title: Genetic disruption of AMPK signaling abolishes both contraction- and insulin-stimulated TBC1D1 phosphorylation and 14-3-3 binding in mouse skeletal muscle.
abstract: TBC1D1 is a Rab-GTPase-activating protein (GAP) known to be phosphorylated in response to insulin, growth factors, pharmacological agonists that activate 5'-AMP-activated protein kinase (AMPK), and muscle contraction. Silencing TBC1D1 in L6 muscle cells by siRNA increases insulin-stimulated GLUT4 translocation, and overexpression of TBC1D1 in 3T3-L1 adipocytes with low endogenous TBC1D1 expression inhibits insulin-stimulated GLUT4 translocation, suggesting a role of TBC1D1 in regulating GLUT4 translocation. Aiming to unravel the regulation of TBC1D1 during contraction and the potential role of AMPK in intact skeletal muscle, we used EDL muscles from wild-type (WT) and AMPK kinase dead (KD) mice. We explored the site-specific phosphorylation of TBC1D1 Ser(237) and Thr(596) and their relation to 14-3-3 binding, a proposed mechanism for regulation of GAP function of TBC1D1. We show that muscle contraction increases 14-3-3 binding to TBC1D1 as well as phosphorylation of Ser(237) and Thr(596) in an AMPK-dependent manner. AMPK activation by AICAR induced similar Ser(237) and Thr(596) phosphorylation of, and 14-3-3 binding to, TBC1D1 as muscle contraction. Insulin did not increase Ser(237) phosphorylation or 14-3-3 binding to TBC1D1. However, insulin increased Thr(596) phosphorylation, and intriguingly this response was fully abolished in the AMPK KD mice. Thus, TBC1D1 is differentially regulated in response to insulin and contraction. This study provides genetic evidence to support an important role for AMPK in regulating TBC1D1 in response to both of these physiological stimuli.
authors.affiliation: Molecular Physiology Group, Copenhagen Muscle Research Centre, Dept. of Exercise and Sport Sciences, Univ. of Copenhagen, DK-2100, Copenhagen, Denmark.
authors.forename: Christian
authors.initials: C
authors.lastname: Pehmøller
authors.forename: Jonas T
authors.initials: JT
authors.lastname: Treebak
authors.forename: Jesper B
authors.initials: JB
authors.lastname: Birk
authors.forename: Shuai
authors.initials: S
authors.lastname: Chen
authors.forename: Carol
authors.initials: C
authors.lastname: Mackintosh
authors.forename: D Grahame
authors.initials: DG
authors.lastname: Hardie
authors.forename: Erik A
authors.initials: EA
authors.lastname: Richter
authors.forename: Jørgen F P
authors.initials: JF
authors.lastname: Wojtaszewski
mesh_terms: D048948:14-3-3 Proteins / Q000378:metabolism*; D055372:AMP-Activated Protein Kinases / Q000235:genetics* / Q000502:physiology; D000818:Animals; D005260:Female; D020690:GTPase-Activating Proteins; D007328:Insulin / Q000494:pharmacology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009119:Muscle Contraction / Q000502:physiology*; D018482:Muscle, Skeletal / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D016296:Mutagenesis / Q000502:physiology; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics
chemical_list: D048948:14-3-3 Proteins; D020690:GTPase-Activating Proteins; D007328:Insulin; D009687:Nuclear Proteins; C095906:Tbc1d1 protein, mouse; C534978:Prkaa2 protein, mouse; D017346:Protein Serine-Threonine Kinases; D055372:AMP-Activated Protein Kinases