14-3-3 isotypes facilitate coupling of protein kinase C-zeta to Raf-1: negative regulation by 14-3-3 phosphorylation

14-3-3 Proteins may function as adapters or scaffold in signal-transduction pathways. We found previously that protein kinase C-zeta (PKC-zeta) can ph osphorylate and activate Raf-1 in a signalling complex [van Dijk, Hilkmann and van Blitterswijk (1997) Biochem. J. 325, 303-307]. We report now that P KC-zeta-Raf-1 interaction is mediated by 14-3-3 proteins in vitro and in vi uo. Coimmunoprecipitation experiments in COS cells revealed that complex fo rmation between PKC-zeta and Raf-1 is mediated strongly by the 14-3-3 beta and -theta isotypes, but not by 14-3-3 zeta. Far-Western blotting revealed that 14-3-3 binds PKC-zeta directly at its regulatory domain, where a S186A mutation in a putative 14-3-3-binding domain strongly reduced the binding and the complex formation with 14-3-3 beta and Raf-1. Treatment of PKC-zeta with lambda protein phosphatase also reduced its binding to 14-3-3 beta in vitro. Preincubation of an immobilized Raf-1 construct with 14-3-3 beta fa cilitated PKC-zeta binding. Together, the results suggest that 14-3-3 binds both PKC-zeta (at phospho-Ser-186) and Raf-1 in a ternary complex. Complex formation was much stronger with a kinase-inactive PKC-g mutant than with wild-type PKC-zeta, supporting the idea that kinase activity leads to compl ex dissociation. 14-3-3 beta and -theta were substrates for PKC-zeta, where as 143-3 zeta was not. Phosphorylation of 14-3-3 beta by PKC-zeta negativel y regulated their physical association. 14-3-3 beta with its putative PKC-z eta phosphorylation sites mutated enhanced co-precipitation between PKC-zet a and Raf-1, suggesting that phosphorylation of 14-3-3 by PKC-zeta weakens the complex in vivo. We conclude that 14-3-3 facilitates coupling of PKC-ze ta to Raf-1 in an isotype-specific and phosphorylation-dependent manner. We suggest that 14-3-3 is a transient mediator of Raf-1 phosphorylation and a ctivation by PKC-zeta.