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 450-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.
Adult skeletal muscle has the unique capacity to regenerate. Muscle regeneration is always associated with inflammation and notably macrophages (MPs), which play dual role. Soon after injury, inflammatory monocyte‐derived macrophages (M1 phenotype) stimulate myogenic cell proliferation. After phagocytosis of muscle debris, MPs switch their phenotype to acquire an anti‐inflammatory profile (M2) and stimulate myogenic cell differentiation and myofibre growth. Here, we explored the role of AMPK in the resolution of inflammation during muscle repair. AMPKα1 KO muscle shows both a delay and an impairment of post‐injury regeneration. These deficiencies are also observed in LysM‐CRE;AMPKfl/fl muscle, confirming the MP specificity of AMPK requirement. In vitro, AMPKα1 KO MPs hardly acquire a M2 profile upon cytokine stimulation. Their phagocytic activity is also altered. In vivo analysis of MP subpopulations (using the AMPKα1−/−;CX3CR1GFP/+ mouse) during muscle repair shows that the number of intramuscular MPs exhibiting the M2 phenotype is reduced in the AMPKα1 KO compared to the WT mouse. Accordingly, leukocytes from AMPKα1 KO muscle do not increase their expression of markers associated with the resolution of inflammation during muscle regeneration. These results strongly support that AMPKα1 is one key regulator of MP switch at time of resolution of inflammation and is essential for a proper muscle repair.
We have studied the mechanism of A-769662, a new activator of AMP-activated protein kinase (AMPK). Unlike other pharmacological activators, it directly activates native rat AMPK by mimicking both effects of AMP, i.e. allosteric activation and inhibition of dephosphorylation. We found that it has no effect on the isolated ␣ subunit kinase domain, with or without the associated autoinhibitory domain, or on interaction of glycogen with the  subunit glycogen-binding domain. Although it mimics actions of AMP, it has no effect on binding of AMP to the isolated Bateman domains of the ␥ subunit. The addition of A-769662 to mouse embryonic fibroblasts or primary mouse hepatocytes stimulates phosphorylation of acetyl-CoA carboxylase (ACC), effects that are completely abolished in AMPK-␣1 ؊/؊ ␣2 ؊/؊ cells but not in TAK1 ؊/؊ mouse embryonic fibroblasts. Phosphorylation of AMPK and ACC in response to A-769662 is also abolished in isolated mouse skeletal muscle lacking LKB1, a major upstream kinase for AMPK in this tissue. However, in HeLa cells, which lack LKB1 but express the alternate upstream kinase calmodulin-dependent protein kinase kinase-, phosphorylation of AMPK and ACC in response to A-769662 still occurs. These results show that in intact cells, the effects of A-769662 are independent of the upstream kinase utilized. We propose that this direct and specific AMPK activator will be a valuable experimental tool to understand the physiological roles of AMPK.The AMP-activated protein kinase (AMPK) 3 is a regulator of energy balance at both the cellular and the whole body levels (1-3). Once activated, it effects a metabolic switch from an anabolic to a catabolic state, both by acutely phosphorylating metabolic enzymes and, in the longer term, by regulating gene expression. AMPK is a heterotrimer composed of a catalytic ␣ subunit and regulatory  and ␥ subunits. Binding of AMP to the two "Bateman domains" formed by four tandem CBS motifs on the ␥ subunit (4) triggers increased phosphorylation at Thr-172 on the activation loop of the ␣ subunit, causing Ͼ100-fold activation (5). AMP binding was previously thought both to promote phosphorylation (6) and to inhibit dephosphorylation (7), although recent results suggest that the effect is exclusively on dephosphorylation (8). AMP binding also causes a further allosteric activation of the phosphorylated kinase by up to 10-fold (5). Phosphorylation of Thr-172 is in most cells catalyzed by the tumor suppressor kinase LKB1 (6, 9), which appears to be constitutively active (10, 11). In some cells, Thr-172 can also be phosphorylated in a Ca 2ϩ -mediated process catalyzed by calmodulin-dependent protein kinase kinases such as CaMKK (12-14). The protein kinase TGF-activated kinase-1 (TAK1) can also activate the Saccharomyces cerevisiae homologue of AMPK (the SNF1 complex) when overexpressed in yeast, as well as phosphorylating Thr-172 on mammalian AMPK in cell-free assays (15). It remains unclear whether this has any physiological relevance in vivo.Most of the metabolic changes...
Activation of AMPactivated protein kinase (AMPK) by exercise and metformin is beneficial for the treatment of type 2 diabetes. We recently found that, in cultured cells, the LKB1 tumor suppressor protein kinase activates AMPK in response to the metformin analog phenformin and the AMP mimetic drug 5-aminoimidazole-4-carboxamide-1--D-ribofuranoside (AICAR). We have also reported that LKB1 activates 11 other AMPK-related kinases. The activity of LKB1 or the AMPK-related kinases has not previously been studied in a tissue with physiological relevance to diabetes. In this study, we have investigated whether contraction, phenformin, and AICAR influence LKB1 and AMPKrelated kinase activity in rat skeletal muscle. Contraction in situ, induced via sciatic nerve stimulation, significantly increased AMPK␣2 activity and phosphorylation in multiple muscle fiber types without affecting LKB1 activity. Treatment of isolated skeletal muscle with phenformin or AICAR stimulated the phosphorylation and activation of AMPK␣1 and AMPK␣2 without altering LKB1 activity. Contraction, phenformin, or AICAR did not significantly increase activities or expression of the AMPK-related kinases QSK, QIK, MARK2/3, and MARK4 in skeletal muscle. The results of this study suggest that muscle contraction, phenformin, or AICAR activates AMPK by a mechanism that does not involve direct activation of LKB1. They also suggest that the effects of excercise, phenformin, and AICAR on metabolic processes in muscle may be mediated through activation of AMPK rather than activation of LKB1 or the AMPK-related kinases.AMP-activated protein kinase; 5-aminoimidazole-4-carboxamide-1--D-ribofuranoside AMP-ACTIVATED PROTEIN KINASE (AMPK) is a key regulator of cellular pathways that consume and generate cellular energy (4, 10). AMPK is activated by the elevation in cellular 5Ј-AMP that accompanies a fall in the ATP/ADP ratio due to the reaction catalyzed by adenylate kinase (9). One of the best studied physiological processes that activates AMPK is exercise in skeletal muscle, where AMPK stimulates uptake of glucose and the oxidation of fatty acids (27). AMPK is also activated by metformin, the most widely utilized drug for reducing blood glucose levels in type 2 diabetic patients (32). The mechanism by which metformin, or its closely related analog phenformin, activates AMPK is unknown but is not thought to involve changes in intracellular levels of AMP or the ATP/ADP ratio (7, 12).The activation of AMPK by both energy depletion (i.e., during exercise) and phenformin requires phosphorylation of the catalytic subunit of AMPK at its T-loop residue (Thr 172 , in both the ␣1 and ␣2 catalytic subunit isoforms of AMPK) by an upstream kinase(s) (9). Recent work performed in Saccharomyces cerevisiae (14,18,25) and in mammalian cells (11,23,28) has demonstrated that a protein kinase termed LKB1 is the primary kinase that mediates the T-loop phosphorylation of AMPK. LKB1 is a 50-kDa serine/threonine kinase that is the product of the gene mutated in the autosomal dominantly in...
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