: Insulin stimulates the mobilization of glucose transporter 4 (GLUT4) storage vesicles from intracellular pools to the plasma membrane, resulting in an influx of glucose into target tissues such as muscle and fat. This system is disturbed in insulin resistance and type 2 diabetes. Insulin-stimulated insertion of GLUT4 into the plasma membrane is the end product of a series of protein-protein interactions and dynamic cytoskeletal events that are still under investigation. In the first submission, we showed that CLIP-associating protein 2 (CLASP2), a protein previously unassociated with insulin action, is responsive to insulin. Using a mass spectrometry-based proteomics approach combined with phosphoserine antibody immunoprecipitation from L6 myotubes, we detected a 4.8-fold increase of CLASP2 in the insulin stimulated anti-phosphoserine immunoprecipitates as compared to basal. Western blotting of CLASP2 immunoprecipitates with the phospho- antibody confirmed that CLASP2 undergoes insulin-stimulated phosphorylation. Confocal imaging of L6 myotubes revealed that CLASP2 is positioned at the plasma membrane within areas of insulin-mediated cortical actin remodeling. Since insulin-induced cortical actin reorganization is a target for GLUT4 translocation, we tested for detection of both CLASP2 and GLUT4 within the plasma membrane ruffle generated by insulin- stimulated dynamic actin remodeling. Confocal imaging revealed that CLASP2 colocalizes with GLUT4 at insulin-stimulated plasma membrane ridges. CLASP2 is known to direct the distal end of microtubules to the cell cortex, and it has been shown that GLUT4 travels along microtubule tracks. In support of the prospect that CLASP2 directs microtubule-based delivery of GLUT4 to cell cortex landing zones important for insulin action, siRNA mediated knockdown of CLASP2 in L6 myotubes inhibited insulin-stimulated GLUT4 translocation to the plasma membrane. Furthermore, siRNA mediated knockdown of CLASP2 in 3T3-L1 adipocytes inhibits insulin- stimulated glucose transport. The revised proposal presents new preliminary data from the previously proposed endogenous CLASP2 interactome studies from 3T3-L1 adipocytes, in which several known CLASP2 interacting proteins were identified, validating the approach. Mouse tissue was analyzed and an isoform- specific CLASP2 protein expression pattern in insulin-sensitive tissues such as muscle and fat was discovered. New preliminary data has identified insulin-regulated endogenous CLASP2 phosphorylation within L6 myotubes, including phosphorylation that is sensitive to inhibition of either glycogen synthase kinase 3 (GSK3) or PI 3-kinase (PI 3-K). Within systems such as cell migration, CLASP2 is known to be negatively regulated by GSK3. We therefore propose to test the overall hypothesis that CLASP2 is negatively regulated by GSK3- mediated phosphorylation in the basal state. Inactivation of GSK3 by insulin through PI 3-K relieves CLASP2, allowing for the distal end of microtubules to situate at the specific CLASP2-targeted landing zones on the cell cortex, whereby situating GLUT4 containing vesicles proximal to the plasma membrane.
Insulin resistance underlies the major public health problems of obesity, type 2 diabetes mellitus, and cardiovascular disease. Understanding the molecular nature of this abnormality in humans will be a key to developing and assessing the effectiveness of new treatments for these diseases.
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