Submitted on February 20, 2009
Revised on May 29, 2009
Accepted on June 13, 2009
GENETIC DISRUPTION OF AMPK SIGNALING ABOLISHES BOTH CONTRACTION-AND INSULIN-STIMULATED TBC1D1 PHOSPHORYLATION AND 14-3-3 BINDING IN MOUSE SKELETAL MUSCLE
Christian Pehmøller1, Jonas T. Treebak2, Jesper Bratz Birk1, Shaui Chen3, Carol MACKINTOSH4, David Grahame Hardie3, Erik A. Richter1, and Jørgen F.P. Wojtaszewski2*
1 University of Copenhagen
2 Copenhagen Muscle Research Centre
3 University of Dundee
4 MRC Protein Phosphorylation Unit, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, U.K.
* To whom correspondence should be addressed. E-mail: jwojtaszewski{at}aki.ku.dk.
TBC1D1 is a Rab-GTPase activating protein 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 over-expression of TBC1D1 in 3T3-L1 adipocytes, with low endogenous TBC1D1 expression, inhibits insulin-stimulated GLUT4 translocation, suggesting a role of TBC1D1 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. Although insulin did not increase Ser237 phosphorylation or 14-3-3 binding to TBC1D1, intriguingly the observed increase in Thr596 phosphorylation in response to insulin 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 regulating TBC1D1 in response to both of these physiological stimuli.
