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).
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).
LKB1 is mutated in both familial and spontaneous tumors, and acts as a master kinase that activates the PAR-1 polarity kinase and the adenosine 5'monophosphate-activated kinase (AMPK). This has led to the hypothesis that LKB1 acts as a tumor suppressor because it is required to maintain cell polarity and growth control through PAR-1 and AMPK, respectively. However, the genetic analysis of LKB1-AMPK signaling in vertebrates has been complicated by the existence of multiple redundant AMPK subunits. This study describes the identification of mutations in the single Drosophila AMPK catalytic subunit AMPKα. Surprisingly, ampkα mutant epithelial cells lose their polarity and overproliferate under energetic stress. LKB1 is required in vivo for AMPK activation, and lkb1 mutations cause similar energetic stress-dependent phenotypes to ampkα mutations. Furthermore, lkb1 phenotypes are rescued by a phosphomimetic version of AMPKα. Thus, LKB1 signals through AMPK to coordinate epithelial polarity and proliferation with cellular energy status, and this might underlie the tumor suppressor function of LKB1 (Mirouse, 2007).
LKB1 is a serine/threonine kinase that is mutated in most cases of Peutz-Jeghers syndrome, which is an autosomal dominant disorder in which patients develop benign hamartomas and a high frequency of malignant tumors. Furthermore, LKB1 is also mutated in some sporadic cancers, such as 30% of lung adenocarcinomas, and the expression of the kinase is also down-regulated in a substantial proportion of breast cancers. In both cases, tumors associated with LKB1 mutations usually derive from epithelial tissue. LKB1 is a master kinase that can potentially activate several downstream kinases by phosphorylating a conserved threonine in their activation loops. Two of these kinases have been extensively characterized: PAR-1/microtubule affinity-regulating kinases (MARKs) and AMPK (AMP-activated protein kinase). PAR-1 regulates cell polarity in numerous cell types and organisms (see Drosophila Par-1). AMPK acts as a cellular energy sensor because it is activated by AMP, which accumulates when ATP levels are low. AMPK then mediates the cellular response to energetic stress by activating energy-producing activities, while inhibiting energy-consuming ones, such as translation and proliferation. LKB1 regulates both cell polarity and cell growth and division in cell culture and in vivo. One hypothesis envisions LKB1 signaling mediated by PAR-1 regulating cell polarity, whereas LKB1 signaling through AMPK could control cell growth and proliferation. However, recent cell culture experiments suggest that AMPK also plays a role in the polarization of MDCK cells by promoting tight junction assembly. This study shows that LKB1 and AMPK are required to maintain epithelial polarity and integrity under energy-limiting conditions in Drosophila melanogaster. Therefore, these results provide a potential mechanism to coordinate the regulation of cell polarity and proliferation with energy conditions within a multicellular animal (Mirouse, 2007).
AMPK contains three protein subunits, α, β, and γ, which form a heterotrimer. The α subunit (AMPKα) encodes a highly conserved serine/threonine kinase, and the other subunits are regulatory. From a D. melanogaster forward genetic screen for mutants affecting larval neuronal dendrite development, several lethal mutations were identified in AMPKα. The ethylmethanesulfonate mutants, ampkα1 and ampkα2, contain a single amino acid change (S211L, completely conserved) and a premature stop codon (Q295 STOP), respectively, whereas ampkα3 has a 16-bp deletion creating a stop codon (Y141 STOP). All ampkα mutants, whether homozygous or in trans with a deletion covering the locus, displayed a completely penetrant and nearly identical phenotype, with greatly enlarged plasma membrane domains in dendrites, but not in axonal compartments. In addition, ampkα1 and ampkα3 could be rescued to viability with either a chromosomal duplication carrying a wild-type ampkα gene, a wild-type AMPKα transgene, or a transgene that is tagged with the red fluorescent protein mCherry. The requirement for ampkα is cell autonomous because transgene expression within only neurons rescues the phenotype. Therefore, these mutations represent the first knockouts of the single AMPKα catalytic subunit in the Drosophla genome and allow the genetic analysis of AMPK function in vivo (Mirouse, 2007).
Although ampkα mutants display a strong phenotype in larval neuronal dendrites, no phenotype was observed in early larval lkb1 mutants, probably because of the large maternal contribution of this protein. To explore the relationship between AMPKα and LKB1 function without the confounding issues caused by the differing maternal contributions of each protein, follicle cells of the Drosophila ovary were examined. The follicle cells that surround the oocyte have a typical epithelial architecture with a highly polarized actin cytoskeleton in which the apical surface is marked by dense actin bundles in the apical microvilli, the lateral cortex is covered by a thin actin mesh, and the basal side contains a prominent network of parallel actin stress fibers. This polarized organization of actin typifies many epithelia, including the main mammalian tissue culture model for polarized epithelial cells, MDCK cells. No actin phenotypes were observed in ampkα3 mutant follicle cells using standard detection procedures. Because AMPK is maximally activated under low cellular energy levels, the influence of energy stress was tested by strongly reducing the availability of sugar in the Drosophila culture medium. Under these conditions, ampkα3 mutant cells display a strong actin phenotype. The density of basal stress fibers is strongly reduced, whereas the amount of apical F-actin increases. This phenotype is highly penetrant under these starvation conditions and is also observed with the two other alleles of ampkα (Mirouse, 2007).
Because this phenotype reflects a disruption of the apical–basal polarity of the actin cytoskeleton, polarity markers were examined within these cells. ampkα mutant clones induced in adult flies fed with high-sugar diets did not show any polarity phenotypes, which is consistent with the absence of an actin phenotype under these conditions. Under energetic starvation conditions, however, ampkα mutant cells show a fully penetrant loss of polarity. Apical markers, such as atypical PKC (aPKC) and Crumbs (Crb) lose their cortical localization completely and appear to be down-regulated, as do the lateral markers Discs large (Dlg) and Coracle (Cora). In contrast, Dystroglycan (Dg), which is normally enriched at the basal cortex, extends into the lateral domain, and occasionally even reaches the apical membrane. This suggests that the phenotype represents an expansion of the basal domain at the expense of the lateral and apical domains (Mirouse, 2007).
Although most aspects of apical–basal polarity are completely disrupted in ampkα mutant clones under energetic stress, E-cadherin (ECad) is usually still enriched at the adherens junctions, suggesting that the altered polarity is not a secondary consequence of a loss of intercellular adhesion. The subapical localization of Bazooka (Baz) with cadherin is also maintained in most cases. This indicates that Baz is not in a complex with aPKC in columnar follicle cells, but is instead associated with the adherens junctions (Mirouse, 2007).
A considerable proportion of ampkα mutant clones show a more severe phenotype, in which the cells round up and lose their epithelial organization to form multiple layers of cells. In these cases, Baz is now also absent from the cell cortex. Finally, larger mutant clones, particularly at the anterior or the posterior of the egg chamber, show a complete loss of epithelial organization and overproliferate to form small, tumorlike growths (Mirouse, 2007).
As one proposed function for AMPK is to sense and maintain cellular ATP levels, the polarity phenotype observed under starvation conditions could be caused by low cellular ATP concentrations. To test this hypothesis, cells were examined that were mutant for tenured (tend). Tend encodes a mitochondrial cytochrome oxidase subunit; therefore, mutants have reduced intracellular ATP concentrations to levels sufficient to maintain cell survival and growth, but not cell division. This cell cycle block is believed to require AMPK activation. In agreement with a role for Tend in cell cycle progression, tend clones bigger than four to six cells under were not observed energetic starvation conditions. In contrast to ampkα mutant cells, however, tend mutant cells showed no polarity defects, ruling out the possibility that the ampkα phenotype is a secondary effect of low ATP levels. The effect of specific nutrient starvation was also tested by feeding flies only glucose, but these conditions did not induce any polarity phenotypes in ampkα mutant cells. Thus, AMPKα is specifically required to maintain epithelial polarity and growth control under conditions of energetic stress (Mirouse, 2007).
Because the results indicate that ampkα plays a role in epithelial polarity, whether the localization of the protein itself is polarized was assessed. LKB1 localization was also examined, since it is a potential regulator of AMPK. Transgenic wild-type fusion proteins for both AMPKα and LKB1 rescue lethal null mutants to viability, and should therefore mimic the localizations of the endogenous proteins. LKB1-GFP is mainly found at the apical and lateral cortex of the follicle cells, and is absent from the basal domain. This basal exclusion is surprising, as cortical localization of LKB1 requires its membrane targeting by prenylation of a conserved CAAX motif. This suggests that the lipid composition of the basal domain is different from the rest of the plasma membrane and/or that LKB1 posttranslational modifications are asymmetrically controlled. In contrast, mCherry-AMPKα does not show any enrichment or asymmetric localization at the plasma membrane, and it is found distributed throughout the cytoplasm, but absent from the nucleus. The localization of LKB1 suggests that AMPK could be activated specifically at the apical and lateral cortices of the cells. To test this hypothesis, an antibody was used against the LKB1 phosphorylation site of AMPK (phospho-T184). The immunostaining is reduced to background levels in both ampkα and lkb1 mutant clones. This confirms the specificity of the antibody and indicates that LKB1 is the principle AMPK kinase in these cells. In wild-type cells, PhosphoT184-AMPK is found diffusely in the cytoplasm. The effect of AMPK on apical–basal polarity is therefore not related to a polarized distribution of the kinase or its localized activation by LKB1 (Mirouse, 2007).
Because LKB1 activates AMPK, it was asked if similar phenotypes could be observed in lkb1 mutant cells. lkb1 clones can lead to severe polarity defects in follicle cells in normally fed flies. However, these defects are observed only in large clones that are induced in the stem cells that give rise to the follicular epithelium, whereas small lkb1 mutant clones, which are induced after the formation of the epithelium, have no effect on follicle cell polarity or the organization of the actin cytoskeleton. This suggests LKB1 is required for the establishment of epithelial polarity in well-fed flies, but not for its maintenance, as is the case for PAR-1. In contrast, under conditions of glucose starvation, small lkb1 clones that were induced after the formation of the follicular epithelium show a fully penetrant polarity phenotype. Under these conditions, a loss of the polarized localization of Dlg, aPKC, Crb, and Cora was observed. However, Baz distribution is usually not affected by lkb1 loss of function. Dg extends laterally and occasionally localizes to the apical domain. The actin cytoskeleton is also disturbed, with more F-actin apically and a decreased density of stress fibers on the basal side. Finally, large lkb1 clones lose their epithelial organization completely and overproliferate to form small neoplasms. Thus, lkb1 mutant cells exhibit identical phenotypes to ampkα mutant cells under low-energy conditions (Mirouse, 2007).
Because lkb1 and ampkα mutant clones lead to very similar polarity defects and LKB1 phosphorylates AMPKα, it was asked if a constitutively active form of AMPKα could rescue the lkb1 phenotype. Therefore, transgenic lines were generated carrying a UAS-AMPKα construct, in which Threonine184 is replaced by an aspartate, which should mimic the activating phosphorylation of this site by LKB1. The expression of the AMPKα-T184D transgene in lkb1 mutant clones fully rescues their starvation-dependent polarity and overproliferation phenotypes, whereas the Gal4 driver alone has no effect. Furthermore, AMPKα-T184D-expressing mutant clones also have a normal actin cytoskeleton. Thus, the phosphomimetic version of AMPKα completely rescues the lkb1 mutant phenotype under conditions of energetic stress (Mirouse, 2007).
The recovery of null mutations in ampkα has allowed the first in vivo analysis of AMPK function in a multicellular organism, which has revealed an unexpected role for the kinase in the maintenance of epithelial polarity, but only under conditions of energetic stress. This implies that at least one of the pathways that normally maintain cell polarity cannot function when cellular energy levels are too low, and that AMPK activation compensates for this defect (Mirouse, 2007).
A surprising feature of the ampkα polarity phenotype is that it has opposite effects on the actin cytoskeleton and the cortical polarity cues. In mildly affected clones, basal actin is strongly reduced, with a corresponding increase in the amount of apical actin. In contrast, mutant clones show an expansion of the basal markers into the lateral and apical regions, as well as a loss of lateral and apical markers. Thus, the effects on actin may be independent of other polarity defects, suggesting that AMPK acts though different pathways to regulate actin and cortical polarity in opposite ways (Mirouse, 2007).
It is unclear how AMPK regulates the actin cytoskeleton, but it is possible that it acts on only one side of the cell and that the reciprocal changes on the other are caused by a change in the concentration of free G-actin or an actin nucleator, as has been shown for abl mutants during cellularization. For example, loss of AMPK could increase actin polymerization apically, thereby depleting the pool of free actin that can polymerize basally. Alternatively, ampkα mutants may prevent the formation of basal actin stress fibers, and thus increase the concentration of free actin, which enhances apical actin polymerization (Mirouse, 2007).
The cortical polarity defects of ampkα mutant clones also suggest a reciprocal relationship between the basal and apical/lateral membrane domains because the basal domain, marked by Dg, is dramatically expanded, whereas the determinants for the lateral domain (Dlg) and the apical domain (aPKC and Crb) disappear from the cortex. This suggests that there is some form of mutual antagonism between the basal and lateral domains that maintains a sharp boundary between them, as has been described for apical and lateral domains through the inhibitory phosphorylation of Baz (PAR-3) by lateral PAR-1, and of PAR-1 by apical aPKC. If this model is correct, AMPK could be required to restrict the extent of the basal domain, with the expansion of this domain in ampkα mutants leading to the exclusion of lateral and apical markers. Indeed, the overexpression of Dg has been found to cause a similar loss of apical and lateral markers to that seen in ampkα clones. Alternatively, AMPK could be necessary to maintain the localization of the apical and lateral determinants, which in turn prevent the basal domain from extending into these regions (Mirouse, 2007).
Mutations in AMPK not only disrupt the polarity of the follicle cell epithelium, but also cause the cells to overproliferate, giving rise to a tumorous phenotype. One possible explanation for this phenotype is that it is caused by the mislocalization and down-regulation of Dlg. Dlg is a member of a class of tumor suppressors in Drosophila that also includes Lgl and Scribble, and follicle cell clones mutant for any of these genes overproliferate to form invasive tumors that are similar to those formed by ampkα and lkb1 clones under low-energy conditions. Furthermore, the tumor suppressor function of these proteins is probably conserved in humans because Scribble restricts proliferation by repressing the G1/S transition, and is a target of the papilloma virus E6 oncoprotein. This may account for the observation that AMPK is required to trigger the G1/S checkpoint under conditions of energetic stress. However, it has also been shown in mammals that AMPK activates TSC2 to repress the insulin–TOR pathway, and thus it functions as a tumor suppressor that inhibits cell growth and division. Loss of this repression might provide an alternative explanation for the overgrowth of ampkα mutant clones (Mirouse, 2007).
Although the molecular pathways involved remain to be elucidated, these results demonstrate that ampkα mutant cells lose their polarity under low-energy conditions and overproliferate to give rise to tumorlike growths. The activation of AMPK depends on its phosphorylation by LKB1, and loss of LKB1 produces an identical tumorous phenotype. Thus, the novel functions of AMPK reported in this work may provide a basis for the tumor suppressor function of LKB1 (Mirouse, 2007).
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).
Reference names in red indicate recommended papers.
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
Baas, A. F., et al. (2004) Complete polarization of single intestinal epithelial cells upon activation of LKB1 by STRAD. Cell 116: 457-466. 15016379
Bardeesy, N. et al. (2002). Loss of the Lkb1 tumour suppressor provokes intestinal polyposis but resistance to transformation. Nature 419: 162-167. 12226664
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
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
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
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
Mirouse, V., Swick, L. L., Kazgan, N., St Johnston, D. and Brenman, J. E. (2007). LKB1 and AMPK maintain epithelial cell polarity under energetic stress. J. Cell Biol. 177(3): 387-92. PubMed Citation: 17470638
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
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
date revised: 25 June 2011
Home page: The Interactive Fly © 2006 Thomas Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.