Atypical PKCζ is involved in RhoA-dependent mitogenic signaling by the P2Y12 receptor in C6 cells

When nucleotide hydrolysis is prevented, agonists of the P2Y(12) receptor enhance the proliferation of C6 glioma cells by RhoA-dependent, protein kinase C (PKC)-dependent activation of the extracellular signal-regulated kinase (ERK) pathway [Claes P, Grobben B, Van Kolen K, Roymans D & Slegers H (2001) Br J Pharmacol134, 402-408; Grobben B, Claes P, Van Kolen K, Roymans D, Fransen P, Sys SU & Slegers H (2001) J Neurochem78, 1325-1338]. In this study, we show that ERK1/2 phosphorylation was not affected by transfection of the cells with the G beta gamma-subunit-scavenging adrenergic receptor kinase peptide [beta ARK1-(495-689)] or with Rap1GAPII, indicating that P2Y(12) receptor stimulation enhances ERK1/2 phosphorylation by G(i)alpha subunit-mediated signaling independently of Rap1 activation. Inhibition of the RhoA downstream effector Rho-associated coiled-coil-containing kinase (ROCK) with Y-27632 did not affect the P2Y(12) receptor-induced increase in ERK1/2 phosphorylation but abrogated the mitogenic response. Involvement of growth factor receptor transactivation in the signaling towards ERK phosphorylation could be ruled out by the lack of an effect of PP2, AG1024, AG1296 or SU1498, inhibitors of Src, insulin-like growth factor receptor, platelet-derived growth factor receptor and vascular endothelial growth factor receptor kinase activity, respectively. Experiments with bisindolylmaleimide I and IX indicated the requirement of PKC activity. Classical and novel PKC isoforms could be excluded by treatment of the cells with Go6976 and calphostin C, whereas addition of a myristoylated PKC zeta pseudosubstrate inhibitor completely abolished P2Y(12) receptor-induced ERK1/2 activation. Moreover, coimmunoprecipitation experiments revealed PKC zeta/Raf1 and PKC zeta/ERK association, indicating the involvement of PKC zeta. From the data presented, we can conclude that the P2Y(12) receptor enhances cell proliferation by a G(i)alpha-dependent, RhoA-dependent PKC zeta/Raf1/MEK/ERK pathway that requires activation of ROCK, which is not involved in ERK1/2 signaling.