Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.31 (AMP-activated protein kinase)
 13,065 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:LKB1 is a master kinase that activates 13 kinases of the AMPK subfamily, including MARK/PAR-1. 1497 52

Mutations in the LKB1 tumour suppressor threonine kinase cause the inherited Peutz-Jeghers cancer syndrome and are also observed in some sporadic cancers. Recent work indicates that LKB1 exerts effects on metabolism, polarity and proliferation by phosphorylating and activating protein kinases belonging to the AMPK subfamily. In vivo, LKB1 forms a complex with STRAD, an inactive pseudokinase, and MO25, an armadillo repeat scaffolding-like protein. Binding of LKB1 to STRAD-MO25 activates LKB1 and re-localises it from the nucleus to the cytoplasm. To learn more about the inherent properties of the LKB1-STRAD-MO25 complex, we first investigated the activity of 34 point mutants of LKB1 found in human cancers and their ability to interact with STRAD and MO25. Interestingly, 12 of these mutants failed to interact with STRAD-MO25. Performing mutagenesis analysis, we defined two binding sites located on opposite surfaces of MO25alpha, which are required for the assembly of MO25alpha into a complex with STRADalpha and LKB1. In addition, we demonstrate that LKB1 does not require phosphorylation of its own T-loop to be activated by STRADalpha-MO25alpha, and discuss the possibility that this unusual mechanism of regulation arises from LKB1 functioning as an upstream kinase. Finally, we establish that STRADalpha, despite being catalytically inactive, is still capable of binding ATP with high affinity, but that this is not required for activation of LKB1. Taken together, our findings reinforce the functional importance of the binding of LKB1 to STRAD, and provide a greater understanding of the mechanism by which LKB1 is regulated and activated through its interaction with STRAD and MO25.
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PMID:Analysis of the LKB1-STRAD-MO25 complex. 1556 63

Activation of the AMP-activated protein kinase (AMPK) results in acute changes in cellular metabolism and transcriptional events that make the cell more robust when encountering an energy challenge. AMPK is thought to be inhibited by glycogen, the major storage form of intracellular carbohydrate. We hypothesized that long-chain acyl-CoA esters (LCACEs) might also inhibit AMPK signaling. Cytosolic LCACEs are available for immediate transport and oxidation within the mitochondria and accordingly may be representative of the lipid energy charge of the cell. We found that LCACEs inhibited phosphorylation of AMPK by the recombinant AMPK kinase (AMPKK) LKB1/STRAD/MO25 in a concentration-dependent manner. Palmitoyl-CoA (PCoA) did not affect the activity of phosphothreonine-172 AMPK. PCoA potently inhibited AMPKK purified from liver. Conversely, PCoA stimulated the kinase activity of LKB1/STRAD/MO25 toward the peptide substrate LKB1tide. Octanoyl-CoA, palmitate, and palmitoylcarnitine did not inhibit AMPKK activity. Removal of AMP from the reaction mixture resulted in reduced AMPKK activity in the presence of PCoA. In conclusion, these results demonstrate that the AMPKK activity of LKB1/STRAD/MO25 is substrate specific and distinct from the kinase activity of LKB1/STRAD/MO25 toward the peptide substrate LKB1tide. They also demonstrate that LCACEs inhibit the AMPKK activity of LKB1/STRAD/MO25 in a specific manner with a dependence on both a long fatty chain and a CoA moiety. These results suggest that the AMPK signaling cascade may directly sense and respond to the lipid energy charge of the cell.
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PMID:Long-chain acyl-CoA esters inhibit phosphorylation of AMP-activated protein kinase at threonine-172 by LKB1/STRAD/MO25. 1564 53

This study was designed to examine activity of AMP-activated protein kinase kinase (AMPKK) in muscles from nontrained and endurance-trained rats. Rats were trained 5 days/wk, 2 h/day for 8 wk at a final intensity of 32 m/min up a 15% grade with 30-s sprints at 53 m/min every 10 min. Gastrocnemius muscles were stimulated in situ in trained and nontrained rats for 5 min at frequencies of 0.4/s and 1/s. Gastrocnemius LKB1 protein, a putative component of the AMPKK complex (LKB1, STRAD, and MO25), increased approximately twofold in response to training. Phosphorylation of AMP-activated protein kinase (AMPK) determined by Western blot and AMPK activity of immunoprecipitates (both isoforms) was increased at both stimulation rates in both trained and nontrained muscles. AMPKK activity was 73% lower in resuspended polyethylene glycol precipitates of muscle extracts from the trained compared with nontrained rats. AMPKK activity did not increase in either trained or nontrained muscle in response to electrical stimulation, even though phospho-AMPK did increase. These results suggest that AMPKK is activated during electrical stimulation of both trained and nontrained muscle by mechanisms other than covalent modification.
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PMID:AMP-activated protein kinase kinase activity and phosphorylation of AMP-activated protein kinase in contracting muscle of sedentary and endurance-trained rats. 1592 23

Muscle contraction results in phosphorylation and activation of the AMP-activated protein kinase (AMPK) by an AMPK kinase (AMPKK). LKB1/STRAD/MO25 (LKB1) is the major AMPKK in skeletal muscle; however, the activity of LKB1 is not increased by muscle contraction. This finding suggests that phosphorylation of AMPK by LKB1 is regulated by allosteric mechanisms. Creatine phosphate is depleted during skeletal muscle contraction to replenish ATP. Thus the concentration of creatine phosphate is an indicator of cellular energy status. A previous report found that creatine phosphate inhibits AMPK activity. The purpose of this study was to determine whether creatine phosphate would inhibit 1) phosphorylation of AMPK by LKB1 and 2) AMPK activity after phosphorylation by LKB1. We found that creatine phosphate did not inhibit phosphorylation of either recombinant or purified rat liver AMPK by LKB1. We also found that creatine phosphate did not inhibit 1) active recombinant alpha1beta1gamma1 or alpha2beta2gamma2 AMPK, 2) AMPK immunoprecipitated from rat liver extracts by either the alpha1 or alpha2 subunit, or 3) AMPK chromatographically purified from rat liver. Inhibition of skeletal muscle AMPK by creatine phosphate was greatly reduced or eliminated with increased AMPK purity. In conclusion, these results suggest that creatine phosphate is not a direct regulator of LKB1 or AMPK activity. Creatine phosphate may indirectly modulate AMPK activity by replenishing ATP at the onset of muscle contraction.
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PMID:Evidence against regulation of AMP-activated protein kinase and LKB1/STRAD/MO25 activity by creatine phosphate. 1627 46

In mammals, AMP-activated protein kinase (AMPK) is involved in the regulation of cellular energy homeostasis and, on the whole animal level, in regulating energy balance and food intake. Because the chicken is a valuable experimental animal model and considering that a first draft of the chicken genome sequence has recently been completed, we were interested in verifying the genetic basis for the LKB1/AMPK pathway in chickens. We identified distinct gene homologues for AMPK alpha, beta and gamma subunits and for LKB1, MO25 and STRAD. Analysis of gene expression by RT-PCR showed that liver, brain, kidney, spleen, pancreas, duodenum, abdominal fat and hypothalamus from 3 wk-old broiler chickens preferentially expressed AMPK alpha-1, beta-2 and gamma-1 subunit isoforms. Heart predominantly expressed alpha-2, beta-2 and gamma-1, whereas skeletal muscle expressed alpha-2, beta-2 and gamma-3 preferentially. Moreover, the AMPK gamma-3 gene was only expressed in heart and skeletal muscle. Genes encoding LKB1, MO25 alpha, MO25 beta, and STRAD beta were expressed in all examined tissues, whereas STRAD alpha was expressed exclusively in brain, hypothalamus, heart and skeletal muscle. Alterations in energy status (fasting and refeeding) produced little significant change in AMPK subunit gene transcription. We also determined the level of phosphorylated (active) AMPK in different tissues and in different states of energy balance. Immunocytochemical analysis of the chicken hypothalamus showed that activated AMPK was present in hypothalamic nuclei involved in regulation of food intake and energy balance. Together, these findings suggest a functional LKB1/AMPK pathway exists in chickens similar to that observed in mammals.
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PMID:Characterization of the AMP-activated protein kinase pathway in chickens. 1634 65

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. We have 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 was 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. Our 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 impaired the catalytic activity and LKB1-mediated phosphorylation of these enzymes. We also provide evidence 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 suppressed 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.
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PMID:The ubiquitin-associated domain of AMPK-related kinases regulates conformation and LKB1-mediated phosphorylation and activation. 1649 40

This review focuses on remarkable recent findings concerning the mechanism by which the LKB1 protein kinase that is mutated in Peutz-Jeghers cancer syndrome operates as a tumor suppressor. We discuss evidence that the cellular localization and activity of LKB1 is controlled through its interaction with a catalytically inactive protein resembling a protein kinase, termed STRAD, and an armadillo repeat-containing protein, named mouse protein 25 (MO25). The data suggest that LKB1 functions as a tumor suppressor by not only inhibiting proliferation, but also by exerting profound effects on cell polarity and, most unexpectedly, on the ability of a cell to detect and respond to low cellular energy levels. Genetic and biochemical findings indicate that LKB1 exerts its effects by phosphorylating and activating 14 protein kinases, all related to the AMP-activated protein kinase. The work described in this review shows how a study of an obscure cancer syndrome can uncover new and important regulatory pathways, relevant to the understanding of multiple human diseases.
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PMID:LKB1-dependent signaling pathways. 1675 88

The tumour suppressor LKB1 plays a critical role in cell proliferation, polarity and energy metabolism. LKB1 is a Ser/Thr protein kinase that is associated with STRAD and MO25 in vivo. Here, we describe the individual expression of the three components of the LKB1 complex using monocistronic vectors and their co-expression using tricistronic vectors that were constructed from monocistronic vectors using a fully modular cloning approach. The data show that among the three individually expressed components of the LKB1 complex, only MO25alpha can be expressed in soluble form, whereas the other two, LKB1 and STRADalpha are found almost exclusively in inclusion bodies. However, using the tricistronic vector system, functional LKB1-MO25alpha-STRADalpha complex was expressed and purified from soluble extracts by sequential immobilized-metal affinity and heparin chromatography, as shown by Western blotting using specific antibodies. In size exclusion chromatography, MO25alpha and STRADalpha exactly co-elute with LKB1 with an apparent molecular weight of the heterotrimeric complex of 160 kDa. The specific activity in the peak fraction of the size exclusion chromatography was 250 U/mg at approximately 25% purity. As shown by autoradiography, LKB1 and STRADalpha, both strongly autophosphorylate in vitro. Moreover, recombinant LKB1 complex activates AMPK by phosphorylation of the alpha-subunit at the Thr-172 site as shown (i) by Western blotting using phospho-specific antibodies after LKB1-dependent phosphorylation, (ii) by LKB1-dependent incorporation of radioactive phosphate into the alpha-subunit of kinase dead AMPK heterotrimer, and (iii) by activity determination of AMPK. Functional mammalian LKB1 complex is constitutively active, and when enriched from bacteria should prove to be a valuable tool for studying its molecular function and regulation.
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PMID:Co-expression of LKB1, MO25alpha and STRADalpha in bacteria yield the functional and active heterotrimeric complex. 1787 8

STRADalpha is a pseudokinase that forms a heterotrimeric complex with the scaffolding protein MO25 and the tumor suppressor serine threonine protein kinase LKB1. Mutations in the LKB1 gene are responsible for the Peutz-Jeghers Syndrome (PJS) characterized by a predisposition to hamartomatous polyps and hyperpigmentation of the buccal mucosa. Mutations in LKB1 have also been observed in some sporadic tumours unrelated to PJS. The LKB1/STRAD/MO25 complex is involved in the regulation of numerous signaling pathways including metabolism, proliferation and cellular polarity of human intestinal epithelial cells. Cell polarization, together with tissue-restricted transcription, represents the main feature of enterocyte differentiation. Since a full-length STRADalpha transcript has not been identified thus far in these cells, we evaluated the expression of endogenous STRADalpha in five colorectal cancer cell lines characterized by their diverse ability to differentiate in vitro. We report herein the discovery of several novel splice isoforms of STRADalpha that differentially affect the kinase activity, complex assembly, subcellular localization of LKB1 and the activation of the LKB1-dependent AMPK pathway.
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PMID:Novel splice isoforms of STRADalpha differentially affect LKB1 activity, complex assembly and subcellular localization. 1792 99


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