The Rho family of small GTP-binding proteins is involved in the regulation of cytoskeletal structure, gene transcription, specific cell fate development, and transformation. We demonstrate in this report that overexpression of an activated form of Rho enhances AP-1 activity in Jurkat T cells in the presence of phorbol myristate acetate (PMA), but activated Rho (V14Rho) has little or no effect on NFAT, Oct-1, and NF-B enhancer element activities under similar conditions. Overexpression of a V14Rho construct incapable of membrane localization (CAAX deleted) abolishes PMA-induced AP-1 transcriptional activation. The effect of Rho on AP-1 is independent of the mitogen-activated protein kinase pathway, as a dominant-negative MEK and a MEK inhibitor (PD98059) did not affect Rho-induced AP-1 activity. V14Rho binds strongly to protein kinase C␣ (PKC␣) in vivo; however, deletion of the CAAX site on V14Rho severely diminished this association. Evidence for a role for PKC␣ as an effector of Rho was obtained by the observation that coexpression of the Nterminal domain of PKC␣ blocked the effects of activated Rho plus PMA on AP-1 transcriptional activity. These data suggest that Rho potentiates AP-1 transcription during T-cell activation.The Ras-related Rho family members are involved in thymic development, cell transformation, actin cytoskeletal rearrangement, and cell polarity (17,26,35,36,41,47). The Rho family is comprised of several related proteins, including Rac1, Rac2, RhoA, RhoB, RhoC, Cdc42Hs, and TC10 (18, 19), which share structural similarity with Ras. These proteins contain intrinsic GTPase activity and bind GTP and GDP in a manner that is regulated by guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and guanine nucleotide dissociation inhibitors (GDIs) (43, 46). Several GEFs for the Rho family, such as Ost (23), Tiam (29), and the faciogenital dysplasia gene product (FGD1 [39]), have been isolated and shown to promote binding of GTP to Rho. Bcr (11), p190 (8,45), and Cdc42GAP (7) have been demonstrated to act as GAPs for the Rho family, promoting the conversion of GTP to GDP.The importance of Rho family members in cellular activation and growth has been underscored by several recent studies. In NIH 3T3 cells, coexpression of oncogenic Ras (61L) with activated Rho (63L) enhances morphological transformation and cell motility. Overexpression of dominant-negative (DN) mutants of Rac or Rho reduces oncogenic Ras transforming activity, indicating that activation of Rho is required for Ras transformation (26,40). Roles for Rho in gene regulation and cell cycle progression have also been demonstrated. Microinjection of activated forms of Rho, Rac, and Cdc42Hs stimulates cell cycle progression and subsequent DNA synthesis. Serum-induced DNA synthesis and progression through the G 1 phase can be blocked by microinjection of C3 exoenzyme (a specific inhibitor of Rho) or by expression of DN Rac or Cdc42Hs (38). In addition, thymuses lacking functional Rho isolated from transgenic mice that o...