (VEGF), which is a potent prooncogenic factor that increases the growth of blood vessels near tumors. Consistent with this observation, VEGF secretion is enhanced in TSC2-/- cells, and rapamycin effectively inhibits the secretion of VEGF from these cells. LKB1-/- cells are also reported to secrete high levels of VEGF compared with their cognate wild-type cells. Given the biochemical relationship between LKB1 and AMPK, the observation that LKB1-/- and TSC2-/- cells display high VEGF expression, and the histological similarity between the hamartomas of PJS and TSC, the functional relationship between the two tumor suppressors, LKB1 and TSC2, was examined (Corradetti, 2004).

LKB1 is one of the first protein kinases shown to function as a tumor suppressor. Despite extensive biochemical, genetic, and cell biological studies, the molecular mechanism of LKB1 as a tumor suppressor has remained elusive. However, the identification of AMPK as a physiological substrate of LKB1 and the observations described in this report provide an important clue for how LKB1 could negatively regulate cell growth. The following mechanism is proposed to explain how LKB1 activates the TSC2 tumor suppressor: LKB1 directly phosphorylates and activates AMPK. The active AMPK then phosphorylates TSC2 to enhance TSC2 function. TSC2 subsequently inhibits mTOR function via TSC2's GAP activity toward the Rheb small GTPase. Therefore, it is postulated that LKB1 negatively regulates cell growth by inhibiting the phosphorylation of important mTOR targets such as S6K and 4EBP1. It should be noted that, in addition to AMPK, LKB1 can phosphorylate and activate several AMPK-related kinases—although these kinases are not regulated by AMP. It remains to be seen whether these AMPK-related kinases can also contribute to LKB1-mediated phosphorylation of TSC2. However, the results make clear that the LKB1-AMPK kinase cascade likely plays a major role in TSC2-mTOR regulation of cellular energy response. Moreover, these data indicate that mutation in TSC2 or LKB1 produces similar cellular phenotypes (rapamycin-sensitive apoptosis and VEGF production), supporting the idea that the two tumor-suppressor proteins function in the same pathway (Corradetti, 2004).

It should also be noted that mutations in the phosphoinositide phosphatase PTEN are associated with Cowden's disease and the Bannayan-Riley-Ruvalcaba syndrome, two other dominantly inherited hamartoma syndromes. Loss of PTEN leads to an increase in 3-phosphoinositide concentration and subsequent activation of AKT, and AKT has been shown to inhibit TSC2 function. Thus, the pathophysiology of several syndromes associated with benign tumors may converge on the TSC2 and mTOR pathway. It is also especially significant that mTOR is a key downstream target of LKB1 in the regulation of cell growth, because the immunosuppressant drug rapamycin specifically inhibits mTOR. Recently, murine studies and clinical trials have indicated that rapamycin and other mTOR inhibitors may be potent drugs for the treatment of TSC and other neoplasms. These studies suggest that rapamycin and mTOR inhibitors may also be potential drugs for the comprehensive treatment of PJS (Corradetti, 2004).

Germline mutations in LKB1, TSC2, or PTEN tumor suppressor genes result in hamartomatous syndromes with shared tumor biological features. The recent observations of LKB1-mediated activation of AMP-activated protein kinase (AMPK) and AMPK inhibition of mTOR through TSC2 prompted an examination of the biochemical and biological relationship between LKB1 and mTOR regulation. LKB1 is required for repression of mTOR under low ATP conditions in cultured cells in an AMPK- and TSC2-dependent manner, and Lkb1 null MEFs and the hamartomatous gastrointestinal polyps from Lkb1 mutant mice show elevated signaling downstream of mTOR. These findings position aberrant mTOR activation at the nexus of these germline neoplastic conditions and suggest the use of mTOR inhibitors in the treatment of Peutz-Jeghers syndrome (Shaw, 2004b).

Mutation of LKB reveals that it is a tumor suppressor

This study investigates the molecular background of the Peutz-Jeghers syndrome (PJS), a rare hereditary disease in which there is predisposition to benign and malignant tumours of many organ systems. A locus for this condition was recently assigned to chromosome 19p. Truncating germline mutations have been identified in a gene residing on chromosome 19p in multiple individuals affected by PJS. This previously identified but unmapped gene, LKB1, has strong homology to a cytoplasmic Xenopus serine/threonine protein kinase XEEK1, and weaker similarity to many other protein kinases. Peutz-Jeghers syndrome is therefore the first cancer-susceptibility syndrome to be identified that is due to inactivating mutations in a protein kinase (Hemminki, 1998).

Peutz-Jeghers (PJ) syndrome is an autosomal-dominant disorder characterized by melanocytic macules of the lips, multiple gastrointestinal hamartomatous polyps and an increased risk for various neoplasms, including gastrointestinal cancer. The PJ gene maps to chromosome 19p13.3 by linkage analysis, with the highest lod score at marker D19S886. In a distance of 190 kb proximal to D19S886, a novel human gene encoding the serine threonine kinase STK11 was identified and characterized. In a three-generation PJ family, an STK11 allele was found with a deletion of exons 4 and 5 and an inversion of exons 6 and 7 segregating with the disease. Sequence analysis of STK11 exons in four unrelated PJ patients has identified three nonsense and one acceptor splice site mutations. All five germline mutations are predicted to disrupt the function of the kinase domain. It is concluded that germline mutations in STK11, probably in conjunction with acquired genetic defects of the second allele in somatic cells, cause the manifestations of PJ syndrome (Jenne, 1998).

Germ-line mutations of LKB1 lead to Peutz-Jeghers syndrome characterized by gastrointestinal polyps and cancer of different organ systems. The mutations lead to loss or severe impairment of Lkb1 serine/threonine kinase activity. Therefore LKB1 has been implicated as a tumor suppressor gene, but only a few mutations in the coding exons of LKB1 have been detected in sporadic tumors. This study identified tumor cell lines with severely reduced mRNA levels and impaired Lkb1 kinase activity. Reintroducing Lkb1 into these cells suppresses cell growth. The Lkb1-mediated growth inhibition is caused by a G(1) cell cycle block and is not detected with several naturally occurring Lkb1 mutants. These results indicate that LKB1 has functional and specific growth-suppressing activity (Tiainen, 1999).

Germline mutations in LKB1 are associated with Peutz-Jeghers syndrome (PJS), a disorder with predisposition to gastrointestinal polyposis and cancer. PJS polyps are unusual neoplasms characterized by marked epithelial and stromal overgrowth but have limited malignant potential. Here it has been shown that Lkb1+/- mice develop intestinal polyps identical to those seen in individuals affected with PJS. Consistent with this in vivo tumour suppressor function, Lkb1 deficiency prevents culture-induced senescence without loss of Ink4a/Arf or p53. Despite compromised mortality, Lkb1-/- mouse embryonic fibroblasts show resistance to transformation by activated Ha-Ras either alone or with immortalizing oncogenes. This phenotype is in agreement with the paucity of mutations in Ras seen in PJS polyps and suggests that loss of Lkb1 function as an early neoplastic event renders cells resistant to subsequent oncogene-induced transformation. In addition, the Lkb1 transcriptome shows modulation of factors linked to angiogenesis, extracellular matrix remodelling, cell adhesion and inhibition of Ras transformation. Together, these data rationalize several features of PJS polyposis -- notably its peculiar histopathological presentation and limited malignant potential -- and place Lkb1 in a distinct class of tumour suppressors (Bardeesy, 2002).

Frequent losses of chromosome 19p have recently been observed in sporadic lung adenocarcinomas, targeting the location of a critical tumor suppressor gene. Fine mapping of the short arm of chromosome 19 was performed and it was found that the LKB1/STK11 gene maps in the minimal-deleted region. Because germ-line mutations at LKB1/STK11 result in the Peutz-Jeghers syndrome and an increased risk of cancer, a detailed genetic screen of the LKB1/STK11 gene was performed in lung tumors. A high frequency of somatic alterations (mainly nonsense mutations) was detected in primary lung adenocarcinomas and in lung cancer cell lines. Thus, these findings demonstrate for the first time that LKB1/STK11 inactivation is a very common event and may be integrally involved in the development of sporadic lung adenocarcinoma (Sanchez-Cespedes, 2002).

Weak expression of LKB1 occurs at a certain frequency in sporadic breast cancer. This indicates that LKB1 gene may relate to the tumorigenesis of breast cancer. To investigate the function of the LKB1 gene in sporadic breast cancer, LKB1 was reintroduced into breast cancer cell lines which lack the LKB1 gene. Also, the LKB1 protein expression was examined in human breast cancer samples. Reintroducing LKB1 into breast cancer cell lines suppresses cell growth by G(1) cell cycle block. The LKB1-mediated G(1) cell cycle arrest is caused by up-regulation of the expression of p21(WAF1/CIP1) in breast cancer MDA-MB-435 cells. Low LKB1 protein expression correlates with higher histological grade, larger tumor size, progesterone receptor status, and presence of lymph node metastasis. Furthermore, LKB1 low expression is associated with a higher relapse rate and a worse OS. It is concluded LKB1 plays a role in tumor suppressor function in human breast cancer. LKB1 expression may be a useful prognostic marker in human breast cancer (Shen, 2002).

Peutz-Jeghers Syndrome (PJS) is thought to be caused by mutations occurring in the widely expressed serine/threonine protein kinase named LKB1/STK11. Recent work has led to the identification of four mutants (R304W, I177N, K175-D176del, L263fsX286) and two novel aberrant LKB1/STK11 cDNA isoforms (r291-464del, r485-1283del) in a group of PJS Italian patients. Three of the four mutations only change 1 or 2 amino acids in the LKB1/STK11 catalytic domain. All six LKB1/STK11 variants analysed are completely inactive in vitro; they were unable to autophosphorylate at Thr336, the major LKB1/STK11 autophosphorylation site, and to phosphorylate the p53 tumour suppressor protein. Five out of the six variants are entirely localised in the nucleus in contrast to the wild type LKB1/STK11, which is detected in both the nucleus and cytoplasm. Finally it has been demonstrated that all 6 LKB1/STK11 variants, in contrast to wild type LKB1/STK11, are unable to suppress the growth of melanoma G361 cells. Taken together, these results demonstrate that the LKB1 mutations investigated in this study lead to the loss of serine/threonine kinase activity and are therefore likely to be the primary cause of PJS development in the patients that they were isolated from (Boudeau, 2003).

The kinase LKB1 mediates glucose homeostasis in liver and therapeutic effects of metformin

The Peutz-Jegher syndrome tumor-suppressor gene encodes a protein-threonine kinase, LKB1, which phosphorylates and activates AMPK [adenosine monophosphate (AMP)-activated protein kinase]. The deletion of LKB1 in the liver of adult mice results in a nearly complete loss of AMPK activity. Loss of LKB1 function results in hyperglycemia with increased gluconeogenic and lipogenic gene expression. In LKB1-deficient livers, TORC2, a transcriptional coactivator of CREB (cAMP response element-binding protein), is dephosphorylated and enters the nucleus, driving the expression of peroxisome proliferator-activated receptor-gamma coactivator 1alpha (PGC-1alpha), which in turn drives gluconeogenesis. Adenoviral small hairpin RNA (shRNA) for TORC2 reduces PGC-1alpha expression and normalizes blood glucose levels in mice with deleted liver LKB1, indicating that TORC2 is a critical target of LKB1/AMPK signals in the regulation of gluconeogenesis. Finally, metformin, one of the most widely prescribed type 2 diabetes therapeutics, is shown to require LKB1 in the liver to lower blood glucose levels (Shaw, 2005).

LKB1 and cell cycle arrest

Germline mutations of the LKB1 tumor suppressor gene lead to Peutz-Jeghers syndrome (PJS), with a predisposition to cancer. LKB1 encodes for a nuclear and cytoplasmic serine/threonine kinase, which is inactivated by mutations observed in PJS patients. Restoring LKB1 activity into cancer cell lines defective for its expression results in a G(1) cell cycle arrest. Investigated here were molecular mechanisms leading to this arrest. Reintroduced active LKB1 was cytoplasmic and nuclear, whereas most kinase-defective PJS mutants of LKB1 localized predominantly to the nucleus. Moreover, when LKB1 was forced to remain cytoplasmic through disruption of the nuclear localization signal, it retained full growth suppression activity in a kinase-dependent manner. LKB1-mediated G(1) arrest was found to be bypassed by co-expression of the G(1) cyclins cyclin D1 and cyclin E. In addition, the protein levels of the CDK inhibitor p21(WAF1/CIP1) and p21 promoter activity were specifically upregulated in LKB1-transfected cells. Both the growth arrest and the induction of the p21 promoter were found to be p53-dependent. These results suggest that growth suppression by LKB1 is mediated through signaling of cytoplasmic LKB1 to induce p21 through a p53-dependent mechanism (Tiainen, 2002).

The LKB1/STK11 serine/threonine kinase is mutated in Peutz-Jeghers syndrome and various sporadic cancers such as lung adenocarcinoma. LKB1 forms a complex with LMO4, GATA-6, and Ldb1, and enhances GATA-mediated transactivation in a kinase-dependent manner. LKB1 has the potential to induce p21 expression in collaboration with LMO4, GATA-6, and Ldb1 through the p53-independent mechanism. These findings suggest that LKB1 regulates GATA-mediated gene expression and that this activity of LKB1 may be important for its tumor suppressor function (Setogawa, 2006).

LKB1 is a mediator of p53-dependent cell death

This study investigated the mechanism and function of LKB1, a Ser/Thr kinase mutated in Peutz-Jegher syndrome (PJS). LKB1 physically associates with p53 and regulates specific p53-dependent apoptosis pathways. LKB1 protein is present in both the cytoplasm and nucleus of living cells and translocates to mitochondria during apoptosis. In vivo, LKB1 is highly upregulated in pyknotic intestinal epithelial cells. In contrast, polyps arising in Peutz-Jegher patients are devoid of LKB1 staining and have reduced numbers of apoptotic cells. It is proposed that a deficiency in apoptosis is a key factor in the formation of multiple benign intestinal polyps in PJS patients, and possibly for the subsequent development of malignant tumors in these patients (Karuman, 2001).

LKB1 associates with Brg1 and is necessary for Brg1-induced growth arrest

Inactivating mutations in the serine-threonine kinase LKB1 (STK11) are found in most patients with Peutz-Jeghers syndrome; however the function of LKB1 is unknown. This study found that LKB1 binds to and regulates brahma-related gene 1 (Brg1), an essential component of chromatin remodeling complexes. The association requires the N terminus of LKB1 and the helicase domain of Brg1 and LKB1 stimulates the ATPase activity of Brg1. Brg1 expression in SW13 cells induces the formation of flat cells indicative of cell cycle arrest and senescence. Expression of a kinase-dead mutant of LKB1, SL26, in SW13 cells blocks the formation of Brg1-induced flat cells, indicating that LKB1 is required for Brg1-dependent growth arrest. The inability of mutants of LKB1 to mediate Brg1-dependent growth arrest may explain the manifestations of Peutz-Jeghers syndrome (Marignani, 2001).

LKB1/PAR4 protein is asymmetrically localized in mouse oocytes and associates with meiotic spindle

The mouse secondary oocyte is polarized at the ultrastructural and molecular level, but very little is known about mechanisms involved in the establishment of this polarity. LKB1 kinase, a mouse homologue of C. elegans PAR4 protein is asymmetrically localized to the animal pole of the mouse oocyte and during oocyte maturation associates with the microtubules of metaphase I and metaphase II meiotic spindles. Therefore, it is suggested that LKB1/PAR4 protein, may participate in the polarization of the oocyte and in the regulation of the asymmetry of meiotic divisions during mouse oogenesis (Szczepanska, 2005).

Activation of PAR-1 kinase and stimulation of tau phosphorylation by diverse signals require the tumor suppressor protein LKB1.

Aberrant phosphorylation of tau is associated with a number of neurodegenerative diseases, including Alzheimer's disease (AD). The molecular mechanisms by which tau phosphorylation is regulated under normal and disease conditions are not well understood. Microtubule affinity regulating kinase (MARK) and PAR-1 have been identified as physiological tau kinases, and aberrant phosphorylation of MARK/PAR-1 target sites in tau has been observed in AD patients and animal models. This study shows that phosphorylation of PAR-1 by the tumor suppressor protein LKB1 is required for PAR-1 activation, which in turn promotes tau phosphorylation in Drosophila. Diverse stress stimuli, such as high osmolarity and overexpression of the human beta-amyloid precursor protein, can promote PAR-1 activation and tau phosphorylation in an LKB1-dependent manner. These results reveal a new function for the tumor suppressor protein LKB1 in a signaling cascade through which the phosphorylation and function of tau is regulated by diverse signals under physiological and pathological conditions (Wang, 2007).

Primary cilia regulate mTORC1 activity and cell size through Lkb1

The mTOR pathway is the central regulator of cell size. External signals from growth factors and nutrients converge on the mTORC1 multi-protein complex to modulate downstream targets, but how the different inputs are integrated and translated into specific cellular responses is incompletely understood. Deregulation of the mTOR pathway occurs in polycystic kidney disease (PKD), where cilia (filiform sensory organelles) fail to sense urine flow because of inherited mutations in ciliary proteins. It was therefore investigated if cilia have a role in mTOR regulation. This study shows that ablation of cilia in transgenic mice results in enlarged cells when compared with control animals. In vitro analysis demonstrated that bending of the cilia by flow is required for mTOR downregulation and cell-size control. Surprisingly, regulation of cell size by cilia is independent of flow-induced calcium transients, or Akt. However, the tumour-suppressor protein Lkb1 localises in the cilium, and flow results in increased AMPK phosphorylation at the basal body. Conversely, knockdown of Lkb1 prevents normal cell-size regulation under flow conditions. These results demonstrate that the cilium regulates mTOR signalling and cell size, and identify the cilium-basal body compartment as a spatially restricted activation site for Lkb1 signalling (Boehlke, 2010).

Lkb1 regulates cell cycle and energy metabolism in haematopoietic stem cells

Little is known about metabolic regulation in stem cells and how this modulates tissue regeneration or tumour suppression. The Lkb1 tumour suppressor and its substrate AMP-activated protein kinase (AMPK), kinases that coordinate metabolism with cell growth, were studied. Deletion of the Lkb1 (also called Stk11) gene in mice caused increased haematopoietic stem cell (HSC) division, rapid HSC depletion and pancytopenia. HSCs depended more acutely on Lkb1 for cell-cycle regulation and survival than many other haematopoietic cells. HSC depletion did not depend on mTOR activation or oxidative stress. Lkb1-deficient HSCs, but not myeloid progenitors, had reduced mitochondrial membrane potential and ATP levels. HSCs deficient for two catalytic α-subunits of AMPK (AMPK-deficient HSCs) showed similar changes in mitochondrial function but remained able to reconstitute irradiated mice. Lkb1-deficient HSCs, but not AMPK-deficient HSCs, exhibited defects in centrosomes and mitotic spindles in culture, and became aneuploid. Lkb1 is therefore required for HSC maintenance through AMPK-dependent and AMPK-independent mechanisms, revealing differences in metabolic and cell-cycle regulation between HSCs and some other haematopoietic progenitors (Nakada, 2010).

The Lkb1 metabolic sensor maintains haematopoietic stem cell survival

Haematopoietic stem cells (HSCs) can convert between growth states that have marked differences in bioenergetic needs. Although often quiescent in adults, these cells become proliferative upon physiological demand. Balancing HSC energetics in response to nutrient availability and growth state is poorly understood, yet essential for the dynamism of the haematopoietic system. This study shows that the Lkb1 tumour suppressor is critical for the maintenance of energy homeostasis in haematopoietic cells. Lkb1 inactivation in adult mice causes loss of HSC quiescence followed by rapid depletion of all haematopoietic subpopulations. Lkb1-deficient bone marrow cells exhibit mitochondrial defects, alterations in lipid and nucleotide metabolism, and depletion of cellular ATP. The haematopoietic effects are largely independent of Lkb1 regulation of AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) signalling. Instead, these data define a central role for Lkb1 in restricting HSC entry into cell cycle and in broadly maintaining energy homeostasis in haematopoietic cells through a novel metabolic checkpoint (Gurumurthy, 2010).

Lkb1 regulates quiescence and metabolic homeostasis of haematopoietic stem cells

The capacity to fine-tune cellular bioenergetics with the demands of stem-cell maintenance and regeneration is central to normal development and ageing, and to organismal survival during periods of acute stress. How energy metabolism and stem-cell homeostatic processes are coordinated is not well understood. Lkb1 acts as an evolutionarily conserved regulator of cellular energy metabolism in eukaryotic cells and functions as the major upstream kinase to phosphorylate AMP-activated protein kinase (AMPK) and 12 other AMPK-related kinases. Whether Lkb1 regulates stem-cell maintenance remains unknown. This study shows that Lkb1 has an essential role in haematopoietic stem cell (HSC) homeostasis. Ablation of Lkb1 in adult mice results in severe pancytopenia and subsequent lethality. Loss of Lkb1 leads to impaired survival and escape from quiescence of HSCs, resulting in exhaustion of the HSC pool and a marked reduction of HSC repopulating potential in vivo. Lkb1 deletion has an impact on cell proliferation in HSCs, but not on more committed compartments, pointing to context-specific functions for Lkb1 in haematopoiesis. The adverse impact of Lkb1 deletion on haematopoiesis was predominantly cell-autonomous and mTOR complex 1 (mTORC1)-independent, and involves multiple mechanisms converging on mitochondrial apoptosis and possibly downregulation of PGC-1 coactivators and their transcriptional network, which have critical roles in mitochondrial biogenesis and function. Thus, Lkb1 serves as an essential regulator of HSCs and haematopoiesis, and more generally, points to the critical importance of coupling energy metabolism and stem-cell homeostasis (Gan, 2010).

The Par3-like polarity protein Par3L is essential for mammary stem cell maintenance

The Par polarity proteins play key roles in asymmetric division of Drosophila melanogaster stem cells; however, whether the same mechanisms control stem cells in mammals is controversial. Although necessary for mammary gland morphogenesis, Par3 is not essential for mammary stem cell function. This study discovered that, instead, a previously uncharacterized protein, Par3-like (Par3L), is vital for mammary gland stem cell maintenance. Par3L function has been mysterious because, unlike Par3, it does not interact with atypical protein kinase C or the Par6 polarity protein. Par3L was found to be expressed by multipotent stem cells in the terminal end buds of murine mammary glands. Ablation of Par3L resulted in rapid and profound stem cell loss. Unexpectedly, Par3L, but not Par3, binds to the tumour suppressor protein Lkb1 and inhibits its kinase activity. This interaction is key for the function of Par3L in mammary stem cell maintenance. These data reveal insights into a link between cell polarity proteins and stem cell survival, and uncover a biological function for Par3L (Huo, 2014).

LKB1 and AMPK regulate synaptic remodeling in old age

Age-related decreases in neural function result in part from alterations in synapses. To identify molecular defects that lead to such changes, this study focused on the outer retina, in which synapses are markedly altered in old rodents and humans. The serine/threonine kinase LKB1 and one of its substrates, AMPK (see Drosophila Ampk), regulate this process. In old mice, synaptic remodeling was accompanied by specific decreases in the levels of total LKB1 and active (phosphorylated) AMPK. In the absence of either kinase, young adult mice developed retinal defects similar to those that occurred in old wild-type animals. LKB1 and AMPK function in rod photoreceptors where their loss leads to aberrant axonal retraction, the extension of postsynaptic dendrites and the formation of ectopic synapses. Conversely, increasing AMPK activity genetically or pharmacologically attenuates and may reverse age-related synaptic alterations. Together, these results identify molecular determinants of age-related synaptic remodeling and suggest strategies for attenuating these changes (Samuel, 2014).


Search PubMed for articles about Drosophila Lkb1 kinase

Al-Hakim, A. K., et al. (2005). 14-3-3 cooperates with LKB1 to regulate the activity and localization of QSK and SIK. J. Cell Sci. 118(Pt 23): 5661-73. 16306228

Andersen, R. O., Turnbull, D. W., Johnson, E. A. and Doe, C. Q. (2012). Sgt1 acts via an LKB1/AMPK pathway to establish cortical polarity in larval neuroblasts. Dev Biol 363: 258-265. Pubmed: 22248825

Baas, A. F., et al. (2004) Complete polarization of single intestinal epithelial cells upon activation of LKB1 by STRAD. Cell 116: 457-466. 15016379

Bansal, P. K., Mishra, A., High, A. A., Abdulle, R. and Kitagawa, K. (2009). Sgt1 dimerization is negatively regulated by protein kinase CK2-mediated phosphorylation at Ser361. J Biol Chem 284: 18692-18698. Pubmed: 19398558

Bardeesy, N. et al. (2002). Loss of the Lkb1 tumour suppressor provokes intestinal polyposis but resistance to transformation. Nature 419: 162-167. 12226664

Benkemoun, L., Descoteaux, C., Chartier, N. T., Pintard, L. and Labbe, J. C. (2014). PAR-4/LKB1 regulates DNA replication during asynchronous division of the early C. elegans embryo. J Cell Biol 205: 447-455. PubMed ID: 24841566

Boehlke, C., et al. (2010). Primary cilia regulate mTORC1 activity and cell size through Lkb1. Nat. Cell Biol. 12(11): 1115-22. PubMed Citation: 20972424

Boudeau, J., et al. (2003). Functional analysis of LKB1/STK11 mutants and two aberrant isoforms found in Peutz-Jeghers Syndrome patients. Hum. Mutat. 21: 172. 12552571

Castanieto, A., Johnston, M. J. and Nystul, T. G. (2014). EGFR signaling promotes the identity of follicle stem cells via maintenance of partial cell polarity. Elife 3. PubMed ID: 25437306

Chartier, N. T., et al. (2011). PAR-4/LKB1 mobilizes nonmuscle myosin through anillin to regulate C. elegans embryonic polarization and cytokinesis. Curr. Biol. 21: 259-269. PubMed Citation: 21276723

Choi, S., Lim, D. S. and Chung, J. (2015). Feeding and fasting signals converge on the LKB1-SIK3 pathway to regulate lipid metabolism in Drosophila. PLoS Genet 11(5): e1005263. PubMed ID: 25996931

Collins, S. P., Reoma, J. L., Gamm, D. M. & Uhler, M. D. (2000). LKB1, a novel serine/threonine protein kinase and potential tumour suppressor, is phosphorylated by cAMP-dependent protein kinase (PKA) and prenylated in vivo. Biochem. J. 345: 673-680. 10642527

Corradetti, M. N., et al. (2004). Regulation of the TSC pathway by LKB1: evidence of a molecular link between tuberous sclerosis complex and Peutz-Jeghers syndrome. Genes Dev. 18: 1533-1538. 15231735

Forcet, C., et al. (2005). Functional analysis of Peutz-Jeghers mutations reveals that the LKB1 C-terminal region exerts a crucial role in regulating both the AMPK pathway and the cell polarity. Hum. Mol. Genet. 14(10): 1283-92. 15800014

Gan, B., et al. (2010). Lkb1 regulates quiescence and metabolic homeostasis of haematopoietic stem cells. Nature 468(7324): 701-4. PubMed Citation: 21124456

Guo, S. and Kemphues, K. J. (1995). par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed. Cell 81: 611-620. PubMed Citation: 7758115

Gurumurthy, S, et al. (2010). The Lkb1 metabolic sensor maintains haematopoietic stem cell survival. Nature 468(7324): 659-63. PubMed Citation: 21124451

Hemminki, A., et al. (1998) A serine/threonine kinase gene defective in Peutz-Jeghers syndrome. Nature 391: 184-187. 9428765

Hemminki, A. (1999). The molecular basis and clinical aspects of Peutz-Jeghers syndrome. Cell. Mol. Life Sci. 55, 735-750. 10379360

Hong, S. P., Momcilovic, M. and Carlson, M. (2005). Function of mammalian LKB1 and Ca2+/calmodulin-dependent protein kinase kinase alpha as Snf1-activating kinases in yeast. J. Biol. Chem. 280(23): 21804-9. 15831494

Huang, N. M., et al. (2002). MEX-3 interacting proteins link cell polarity to asymmetric gene expression in Caenorhabditis elegans. Development 129: 747-759. 11830574

Huo, Y. and Macara, I. G. (2014). The Par3-like polarity protein Par3L is essential for mammary stem cell maintenance. Nat Cell Biol 16: 529-537. PubMed ID: 24859006

Jaleel, M., et al. (2006). The ubiquitin-associated domain of AMPK-related kinases regulates conformation and LKB1-mediated phosphorylation and activation. Biochem J. 394(Pt 3): 545-55. 16396636

Jenne, D. E., et al. (1998). Peutz-Jeghers syndrome is caused by mutations in a novel serine threonine kinase. Nat. Genet. 18: 38-43. 9425897

Karuman, P., et al. (2001). The Peutz-Jegher gene product LKB1 is a mediator of p53-dependent cell death. Mol. Cell. 7: 1307-1319. 11430832

Kemphues, K. J., Priess, J. R., Morton, D. G. and Cheng, N. S. (1988). Identification of genes required for cytoplasmic localization in early C. elegans embryos. Cell 52: 311-320. 88151036

Lee, J. H., et al. (2005). JNK pathway mediates apoptotic cell death induced by tumor suppressor LKB1 in Drosophila. Cell Death and Differentiation 13(7): 1110-22. 16273080

Lee, J. H., et al. (2007). Energy-dependent regulation of cell structure by AMP-activated protein kinase. Nature 447(7147): 1017-20. PubMed Citation: 17486097

Marignani, P. A., Kanai, F. and Carpenter, C. L. (2001). LKB1 associates with Brg1 and is necessary for Brg1-induced growth arrest. J. Biol. Chem. 276: 32415-32418. 11445556

Martin, S. G. and St Johnston, D. (2003). A role for Drosophila LKB1 in anterior-posterior axis formation and epithelial polarity. Nature 421(6921): 379-84. 12540903

Nakada, D., Saunders, T. L. and Morrison, S. J. (2010). Lkb1 regulates cell cycle and energy metabolism in haematopoietic stem cells. Nature 468(7324): 653-8. PubMed Citation: 21124450

Narbonne, P. and Roy, R. (2006). Inhibition of germline proliferation during C. elegans dauer development requires PTEN, LKB1 and AMPK signalling. Development 133(4): 611-9. 16407400

Narbonne, P., Hyenne, V., Li, S., Labbé, J. C. and Roy, R. (2010). Differential requirements for STRAD in LKB1-dependent functions in C. elegans. Development 137(4): 661-70. PubMed Citation: 20110331

Sahin, F., et al. (2003). Loss of Stk11/Lkb1 expression in pancreatic and biliary neoplasms. Mod. Pathol. 16: 686-691. 12861065

Sakamoto, K., et al. (2005). Deficiency of LKB1 in skeletal muscle prevents AMPK activation and glucose uptake during contraction. EMBO J. 24(10): 1810-20. 15889149

Samuel, M. A., Voinescu, P. E., Lilley, B. N., de Cabo, R., Foretz, M., Viollet, B., Pawlyk, B., Sandberg, M. A., Vavvas, D. G. and Sanes, J. R. (2014). LKB1 and AMPK regulate synaptic remodeling in old age. Nat. Neurosci 17: 1190-1197. PubMed ID: 25086610

Sanchez-Cespedes, M. et al. (2002) . Inactivation of LKB1/STK11 is a common event in adenocarcinomas of the lung. Cancer Res. 62: 3659-3662. 12097271

Sapkota, G. P. et al. (2001). Phosphorylation of the protein kinase mutated in Peutz-Jeghers cancer syndrome, LKB1/STK11, at Ser431 by p90RSK and cAMP-dependent protein kinase, but not its farnesylation at Cys433, is essential for LKB1 to suppress cell growth. J. Biol. Chem. 276: 19469-19482. 11297520

Setogawa, T., et al. (2006). The tumor suppressor LKB1 induces p21 expression in collaboration with LMO4, GATA-6, and Ldb1. Biochem. Biophys. Res. Commun. 343(4): 1186-90. 16580634

Shaw, R. J., et al. (2004a). The tumor suppressor LKB1 kinase directly activates AMP-activated kinase and regulates apoptosis in response to energy stress. Proc. Natl. Acad. Sci. 101: 3329-3335. Medline abstract: 14985505

Shaw, R. J., et al. (2004b). The LKB1 tumor suppressor negatively regulates mTOR signaling. Cancer Cell 6: 91-99. 15261145

Shaw, R. J., et al. (2005). The kinase LKB1 mediates glucose homeostasis in liver and therapeutic effects of metformin. Science 310(5754): 1642-6. 16308421

Shen, Z., Wen, X. F., Lan, F., Shen, Z. Z. and Shao, Z. M. (2002). The tumor suppressor gene LKB1 is associated with prognosis in human breast carcinoma. Clin. Cancer Res. 8: 2085-2090. 12114407

Szczepanska, K. and Maleszewski M. (2005). LKB1/PAR4 protein is asymmetrically localized in mouse oocytes and associates with meiotic spindle. Gene Expr. Patterns 6(1): 86-93. 15963768

ten Klooster, J. P., et al. (2009). Mst4 and Ezrin induce brush borders downstream of the Lkb1/Strad/Mo25 polarization complex. Dev. Cell 16(4): 551-62. PubMed Citation: 19386264

Tenlen, J. R., et al. (2008). MEX-5 asymmetry in one-cell C. elegans embryos requires PAR-4- and PAR-1-dependent phosphorylation. Development 135(22): 3665-75. PubMed Citation: 18842813

Tiainen, M., Ylikorkala, A. and Makela, T. P. (1999). Growth suppression by Lkb1 is mediated by a G(1) cell cycle arrest. Proc. Natl. Acad. Sci. 96: 9248-9251. 10430928

Tiainen, M., Vaahtomeri, K., Ylikorkala, A. and Makela, T. P. (2002). Growth arrest by the LKB1 tumor suppressor: induction of p21(WAF1/CIP1). Hum. Mol. Genet. 11: 1497-1504. 12045203

Wang, J. W., Imai, Y. and Lu, B. (2007). Activation of PAR-1 kinase and stimulation of tau phosphorylation by diverse signals require the tumor suppressor protein LKB1. J. Neurosci. 27: 574-581. PubMed Citation: 17234589

Watts, J. L., Morton, D. G., Bestman, J. and Kemphues, K. J. (2000). The C. elegans par-4 gene encodes a putative serine-threonine kinase required for establishing embryonic asymmetry. Development 127: 1467-1475. 10704392

Xie, Z., et al. (2006). Activation of protein kinase C zeta by peroxynitrite regulates LKB1-dependent AMP-activated protein kinase in cultured endothelial cells. J. Biol. Chem. 281(10): 6366-75. 16407220

Yoo, L. I., Chung, D. C. and Yuan, J. (2002). LKB1--a master tumour suppressor of the small intestine and beyond. Nature Rev. Cancer 2: 529-535. 12094239

Zuehlke, A. and Johnson, J. L. (2010). Hsp90 and co-chaperones twist the functions of diverse client proteins. Biopolymers 93: 211-217. Pubmed: 19697319

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date revised: 10 February 2013

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