1779-P: Characterization of Insulin-Regulated CLASP2 Phosphorylation

Abstract

During an unbiased proteomics screen of rat L6 myotubes, we previously discovered the first member of the microtubule plus-end interacting protein (+TIP) family that undergoes insulin-stimulated phosphorylation, CLIP-associating protein 2 (CLASP2). Here we report that CLASP2 protein is expressed in several muscle groups as well as epididymal fat in mouse and we have successfully confirmed the expression of CLASP2 protein in human vastus lateralis muscle by mass spectrometry. In addition, insulin resistant, 60% fat diet-fed mice exhibit a loss of CLASP2 expression in epididymal adipose tissue versus control animals. We performed an insulin time course and discovered that insulin stimulates the phosphorylation of both endogenous and overexpressed CLASP2 in 3T3-L1 adipocytes in a manner to that similarly observed in L6 myotubes. Inhibition of either PI 3-kinase with wortmannin or mTOR with rapamycin had no effect on insulin-stimulated, proline-directed serine phosphorylation of CLASP2, whereas inhibition of the MAPK pathway reduced proline-directed serine phosphorylation of endogenous CLASP2 to basal levels. Quantitative phosphoproteomics identified Ser884/Ser1004/Ser1005/Ser1021 as significant insulin-stimulated CLASP2 phosphorylation sites that can be decreased after inhibition of the MAPK pathway. These findings indicate that CLASP2 undergoes insulin-stimulated proline-directed serine phosphorylation that is under control of the MAPK pathway in 3T3-L1 adipocytes. In addition, we also present new evidence of CLASP2 protein expression in mammalian tissues that are specifically targeted for insulin-stimulated glucose uptake and suggest that CLASP2 is altered within in vivo models of insulin resistance and type 2 diabetes. Disclosure N.K. Barker: None. J.L. Krantz: None. P.R. Langlais: None. Funding National Institutes of Health