title: 14-3-3 isotypes facilitate coupling of protein kinase C-zeta to Raf-1: negative regulation by 14-3-3 phosphorylation.
abstract: 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 phosphorylate and activate Raf-1 in a signalling complex [van Dijk, Hilkmann and van Blitterswijk (1997) Biochem. J. 325, 303-307]. We report now that PKC-zeta-Raf-1 interaction is mediated by 14-3-3 proteins in vitro and in vivo. Co-immunoprecipitation experiments in COS cells revealed that complex formation between PKC-zeta and Raf-1 is mediated strongly by the 14-3-3beta and -theta; isotypes, but not by 14-3-3zeta. 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 14-3-3beta and Raf-1. Treatment of PKC-zeta with lambda protein phosphatase also reduced its binding to 14-3-3beta in vitro. Preincubation of an immobilized Raf-1 construct with 14-3-3beta facilitated 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-zeta mutant than with wild-type PKC-zeta, supporting the idea that kinase activity leads to complex dissociation. 14-3-3beta and -&theta; were substrates for PKC-zeta, whereas 14-3-3zeta was not. Phosphorylation of 14-3-3beta by PKC-zeta negatively regulated their physical association. 14-3-3beta with its putative PKC-zeta phosphorylation sites mutated enhanced co-precipitation between PKC-zeta 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-zeta 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 activation by PKC-zeta.
authors.affiliation: Division of Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
authors.forename: P C
authors.initials: PC
authors.lastname: Van Der Hoeven
authors.forename: J C
authors.initials: JC
authors.lastname: Van Der Wal
authors.forename: P
authors.initials: P
authors.lastname: Ruurs
authors.forename: M C
authors.initials: MC
authors.lastname: Van Dijk
authors.forename: J
authors.initials: J
authors.lastname: Van Blitterswijk
mesh_terms: D048948:14-3-3 Proteins; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D020836:Protein Structure, Quaternary; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D013379:Substrate Specificity; D014446:Tyrosine 3-Monooxygenase; D000099221:Protein Kinase C zeta
chemical_list: D048948:14-3-3 Proteins; D010768:Phosphoserine; D020033:Protein Isoforms; D011493:Protein Kinase C; D011506:Proteins; D019908:Proto-Oncogene Proteins c-raf; D011994:Recombinant Proteins; D014446:Tyrosine 3-Monooxygenase; D000099221:Protein Kinase C zeta