Aims: We previously showed that primary cilia coordinate PDGFRa-mediated activation of the Akt and Mek1/2-Erk1/2 pathways in regulation of cell cycle entry and directional migration of fibroblasts (1,2). Further, ciliary PDGFR signaling activates the Na⁺/H⁺ exchanger NHE1 to control cell migration (3), indicating that Akt and/or Mek1/2-Erk1/2 could act upstream of NHE1. The aim of this study was to delineate the roles of Akt and Ribosomal S kinase (Rsk) in the signaling pathways that control PDGFRa- mediated NHE1 activation. Methods: Quiescent NIH3T3 cells were stimulated with PDGF-AA (a specific PDGFRa ligand) for 0-30 min, followed by western blotting of total and activated forms of the relevant signaling components. To analyze the roles of Akt and Mek1/2-Erk1/2 in activation of Rsk, we used specific inhibitors of Akt (Akti1/2) and Mek1/2 (U0126) at 0.3-10 mM. Results: PDGFRa was phosphorylated on Tyr754 after 3 min and remained so for at least 30 min, consistent with receptor activation. With a similar time course, Akt was phosphorylated on Ser473, Erk1/2 on Thr202 and Tyr204, and Rsk on Ser380 and Thr573, consistent with activation of these kinases. Thus, Akt, Mek1/2-Erk1/2 and Rsk, all of which are known to regulate NHE1, are activated downstream from PDGFRa. Phoshorylation of Akt and Erk1/2 was inhibited dose-dependently by Akti1/2 and U0126, respectively, with full inhibition at 10 mM and no apparent cross- reaction with other pathways. Importantly, phosphorylation of Rsk was inhibited only by U0126 and in a dose-dependent manner comparable to that of Erk1/2. Conclusion: Stimulation of the ciliary PDGFRa leads to activation of Rsk downstream of Mek1/2-Erk1/2 and independently of Akt. Conditions to determine the roles of these pathways in NHE1 activation in migrating cells were established. (1) Schneider, L. et al. 2005. Curr. Biol. 15:1861-6 (2) Schneider, L. et al. 2009. Cell. Physiol. Biochem. In press (3) Schneider, L. et al. 2009. J. Cell Biol. 185:163-76