The ERM (ezrin, radixin, moesin) proteins function as cross-linkers between cell membrane and cytoskeleton by binding to membrane proteins via their N-terminal domain and to F-actin via their C-terminal domain. Previous studies from our laboratory have shown that the alpha-subunit of heterotrimeric G(13) protein induces conformational activation of radixin via interaction with its N-terminal domain (Vaiskunaite, R., Adarichev, V., Furthmayr, H., Kozasa, T., Gudkov, A., and Voyno-Yasenetskaya, T. A. (2000) J. Biol. Chem. 275, 26206-26212). In the present study, we tested whether radixin can regulate Galpha(13)-mediated signaling pathways. We determined the effects of the N-terminal domain (amino acids 1-318) and C-terminal domain (amino acids 319-583) of radixin on serum response element (SRE)-dependent gene transcription initiated by a constitutively activated Galpha(13)Q226L. The N-terminal domain potentiated SRE activation induced by Galpha(13)Q226L; RhoGDI inhibited this effect. Surprisingly, the C-terminal domain also stimulated the SRE-dependent gene transcription. When co-transfected with Galpha(13)Q226L, the C-terminal domain of radixin synergistically stimulated the SRE activation; RhoGDI inhibited this effect. Using in vivo pull-down assays, we have determined that the C-terminal domain of radixin activated Rac1 but not RhoA or Cdc42 proteins. By contrast, Galpha(13)Q226L activated RhoA but not Rac1 or Cdc42. We have also shown that both the C-terminal domain of radixin and Galpha(13)Q226L can stimulate Ca(2+)/calmodulin-dependent kinase, CaMKII. Activated mutant that mimics the phosphorylated state of radixin (T564E) stimulated Rac1, induced the phosphorylation of CaMKII, and stimulated SRE-dependent gene transcription. Down-regulation of endogenous radixin using small interference RNA inhibited SRE-dependent gene transcription and phosphorylation of CaMKII induced by Galpha(13)Q226L. Overall, our results indicated that radixin via its C-terminal domain mediates SRE-dependent gene transcription through activation of Rac1 and CaMKII. In addition, the radixin-CaMKII signaling pathway is involved in Galpha(13)-mediated SRE-dependent gene transcription, suggesting that radixin could be involved in novel signaling pathway regulated by G(13) protein.