Investigation of LKB1 Ser431 phosphorylation and Cys433 farnesylation using mouse knockin analysis reveals an unexpected role of prenylation in regulating AMPK activity
| Autor: | Natalia Shpiro, Maria Stella Ritorto, Kei Sakamoto, Dario R. Alessi, Vanessa P. Houde, Robert Gourlay, Joby Varghese, Paul Davies |
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| Rok vydání: | 2014 |
| Předmět: |
AICAR
5-amino-4-imidazolecarboxamide riboside PDK1 phosphoinositide-dependent kinase 1 ACC acetyl-CoA carboxylase AMP-Activated Protein Kinases Mitogen-activated protein kinase kinase Biochemistry 5-amino-4-imidazolecarboxamide riboside (AICAR) MAP2K7 Ribosomal s6 kinase Mice PKA cAMP-dependent protein kinase Serine ASK1 Phosphorylation MAPKAPK MAPKAP (MAPK-activated protein) kinase HA haemagglutinin DMEM Dulbecco's modified Eagle's medium biology EDL extensor digitorum longus HRP horseradish peroxidase RSK ribosomal S6 kinase Cell biology GAPDH glyceraldehyde-3-phosphate dehydrogenase SAD-A/SAD-B kinase BDNF brain-derived neurotrophic factor ACTH adrenocorticotropic hormone NUAK1 NUAK family SNF1-like kinase MARK4 MAP/microtubule affinity-regulating kinase 4 signal transduction PI3K phosphoinositide 3-kinase raptor regulatory associated protein of mTOR Research Article PH pleckstrin homology Molecular Sequence Data Protein Prenylation mTOR mammalian target of rapamycin Protein Serine-Threonine Kinases ERK extracellular-signal-regulated kinase SIK3 salt-inducible kinase 3 ER endoplasmic reticulum HEK human embryonic kidney TBC1D1 TBC (Tre-2/Bub2/Cdc16) domain family member 1 Animals Humans HSP90 heat-shock protein 90 Amino Acid Sequence Cysteine c-Raf ARK5 AMPK-related protein kinase 5 phenformin Molecular Biology BiP immunoglobulin heavy-chain-binding protein MAP kinase kinase kinase AMP-activated protein kinase-related kinase (AMPK-related kinase) Adenylate Kinase AMPK Cell Biology MEF mouse embryonic fibroblast Molecular biology STRAD STE20-related kinase adaptor AMPK AMP-activated protein kinase HEK293 Cells monoclonal antibody biology.protein Cyclin-dependent kinase 9 BRSK BR serine/threonine kinase MAPK mitogen-activated protein kinase |
| Zdroj: | Biochemical Journal |
| ISSN: | 1470-8728 0264-6021 |
| DOI: | 10.1042/bj20131324 |
| Popis: | The LKB1 tumour suppressor protein kinase functions to activate two isoforms of AMPK (AMP-activated protein kinase) and 12 members of the AMPK-related family of protein kinases. The highly conserved C-terminal residues of LKB1 are phosphorylated (Ser431) by PKA (cAMP-dependent protein kinase) and RSK (ribosomal S6 kinase) and farnesylated (Cys433) within a CAAX motif. To better define the role that these post-translational modifications play, we created homozygous LKB1S431A/S431A and LKB1C433S/C433S knockin mice. These animals were viable, fertile and displayed no overt phenotypes. Employing a farnesylation-specific monoclonal antibody that we generated, we established by immunoprecipitation that the vast majority, if not all, of the endogenous LKB1 is prenylated. Levels of LKB1 localized at the membrane of the liver of LKB1C433S/C433S mice and their fibroblasts were reduced substantially compared with the wild-type mice, confirming that farnesylation plays a role in mediating membrane association. Although AMPK was activated normally in the LKB1S431A/S431A animals, we unexpectedly observed in all of the examined tissues and cells taken from LKB1C433S/C433S mice that the basal, as well as that induced by the AMP-mimetic AICAR (5-amino-4-imidazolecarboxamide riboside), AMPK activation, phenformin and muscle contraction were significantly blunted. This resulted in a reduced ability of AICAR to inhibit lipid synthesis in primary hepatocytes isolated from LKB1C433S/C433S mice. The activity of several of the AMPK-related kinases analysed [BRSK1 (BR serine/threonine kinase 1), BRSK2, NUAK1 (NUAK family, SNF1-like kinase 1), SIK3 (salt-inducible kinase 3) and MARK4 (MAP/microtubule affinity-regulating kinase 4)] was not affected in tissues derived from LKB1S431A/S431A or LKB1C433S/C433S mice. Our observations reveal for the first time that farnesylation of LKB1 is required for the activation of AMPK. Previous reports have indicated that a pool of AMPK is localized at the plasma membrane as a result of myristoylation of its regulatory AMPKβ subunit. This raises the possibility that LKB1 farnesylation and myristoylation of AMPKβ might promote the interaction and co-localization of these enzymes on a two-dimensional membrane surface and thereby promote efficient activation of AMPK. Our observations reveal a significant role for the farnesylation of LKB1 being required for the efficient activation of AMPK. Our findings also cast doubt on previous claims that phosphorylation of Ser431 was essential for orchestrating neuronal cell polarity and embryo development. |
| Databáze: | OpenAIRE |
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