Novel roles of specific isoforms of protein kinase C in activation of the c-fos serum response element

Protein kinase C (PKC) is a multigene family of enzymes consisting of at le ast 11 isoforms. It has been implicated in the induction of c-fos and other immediate response genes by various mitogens. The serum response element ( SRE) in the c-fos promoter is necessary and sufficient for induction of tra nscription of c-fos by serum, growth factors, and the phorbol ester 12-O-te tradecanoylphorbol-13-acetate (TPA). It forms a complex with the ternary co mplex factor (TCF) and,vith a dimer of the serum response factor (SRF), TCF is the target of several signal transduction pathways and SRF is the targe t of the rhoA pathway, In this study,re generated dominant-negative and con stitutively active mutants of PKC-alpha, PKC-delta, PKC-epsilon, and PKC-ze ta to determine the roles of individual isoforms of PKC in activation of th e SRE, Transient-transfection assays with NIH 3T3 cells, using an SRE-drive n luciferase reporter plasmid, indicated that PKC-alpha and PKC-epsilon, bu t not PKC-delta or PKC-zeta, mediate SRE activation. TPA-induced activation of the SRE was partially inhibited by dominant negative c-Raf, ERK1, or ER K2, and constitutively active mutants of PKC-alpha and PKC-F activated the transactivation domain of Elk-1. TPA-induced activation of the SRE was also partially inhibited by a dominant-negative MEKK1. Furthermore, TPA treatme nt of serum-starved NIH 3T3 cells led to phosphorylation of SEK1, and const itutively active mutants of PKC-alpha and PKC-epsilon activated the transac tivation domain of c-Jun, a major substrate of JNK. Constitutively active m utants of PKC-alpha and PKC-epsilon could also induce a mutant c-fos promot er which lacks the TCF binding site, and they also induce transactivation a ctivity of the SRF. Furthermore, rhoA-mediated SRE activation was blocked b y dominant negative mutants of PKC-alpha or PKC-epsilon. Taken together, th ese findings indicate that PKC-alpha and PKC-epsilon can enhance the activi ties of at least three signaling pathways that converge on the SRE: c-Raf-M EK1-ERK-TCF, MEKK1-SEK1-JNK-TCF, and rhoA-SRF. Thus, specific isoforms of P KC may play a role in integrating networks of signal transduction pathways that control gene expression.