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Calmodulin-Binding Domain of AS160 Regulates Contraction- but Not Insulin-Stimulated Glucose Uptake in Skeletal Muscle

¹ Department of Metabolism, Joslin Diabetes Center Research Division, Boston, Massachusetts
² Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts

Address correspondence and reprint requests to Laurie J. Goodyear, Metabolism, Joslin Diabetes Center, One Joslin Place, Boston, MA 02215. E-mail: laurie.goodyear{at}joslin.harvard.edu

Abbreviations: AMPK, AMP-activated protein kinase; AS160, Akt substrate of 160 kDa;CaMK, Ca²⁺/calmodulin-activated protein kinase; CBD, calmodulin-binding domain; GAP, GTPase-activating protein; PAS, phospho-Akt substrate; PKC, protein kinase C

OBJECTIVE—Insulin and contraction increase skeletal muscle glucose uptake through distinct and additive mechanisms. However, recent reports have demonstrated that both signals converge on the Akt substrate of 160 kDa (AS160), a protein that regulates GLUT4 translocation. Although AS160 phosphorylation is believed to be the primary factor affecting its activity, AS160 also possesses a calmodulin-binding domain (CBD). This raises the possibility that contraction-stimulated increases in Ca²⁺/calmodulin could also modulate AS160 function.

RESEARCH DESIGN AND METHODS—To evaluate the AS160 CBD in skeletal muscle, empty-vector, wild-type, or CBD-mutant AS160 cDNAs were injected into mouse muscles followed by in vivo electroporation. One week later, AS160 was overexpressed by 14-fold over endogenous protein.

RESULTS—Immunoprecipitates of wild-type and CBD-mutant AS160 were incubated with biotinylated calmodulin in the presence of Ca²⁺. Wild-type AS160, but not the CBD-mutant AS160, associated with calmodulin. Next, we measured insulin- and contraction-stimulated glucose uptake in vivo. Compared with empty-vector and wild-type AS160, insulin-stimulated glucose uptake was not altered in muscles expressing CBD-mutant AS160. In contrast, contraction-stimulated glucose uptake was significantly decreased in CBD-mutant–expressing muscles. This inhibitory effect on glucose uptake was not associated with aberrant contraction-stimulated AS160 phosphorylation. Interestingly, AS160 expressing both calmodulin-binding and Rab-GAP (GTPase-activating protein) domain point mutations (CBD + R/K) fully restored contraction-stimulated glucose uptake.

CONCLUSIONS—Our results suggest that the AS160 CBD directly regulates contraction-induced glucose uptake in mouse muscle and that calmodulin provides an additional means of modulating AS160 Rab-GAP function independent of phosphorylation. These findings define a novel AS160 signaling component, unique to contraction and not insulin, leading to glucose uptake in skeletal muscle.

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