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).
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).
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).
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).
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 kinasesóalthough 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).
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 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).
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).
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).
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).
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).
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