The Formin mDia Regulates GSK3β through Novel PKCs to Promote Microtubule Stabilization but Not MTOC Reorientation in Migrating Fibroblasts

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Abstract
In migrating cells, external signals polarize the microtubule (MT) cytoskeleton by stimulating the formation of oriented, stabilized MTs and inducing the reorientation of the MT organizing center (MTOC). Glycogen synthase kinase 3β (GSK3β) has been implicated in each of these processes, although whether it regulates both processes in a single system and how its activity is regulated are unclear. We examined these issues in wound-edge, serum-starved NIH 3T3 fibroblasts where MT stabilization and MTOC reorientation are triggered by lysophosphatidic acid (LPA), but are regulated independently by distinct Rho GTPase-signaling pathways. In the absence of other treatments, the GSK3β inhibitors, LiCl or SB216763, induced the formation of stable MTs, but not MTOC reorientation, in starved fibroblasts. Overexpression of GSK3β in starved fibroblasts inhibited LPA-induced stable MTs without inhibiting MTOC reorientation. Analysis of factors involved in stable MT formation (Rho, mDia, and EB1) showed that GSK3β functioned upstream of EB1, but downstream of Rho-mDia. mDia was both necessary and sufficient for inducing stable MTs and for up-regulating GSK3β phosphorylation on Ser9, an inhibitory site. mDia appears to regulate GSK3β through novel class PKCs because PKC inhibitors and dominant negative constructs of novel PKC isoforms prevented phosphorylation of GSK3β Ser9 and stable MT formation. Novel PKCs also interacted with mDia in vivo and in vitro. These results identify a new activity for the formin mDia in regulating GSK3β through novel PKCs and implicate novel PKCs as new factors in the MT stabilization pathway.