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This Article Full Text Full Text (PDF) All Versions of this Article: 297/3/E665    most recent 00115.2009v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Pehmøller, C. Articles by Wojtaszewski, J. F. P. PubMed PubMed Citation Articles by Pehmøller, C. Articles by Wojtaszewski, J. F. P.

Genetic disruption of AMPK signaling abolishes both contraction- and insulin-stimulated TBC1D1 phosphorylation and 14-3-3 binding in mouse skeletal muscle

¹Molecular Physiology Group, Copenhagen Muscle Research Centre, Department of Exercise and Sport Sciences, University of Copenhagen, Copenhagen, Denmark; and ²MRC Protein Phosphorylation Unit and ³Division of Molecular Physiology, College of Life Sciences, University of Dundee, Dundee, Scotland, United Kingdom

Submitted 20 February 2009 ; accepted in final form 13 June 2009

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 Ser²³⁷ and Thr⁵⁹⁶ 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 Ser²³⁷ and Thr⁵⁹⁶ in an AMPK-dependent manner. AMPK activation by AICAR induced similar Ser²³⁷ and Thr⁵⁹⁶ phosphorylation of, and 14-3-3 binding to, TBC1D1 as muscle contraction. Insulin did not increase Ser²³⁷ phosphorylation or 14-3-3 binding to TBC1D1. However, insulin increased Thr⁵⁹⁶ 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.

5'-AMP-activated protein kinase; wortmannin; 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside; AMPK kinase dead; extensor digitorum longus muscle