Insulin-stimulated phosphorylation of CLASP2 by ERK regulates microtubule plus-end binding of CLASP2 and insulin-regulated microtubule stabilization.

CLASP2, a microtubule plus-end tracking protein, exhibits changes in phosphorylation as well as localization along the microtubule in response to insulin stimulation. We report that CLASP2 is expressed in insulin target tissues such as skeletal muscle and adipose tissue and that CLASP2 protein abundance is decreased in epididymal fat of insulin resistant mice. Using a commercially available proline-directed phospho-antibody together with site-directed mutagenesis and quantitative proteomics, we establish that mouse CLASP2 (Uniprot ID Q8BRT1) undergoes insulin-stimulated phosphorylation at serines 884/893/1004/1005/1021 in 3T3-L1 adipocytes. Treatment of cells with the ERK inhibitor PD98059 inhibits the insulin-stimulated phosphorylation of CLASP2 at the proline-directed serines 893/1004/1005/1021. In vitro incubation of CLASP2 with ERK2 increases CLASP2 phosphorylation, whereas phosphorylation of a Ser884/893/1004/1005/1021Ala CLASP2 penta-mutant is decreased, indicating ERK2 is capable of directly phosphorylating CLASP2. When expressed in 3T3-L1 adipocytes, the CLASP2 penta-mutant presents significantly diminished insulin-stimulated microtubule plus-end trailing versus wildtype CLASP2. The lack of proline-directed, insulin-stimulated CLASP2 phosphorylation results in reduced insulin-induced microtubule stabilization. We introduce insulin-stimulated, proline-directed CLASP2 phosphorylation under control of the ERK pathway as a contributing factor for insulin-induced microtubule stabilization.