Rho GTPases regulate a multitude of cellular processes from cytoskeletal reorganization to gene transcription and are negatively regulated by GTPase-activating proteins (GAPs). Cdc42 GTPase-activating protein (CdGAP) is a ubiquitously expressed GAP for Rac1 and Cdc42. In this study, we set out to identify CdGAP-binding partners and, using a yeast two-hybrid approach, glycogen synthase kinase 3␣ (GSK-3␣) was identified as a partner for CdGAP. GSK-3 exists in two isoforms, ␣ and ␤, and is

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... in regulating many cellular functions from insulin response to tumorigenesis. We show that GSK-3␣ and -␤ interact with CdGAP in mammalian cells. We also demonstrate that GSK-3 phosphorylates CdGAP both in vitro and in vivo on Thr-776, which we have previously shown to be an ERK 1/2 phosphorylation site involved in CdGAP regulation. We report that the mRNA and protein levels of CdGAP are increased upon serum stimulation and that GSK-3 activity is necessary for the up-regulation of the protein levels of CdGAP but not for the increase in mRNA. We conclude that GSK-3 is an important regulator of CdGAP and that regulation of CdGAP protein levels by serum presents a novel mechanism for cells to control Cdc42/Rac1 GTPase signaling pathways. 5 The abbreviations used are: GAP, GTPase-activating protein; CdGAP, Cdc42 GTPase-activating protein; CdGAP-l, CdGAP long isoform; CdGAP-s, CdGAP short isoform; PRD, proline-rich domain; GSK-3, glycogen synthase kinase 3; ActD, actinomycin D; DRB, 5,6-dichlorobenzimidazole 1-␤-D-ribofuranoside; MBP, myelin basic protein; MOPS, 4-morpholinepropanesulfonic acid.