Ca2+ signalling: a new route to NAADP

The LKB1 serine/threonine kinase is a tumour suppressor responsible for the inherited familial cancer disorder Peutz-Jeghers syndrome and is inactivated in a large percentage of human lung cancers. LKB1 acts a master kinase, directly phosphorylating and activating a family of 14 AMPK (AMP-activated protein kinase)-related kinases which control cell metabolism, cell growth and cell polarity. In this issue of the Biochemical Journal, Hardie and colleagues discover an alternative splice form of LKB1 that alters the C-terminus of the protein containing a few known sites of post-translational regulation. Although widely expressed, the short isoform (LKB1(s)) is the sole splice isoform expressed in testes, and its expression peaks at the time of spermatid maturation. Male mice lacking the LKB1(s) isoform have dramatic defects in spermatozoa, resulting in sterility.     

Characterization of an alternative splice variant of LKB1

LKB1 is an upstream activating kinase for the AMP-activated protein kinase (AMPK) and at least 12 other AMPK-related kinases. LKB1 therefore acts as a master kinase regulating the activity of a wide range of downstream kinases, which themselves have diverse physiological roles. Here we identify a second form of LKB1 generated by alternative splicing of the LKB1 gene. The two LKB1 proteins have different C-terminal sequences generating a 50-kDa form (termed LKB1L) and a 48-kDa form (LKB1S). LKB1L is widely expressed in mouse tissues, whereas LKB1S has a restricted tissue distribution with predominant expression in the testis. LKB1S, like LKB1L, forms a complex with MO25 and STRAD, and phosphorylates and activates AMPK both in vitro and in intact cells. A phosphorylation site (serine 431 in mouse) and a farnesylation site (cysteine 433 in mouse) within LKB1L are not conserved in LKB1S raising the possibility that these sites might be involved in differential regulation and/or localization of the two forms of LKB1. However, we show that phosphorylation of serine 431 has no effect on LKB1L activity and that both LKB1L and LKB1S have similar patterns of subcellular localization. These results indicate that the physiological significance of the different forms of LKB1 is not related directly to differences in the C-terminal sequences but may be due to their differential patterns of tissue distribution.     

C-terminal phosphorylation of LKB1 is not required for regulation of AMP-activated protein kinase, BRSK1, BRSK2, or cell cycle arrest

The tumor suppressor protein kinase LKB1 exerts its effects by phosphorylating and activating AMP-activated protein kinase (AMPK) and members of the AMPK-related kinase family, such as the brain-specific kinases BRSK1/BRSK2 (SAD-B/SAD-A). LKB1 contains a conserved serine residue near the C terminus (Ser-431 in mouse LKB1) that is phosphorylated by cyclic AMP-dependent protein kinase and p90-RSK. Although some studies suggest that LKB1 is constitutively active and is not rate-limiting for activation of AMPK, others have suggested that phosphorylation of Ser-431 is necessary to allow LKB1 to phosphorylate and activate AMPK and other downstream kinases. Prompted by our discovery of an LKB1 splice variant (LKB1S) that lacks Ser-431, we have reinvestigated this question. In HeLa cells (which lack endogenous LKB1), co-expression with STRADalpha and MO25alpha of wild type LKB1, the S431A or S431E mutants of LKB1, or LKB1(S) gave equal levels of activation of endogenous AMPK. Similarly, recombinant STRADalpha.MO25alpha complexes containing these LKB1 variants were equally effective at phosphorylating and activating AMPK, BRSK1, and BRSK2 in cell-free assays. Finally, all four LKB1 variants and a truncated LKB1 lacking the C-terminal region altogether were equally effective at causing cell cycle arrest when co-expressed with STRADalpha and MO25alpha in the G361 melanoma cell line. Our results do not support the idea that phosphorylation of Ser-431 increases the ability of LKB1 to phosphorylate downstream targets.     

Protein kinase Czeta-dependent LKB1 serine 428 phosphorylation increases LKB1 nucleus export and apoptosis in endothelial cells

LKB1 is a serine-threonine protein kinase that, when inhibited, may result in unregulated cell growth and tumor formation. However, how LKB1 is regulated remains poorly understood. The aim of the present study was to define the upstream signaling events responsible for peroxynitrite (ONOO(-))-induced LKB1 activation. Exposure of cultured human umbilical vein endothelial cells to a low concentration of ONOO(-) (5 microM) significantly increased the phosphorylation of LKB1 at Ser(428) and protein kinase Czeta (PKCzeta) at Thr(410). These effects were accompanied by increased activity of the lipid phosphatase PTEN, decreased activity and phosphorylation (Ser(473)) of Akt, and induction of apoptosis. ONOO(-) enhanced Akt-Ser(473) phosphorylation in LKB1-deficient HeLa S3 cells or in HeLa S3 cells transfected with kinase-dead LKB1. Conversely, ONOO(-) inhibited Akt Ser(473) phosphorylation when wild type LKB1 were reintroduced in HeLa S3 cells. Further analysis revealed that PKCzeta directly phosphorylated LKB1 at Ser(428) in vitro and in intact cells, resulting in increased PTEN phosphorylation at Ser(380)/Thr(382/383). Finally, ONOO(-) enhanced PKCzeta nuclear import and LKB1 nuclear export. We conclude that PKCzeta mediates LKB1-dependent Akt inhibition in response to ONOO(-), resulting in endothelial apoptosis.     

Activation of the tumour suppressor kinase LKB1 by the STE20-like pseudokinase STRAD

The LKB1 gene encodes a serine/threonine kinase mutated in Peutz-Jeghers cancer syndrome. Despite several proposed models for LKB1 function in development and in tumour suppression, the detailed molecular action of LKB1 remains undefined. Here, we report the identification and characterization of an LKB1-specific adaptor protein and substrate, STRAD (STe20 Related ADaptor). STRAD consists of a STE20- like kinase domain, but lacks several residues that are indispensable for intrinsic catalytic activity. Endogenous LKB1 and STRAD form a complex in which STRAD activates LKB1, resulting in phosphorylation of both partners. STRAD determines the subcellular localization of wild-type, but not mutant LKB1, translocating it from nucleus to cytoplasm. One LKB1 mutation previously identified in a Peutz-Jeghers family that does not compromise its kinase activity is shown here to interfere with LKB1 binding to STRAD, and hence with STRAD-dependent regulation. Removal of endogenous STRAD by siRNA abrogates the LKB1-induced G(1) arrest. Our results imply that STRAD plays a key role in regulating the tumour suppressor activities of LKB1.     

Characterization of the liver kinase B1-mouse protein-25 -Ste-20-related adaptor protein complex in adult mouse skeletal muscle

In liver, the AMP-activated protein kinase kinase (AMPKK) complex was identified as the association of liver kinase B1 (LKB1), mouse protein 25 (MO25α/β), and Ste-20-related adaptor protein (STRADα/β); however, this complex has yet to be characterized in skeletal muscle. We demonstrate the expression of the LKB1-MO25-STRAD complex in skeletal muscle, confirm the absence of mRNA splice variants, and report the relative mRNA expression levels of these proteins in control and muscle-specific LKB1 knockout (LKB1(-/-)) mouse muscle. LKB1 detection in untreated control and LKB1(-/-) muscle lysates revealed two protein bands (50 and 60 kDa), although only the heavier band was diminished in LKB1(-/-) samples [55 ± 2.5 and 13 ± 1.5 arbitrary units (AU) in control and LKB1(-/-), respectively, P < 0.01], suggesting that LKB1 is not represented at 50 kDa, as previously cited. The 60-kDa LKB1 band was further confirmed following purification using polyethylene glycol (43 ± 5 and 8.4 ± 4 AU in control and LKB1(-/-), respectively, P < 0.01) and ion-exchange fast protein liquid chromatography. Mass spectrometry confirmed LKB1 protein detection in the 60-kDa protein band, while none was detected in the 50-kDa band. Coimmunoprecipitation assays demonstrated LKB1-MO25-STRAD complex formation. Quantitative PCR revealed significantly reduced LKB1, MO25α, and STRADβ mRNA in LKB1(-/-) muscle. These findings demonstrate that the LKB1-MO25-STRAD complex is the principal AMPKK in skeletal muscle.     

Cardiac-specific Deletion of LKB1 Leads to Hypertrophy and Dysfunction

LKB1 encodes a serine/threonine kinase, which functions upstream of the AMP-activated protein kinase (AMPK) superfamily. To clarify the role of LKB1 in heart, we generated and characterized cardiac myocyte-specific LKB1 knock-out (KO) mice using α-myosin heavy chain-Cre deletor strain. LKB1-KO mice displayed biatrial enlargement with atrial fibrillation and cardiac dysfunction at 4 weeks of age. Left ventricular hypertrophy was observed in LKB1-KO mice at 12 weeks but not 4 weeks of age. Collagen I and III mRNA expression was elevated in atria at 4 weeks, and atrial fibrosis was seen at 12 weeks. LKB1-KO mice displayed cardiac dysfunction and atrial fibrillation and died within 6 months of age. Indicative of a prohypertrophic environment, the phosphorylation of AMPK and eEF2 was reduced, whereas mammalian target of rapamycin (mTOR) phosphorylation and p70S6 kinase phosphorylation were increased in both the atria and ventricles of LKB1-deficient mice. Consistent with vascular endothelial growth factor mRNA and protein levels being significantly reduced in LKB1-KO mice, these mice also exhibited a reduction in capillary density of both atria and ventricles. In cultured cardiac myocytes, LKB1 silencing induced hypertrophy, which was ameliorated by the expression of a constitutively active form AMPK or by treatment with the inhibitor of mTOR, rapamycin. These findings indicate that LKB1 signaling in cardiac myocytes is essential for normal development of the atria and ventricles. Cardiac hypertrophy and dysfunction in LKB1-deficient hearts are associated with alterations in AMPK and mTOR/p70S6 kinase/eEF2 signaling and with a reduction in vascular endothelial growth factor expression and vessel rarefaction.     

Identification of the sucrose non-fermenting related kinase SNRK, as a novel LKB1 substrate

Recent work has shown that the LKB1 tumour suppressor protein kinase phosphorylates and activates protein kinases belonging to the AMP activated kinase (AMPK) subfamily. In this study, we identify the sucrose non-fermenting protein (SNF1)-related kinase (SNRK), a largely unstudied AMPK subfamily member, as a novel substrate for LKB1. We demonstrate that LKB1 activates SNRK by phosphorylating the T-loop residue (Thr173), and that the LKB1 regulatory subunits STRAD and MO25 are required for LKB1 to activate SNRK. We find that SNRK is not active when expressed in HeLa cells that lack expression of LKB1, and its activity is restored by expression of wild type LKB1, but not catalytically deficient LKB1. We also present evidence that two other AMPK-related kinases more distantly related to AMPK than SNRK, namely NIM1 and testis-specific serine/threonine kinase-1 (TSSK1) are not substrates for LKB1. Tissue distribution analysis indicates that SNRK protein is mainly expressed in testis, similar to TSSK isoforms, whereas NIM1 is more widely expressed. These results provide evidence that SNRK could mediate some of the physiological effects of LKB1.     

LKB1 is an essential regulator of spermatozoa release during spermiation in the mammalian testis

LKB1 acts as a master upstream protein kinase regulating a number of kinases involved in diverse cellular functions. Recent studies have suggested a role for LKB1 in male fertility. Male mice with reduced total LKB1 expression, including the complete absence of the major splice variant in testis (LKB1(S)), are completely infertile. We sought to further characterise these mice and determine the mechanism underlying this infertility. This involved expression studies of LKB1 in developing germ cells, morphological analysis of mature spermatozoa and histological studies of both the testis and epididymis using light microscopy and transmission electron microscopy. We conclude that a defect in the release of mature spermatids from the seminiferous epithelium (spermiation) during spermatozoan development is a major cause of the infertility phenotype. We also present evidence that this is due, at least in part, to defects in the breakdown of the junctions, known as ectoplasmic specialisations, between the sertoli cells of the testis epithelium and the heads of the maturing spermatids. Overall this study uncovers a critical role for LKB1 in spermiation, a highly regulated, but poorly understood process vital for male fertility.     

Expression of an active LKB1 complex in cardiac myocytes results in decreased protein synthesis associated with phenylephrine-induced hypertrophy

AMP-activated protein kinase (AMPK) is a major metabolic regulator in the cardiac myocyte. Recently, LKB1 was identified as a kinase that regulates AMPK. Using immunoblot analysis, we confirmed high expression of LKB1 in isolated rat cardiac myocytes but show that, under basal conditions, LKB1 is primarily localized to the nucleus, where it is inactive. We examined the role of LKB1 in cardiac myocytes, using adenoviruses that express LKB1, and its binding partners Ste20-related adaptor protein (STRADalpha) and MO25alpha. Infection of neonatal rat cardiac myocytes with all three adenoviruses substantially increased LKB1/STRADalpha/MO25alpha expression, LKB1 activity, and AMPKalpha phosphorylation at its activating phosphorylation site (threonine-172). Since activation of AMPK can inhibit hypertrophic growth and since LKB1 is upstream of AMPK, we hypothesized that expression of an active LKB1 complex would also inhibit protein synthesis associated with hypertrophic growth. Expression of the LKB1/STRADalpha/MO25alpha complex in neonatal rat cardiac myocytes inhibited the increase in protein synthesis observed in cells treated with phenylephrine (measured via [(3)H]phenylalanine incorporation). This was associated with a decreased phosphorylation of p70S6 kinase and its substrate S6 ribosomal protein, key regulators of protein synthesis. In addition, we show that the pathological cardiac hypertrophy in transgenic mice with cardiac-specific expression of activated calcineurin is associated with a significant decrease in LKB1 expression. Together, our data show that increased LKB1 activity in the cardiac myocyte can decrease hypertrophy-induced protein synthesis and suggest that LKB1 activation may be a method for the prevention of pathological cardiac hypertrophy.     

LKB1 is the upstream kinase in the AMP-activated protein kinase cascade

Inactivating mutations in the protein kinase LKB1 lead to a dominantly inherited cancer in humans termed Peutz-Jeghers syndrome. The role of LKB1 is unclear, and only one target for LKB1 has been identified in vivo [3]. AMP-activated protein kinase (AMPK) is the downstream component of a protein kinase cascade that plays a pivotal role in energy homeostasis. AMPK may have a role in protecting the body from metabolic diseases including type 2 diabetes, obesity, and cardiac hypertrophy. We previously reported the identification of three protein kinases (Elm1, Pak1, and Tos3 [9]) that lie upstream of Snf1, the yeast homologue of AMPK. LKB1 shares sequence similarity with Elm1, Pak1, and Tos3, and we demonstrated that LKB1 phosphorylates AMPK on the activation loop threonine (Thr172) within the catalytic subunit and activates AMPK in vitro [9]. Here, we have investigated whether LKB1 corresponds to the major AMPKK activity present in cell extracts. AMPKK purified from rat liver corresponds to LKB1, and blocking LKB1 activity in cells abolishes AMPK activation in response to different stimuli. These results identify a link between two protein kinases, previously thought to lie in unrelated, distinct pathways, that are associated with human diseases.     

Phosphorylation of the protein kinase mutated in Peutz-Jeghers cancer syndrome, LKB1/STK11, at Ser431 by p90(RSK) and cAMP-dependent protein kinase, but not its farnesylation at Cys(433), is essential for LKB1 to suppress cell vrowth

Peutz-Jeghers syndrome is an inherited cancer syndrome that results in a greatly increased risk of developing tumors in those affected. The causative gene is a protein kinase termed LKB1, predicted to function as a tumor suppressor. The mechanism by which LKB1 is regulated in cells is not known. Here, we demonstrate that stimulation of Rat-2 or embryonic stem cells with activators of ERK1/2 or of cAMP-dependent protein kinase induced phosphorylation of endogenously expressed LKB1 at Ser(431). We present pharmacological and genetic evidence that p90(RSK) mediated this phosphorylation in response to agonists that activate ERK1/2 and that cAMP-dependent protein kinase mediated this phosphorylation in response to agonists that activate adenylate cyclase. Ser(431) of LKB1 lies adjacent to a putative prenylation motif, and we demonstrate that full-length LKB1 expressed in 293 cells was prenylated by addition of a farnesyl group to Cys(433). Our 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) did 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 was still able to suppress the growth of cells.     

Function of mammalian LKB1 and Ca2+/calmodulin-dependent protein kinase kinase alpha as Snf1-activating kinases in yeast

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. We show that LKB1 functions heterologously in yeast. In pak1Delta tos3Delta elm1Delta cells, LKB1 activated Snf1 catalytic activity and conferred a Snf(+) growth phenotype. Coexpression of STRADalpha and MO25alpha, which form a complex with LKB1, enhanced 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 phosphorylated 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 provided insight into the regulation of Snf1. When activated by LKB1 or CaMKKalpha, Snf1 activity was 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.     

Complete polarization of single intestinal epithelial cells upon activation of LKB1 by STRAD

The LKB1 gene encodes a serine/threonine kinase that is mutated in the Peutz-Jeghers cancer syndrome. LKB1 is homologous to the Par-4 polarity genes in C. elegans and D. melanogaster. We have previously reported the identification and characterization of an LKB1-specific adaptor protein, STRAD, which activates LKB1 and translocates it from nucleus to cytoplasm. We have now constructed intestinal epithelial cell lines 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. We conclude 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.     

Phosphorylation of Serine 399 in LKB1 Protein Short Form by Protein Kinase Cζ Is Required for Its Nucleocytoplasmic Transport and Consequent AMP-activated Protein Kinase (AMPK) Activation

Two splice variants of LKB1 exist: LKB1 long form (LKB1_L) and LKB1 short form (LKB1_S). In a previous study, we demonstrated that phosphorylation of Ser-428/431 (in LKB1_L) by protein kinase Cζ (PKCζ) was essential for LKB1-mediated activation of AMP-activated protein kinase (AMPK) in response to oxidants or metformin. Paradoxically, LKB1_S also activates AMPK although it lacks Ser-428/431. Thus, we hypothesized that LKB1_S contained additional phosphorylation sites important in AMPK activation. Truncation analysis and site-directed mutagenesis were used to identify putative PKCζ phosphorylation sites in LKB1_S. Substitution of Ser-399 to alanine did not alter the activity of LKB1_S, but abolished peroxynitrite- and metformin-induced activation of AMPK. Furthermore, the phosphomimetic mutation (S399D) increased the phosphorylation of AMPK and its downstream target phospho-acetyl-coenzyme A carboxylase (ACC). PKCζ-dependent phosphorylation of Ser-399 triggered nucleocytoplasmic translocation of LKB1_S in response to metformin or peroxynitrite treatment. This effect was ablated by pharmacological and genetic inhibition of PKCζ, by inhibition of CRM1 activity and by substituting Ser-399 with alanine (S399A). Overexpression of PKCζ up-regulated metformin-mediated phosphorylation of both AMPK (Thr-172) and ACC (Ser-79), but the effect was ablated in the S399A mutant. We conclude that, similar to Ser-428/431 (in LKB1_L), Ser-399 (in LKB1_S) is a PKCζ-dependent phosphorylation site essential for nucleocytoplasmic export of LKB1_S and consequent AMPK activation.     

Post-translational Modification of Serine/Threonine Kinase LKB1 via Adduction of the Reactive Lipid Species 4-Hydroxy-trans-2-nonenal (HNE) at Lysine Residue 97 Directly Inhibits Kinase Activity

Oxidative stress is pathogenic in a variety of diseases, but the mechanism by which cellular signaling is affected by oxidative species has yet to be fully characterized. Lipid peroxidation, a secondary process that occurs during instances of free radical production, may play an important role in modulating cellular signaling under conditions of oxidative stress. 4-Hydroxy-trans-2-nonenal (HNE) is an electrophilic aldehyde produced during lipid peroxidation that forms covalent adducts on proteins, altering their activity and function. One such target, LKB1, has been reported to be inhibited by HNE adduction. We tested the hypothesis that HNE inhibits LKB1 activity through adduct formation on a specific reactive residue of the protein. To elucidate the mechanism of the inhibitory effect, HEK293T cells expressing LKB1 were treated with HNE (10 μm for 1 h) and assayed for HNE-LKB1 adduct formation and changes in LKB1 kinase activity. HNE treatment resulted in the formation of HNE-LKB1 adducts and decreased LKB1 kinase activity by 31 ± 9% (S.E.) but had no effect on the association of LKB1 with its adaptor proteins sterile-20-related adaptor and mouse protein 25. Mutation of LKB1 lysine residue 97 reduced HNE adduct formation and attenuated the effect of HNE on LKB1 activity. Taken together, our results suggest that adduction of LKB1 Lys-97 mediates the inhibitory effect of HNE.     

Metastasis suppression by adiponectin

Adiponectin is an adipocytokine involved in the pathogenesis of various obesity-related disorders. Also, it has been shown that adiponectin has therapeutic potential for metabolic syndrome, systemic insulin resistance, cardiovascular disease and more recently carcinogenesis. Adiponectin can modulate breast cancer cell growth and proliferation. Anti-metastatic effects of adiponectin have also been elucidated. It has been shown that adiponectin inhibits important metastatic properties such as adhesion, invasion and migration of breast cancer cells. Examination of the underlying molecular mechanisms has shown that adiponectin treatment increases AMP-activated protein kinase (AMPK) phosphorylation and activity. Adiponectin also increases phosphorylation of downstream target of AMPK, Acetyl-CoA Carboxylase (ACC) and decreases phosphorylation of p70S6 kinase (S6K). Importantly, adiponectin treatment increases the expression of tumor suppressor gene, LKB1 in breast cancer cells. LKB1 is required for adiponectin-mediated modulation of AMPK-S6K axis and more importantly, its biological functions including inhibition of adhesion, migration and invasion of breast cancer cells. Although further studies are required to analyze the effect of adiponectin on LKB1-AMPK-S6K axis, these data present a novel mechanism involving specific upregulation of tumor suppressor gene LKB1 by which adiponectin inhibits adhesion, invasion and migration of breast cancer cells. These results highlight a new role for LKB1 in adiponectin action and may have significant implication for development of novel therapeutic options.     

Liver kinase B1 regulates the centrosome via PLK1

Liver kinase B1 (LKB1) is a tumor suppressor mutationally inactivated in Peutz–Jeghers syndrome (PJS) and various sporadic cancers. Although LKB1 encodes a kinase that possesses multiple functions, no individual hypothesis posed to date has convincingly explained how loss of LKB1 contributes to carcinogenesis. In this report we demonstrated that LKB1 maintains genomic stability through the regulation of centrosome duplication. We found that LKB1 colocalized with centrosomal proteins and was situated in the mitotic spindle pole. LKB1 deficiency-induced centrosome amplification was independent of AMP-activated protein kinase (AMPK), a well-defined substrate of LKB1. Cells lacking LKB1 exhibited an increase in phosphorylated and total Polo-like kinase 1 (PLK-1), NIMA-related kinase 2 (NEK2), and ninein-like protein (NLP). Overexpression of active PLK1 (T210D) reversed the inhibition of LKB1 on centrosome amplification. In contrast, depletion of PLK1 with siRNA or suppression of PLK1 kinase activity with BTO-1 (5-Cyano-7-nitro-2-benzothiazolecarboxamide-3-oxide) abrogated LKB1 deficiency-induced centrosome amplification. We further characterized that LKB1 phosphorylated and activated AMPK-related kinase 5 (NUAK1 or ARK5) that in turn increased the phosphorylation of MYPT1, enhanced the binding between MYPT1–PP1 and PLK1, and conferred an effective dephosphorylation of PLK1. More importantly, we noted that LKB1-deficient cells exhibited multiple nuclear abnormalities, such as mitotic delay, binuclear, polylobed, grape, large, and micronuclear. Immediate depletion of LKB1 resulted in the accumulation of multiploidy cells. Expression of LKB1 is reversely correlated with the levels of PLK1 in human cancer tissues. Thus, we have uncovered a novel function of LKB1 in the maintenance of genomic stability through the regulation of centrosome mediated by PLK1.     

LKB1 is a master kinase that activates 13 kinases of the AMPK subfamily, including MARK/PAR-1

We recently demonstrated that the LKB1 tumour suppressor kinase, in complex with the pseudokinase STRAD and the scaffolding protein MO25, phosphorylates and activates AMP-activated protein kinase (AMPK). A total of 12 human kinases (NUAK1, NUAK2, BRSK1, BRSK2, QIK, QSK, SIK, MARK1, MARK2, MARK3, MARK4 and MELK) are related to AMPK. Here we demonstrate that LKB1 can phosphorylate the T-loop of all the members of this subfamily, apart from MELK, increasing their activity >50-fold. LKB1 catalytic activity and the presence of MO25 and STRAD are required for activation. Mutation of the T-loop Thr phosphorylated by LKB1 to Ala prevented activation, while mutation to glutamate produced active forms of many of the AMPK-related kinases. Activities of endogenous NUAK2, QIK, QSK, SIK, MARK1, MARK2/3 and MARK4 were markedly reduced in LKB1-deficient cells. Neither LKB1 activity nor that of AMPK-related kinases was stimulated by phenformin or AICAR, which activate AMPK. Our results show that LKB1 functions as a master upstream protein kinase, regulating AMPK-related kinases as well as AMPK. Between them, these kinases may mediate the physiological effects of LKB1, including its tumour suppressor function.