GSK3 beta-mediated phosphorylation of the microtubule-associated protein 2C (MAP2C) prevents microtubule bundling

A major determinant of neuronal morphology is the cytoskeleton. And one of the main regulatory mechanisms of cytoskeletal proteins is the modification of their phosphorylation state via changes in the relative activities of p rotein kinases and phosphatases in neurons. In particular the microtubule-a ssociated protein 2 (MAP2) family of proteins are abundant cytoskeletal com ponents predominantly expressed in neurons and have been found to be substr ates for most of protein kinases and phosphatases present in neurons, inclu ding glycogen-synthase kinase 3 (GSK3). It Las been suggested that changes in GSK3-mediated MAP phosphorylation may modify MT stability and could cont rol neuronal development, We have previously shown that MAP2 is phosphoryla ted in vitro and in situ by GSK3 at Thr(1620) and Th-1623, located in the p roline-rich region of MAP2 and recognized by antibody 305. However the func tion of the phosphorylation of this site of MAP2 is still unknown. In this study, non-neuronal COS-T cells have been co-transfected with cDNAs encodin g MAP2C and either wild type or mutated GSK3 beta to analyze possible effec ts on microtubule stability and on the association of MAP2 with microtubule s. We have found that GSK3 beta phosphorylates MAP2C in co-transfected cell s, Moreover, this phosphorylation is inhibited by the specific GSK3 inhibit or lithium chloride. Additionally, the formation of microtubule bundles, wh ich is observed after transfection with MAP2C, was decreased when MAP2C was co-transfected with GSK3 beta wild type. Microtubule bundles were not obse rved in cells expressing MAP2C phosphorylated at the site recognized by ant ibody 305. The absence of microtubule bundles was reverted after treatment of MAP2C/GSK3 beta wild type transfected cells with lithium chloride. Highl y phosphorylated MAP2C species, which were phosphorylated at the site recog nized by antibody 305, appeared in cells co-transfected with MAP2C and GSK3 beta wild type. Interestingly, these MAP2C species were enriched in cytosk eleton-unbound protein preparations. These data suggests that GSK3-mediated phosphorylation of MAP2 may modify its binding to microtubules and regulat e microtubule stability.