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Homo-oligomerization and activation of AMP-activated protein kinase are mediated by the kinase domain alphaG-helix.

ABSTRACT: AMP-activated protein kinase (AMPK) is a heterotrimeric complex playing a crucial role in maintaining cellular energy homeostasis. Recently, homodimerization of mammalian AMPK and yeast ortholog SNF1 was shown by us and others. In SNF1, it involved specific hydrophobic residues in the kinase domain alphaG-helix. Mutation of the corresponding AMPK alpha-subunit residues (Val-219 and Phe-223) to glutamate reduced the tendency of the kinase to form higher order homo-oligomers, as was determined by the following three independent techniques in vitro: (i) small angle x-ray scattering, (ii) surface plasmon resonance spectroscopy, and (iii) two-dimensional blue native/SDS-PAGE. Recombinant protein as well as AMPK in cell lysates of primary cells revealed distinct complexes of various sizes. In particular, the assembly of very high molecular mass complexes was dependent on both the alphaG-helix-mediated hydrophobic interactions and kinase activation. In vitro and when overexpressed in double knock-out (alpha1(-/-), alpha2(-/-)) mouse embryonic fibroblast cells, activation of mutant AMPK was impaired, indicating a critical role of the alphaG-helix residues for AMPK activation via its upstream kinases. Also inactivation by protein phosphatase 2Calpha was affected in mutant AMPK. Importantly, activation of mutant AMPK by LKB1 was restored by exchanging the corresponding and conserved hydrophobic alphaG-helix residues of LKB1 (Ile-260 and Phe-264) to positively charged amino acids. These mutations functionally rescued LKB1-dependent activation of mutant AMPK in vitro and in cell culture. Our data suggest a physiological role for the hydrophobic alphaG-helix residues in homo-oligomerization of heterotrimers and cellular interactions, in particular with upstream kinases, indicating an additional level of AMPK regulation.

SUBMITTER: Scholz R

PROVIDER: S-EPMC2785672 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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Homo-oligomerization and activation of AMP-activated protein kinase are mediated by the kinase domain alphaG-helix.

Scholz Roland R Suter Marianne M Weimann Théodore T Polge Cécile C Konarev Petr V PV Thali Ramon F RF Tuerk Roland D RD Viollet Benoit B Wallimann Theo T Schlattner Uwe U Neumann Dietbert D

The Journal of biological chemistry 20090803 40

AMP-activated protein kinase (AMPK) is a heterotrimeric complex playing a crucial role in maintaining cellular energy homeostasis. Recently, homodimerization of mammalian AMPK and yeast ortholog SNF1 was shown by us and others. In SNF1, it involved specific hydrophobic residues in the kinase domain alphaG-helix. Mutation of the corresponding AMPK alpha-subunit residues (Val-219 and Phe-223) to glutamate reduced the tendency of the kinase to form higher order homo-oligomers, as was determined by ...[more]

PMID: 19651772

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Project description:Background & aimsGlycogen synthase kinase 3β (Gsk3β [Gsk3b]) is a ubiquitously expressed kinase with distinctive functions in different types of cells. Although its roles in regulating innate immune activation and ischaemia and reperfusion injuries (IRIs) have been well documented, the underlying mechanisms remain ambiguous, in part because of the lack of cell-specific tools in vivo.MethodsWe created a myeloid-specific Gsk3b knockout (KO) strain to study the function of Gsk3β in macrophages in a murine liver partial warm ischaemia model.ResultsCompared with controls, myeloid Gsk3b KO mice were protected from IRI, with diminished proinflammatory but enhanced anti-inflammatory immune responses in livers. In bone marrow-derived macrophages, Gsk3β deficiency resulted in an early reduction of Tnf gene transcription but sustained increase of Il10 gene transcription on Toll-like receptor 4 stimulation in vitro. These effects were associated with enhanced AMP-activated protein kinase (AMPK) activation, which led to an accelerated and higher level of induction of the novel innate immune negative regulator small heterodimer partner (SHP [Nr0b2]). The regulatory function of Gsk3β on AMPK activation and SHP induction was confirmed in wild-type bone marrow-derived macrophages with a Gsk3 inhibitor. Furthermore, we found that this immune regulatory mechanism was independent of Gsk3β Ser9 phosphorylation and the phosphoinositide 3-kinase-Akt signalling pathway. In vivo, myeloid Gsk3β deficiency facilitated SHP upregulation by ischaemia-reperfusion in liver macrophages. Treatment of Gsk3b KO mice with either AMPK inhibitor or SHP small interfering RNA before the onset of liver ischaemia restored liver proinflammatory immune activation and IRI in these otherwise protected hosts. Additionally, pharmacological activation of AMPK protected wild-type mice from liver IRI, with reduced proinflammatory immune activation. Inhibition of the AMPK-SHP pathway by liver ischaemia was demonstrated in tumour resection patients.ConclusionsGsk3β promotes innate proinflammatory immune activation by restraining AMPK activation.Lay summaryGlycogen synthase kinase 3β promotes macrophage inflammatory activation by inhibiting the immune regulatory signalling of AMP-activated protein kinase and the induction of small heterodimer partner. Therefore, therapeutic targeting of glycogen synthase kinase 3β enhances innate immune regulation and protects liver from ischaemia and reperfusion injury.

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Homo-oligomerization and activation of AMP-activated protein kinase are mediated by the kinase domain alphaG-helix.

Publications

Homo-oligomerization and activation of AMP-activated protein kinase are mediated by the kinase domain alphaG-helix.

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