PROTEIN-KINASE C-EPSILON ASSOCIATES WITH THE RAF-1 KINASE AND INDUCESTHE PRODUCTION OF GROWTH-FACTORS THAT STIMULATE RAF-1 ACTIVITY

Several observations indicate that the Raf-1 kinase is a downstream ef fector of protein kinase C-epsilon (PKC epsilon). We recently have sho wn that Raf-1 is constitutively activated in PKC epsilon transformed R at6 fibroblasts, and transformation can be reverted by expression of a dominant negative Raf-1, but not a dominant negative Ras mutant (Caca ce et al., 1996), Cai et al, (1997) demonstrated that PKC epsilon indu ced proliferation of NIH3T3 cells is independent of Ras or Src, but de pends on Raf-1. These authors further suggested that PKC epsilon activ ates Raf-1 by direct phosphorylation. Here we have investigated the fu nctional interaction between PKC epsilon and Raf-1, PKCE, but not PKC alpha, was found to bind to the Raf-1 kinase domain, The association a ppeared to be direct, as it could be reconstituted in vitro with purif ied proteins, Raf-1 and PKC epsilon could be co-precipitated from Sf-9 insect cells and PKC epsilon transformed NIH313 cells (NIH/epsilon). The association was negatively regulated by ATP in vitro and by TPA tr eatment in NIH/epsilon cells, but not in Sf-9 insect cells, Raf-1 was constitutively activated in NIH/epsilon cells, How ever, using coexpre ssion experiments in Sf-9 cells and transiently transfected A293 cells we did not obtain any evidence for a direct activation of Raf-1 by PK C epsilon. PKCE did not induce translocation of Raf-1 to the membrane, Furthermore, PKCE did not activate Raf-1 nor enhance the kinase activ ity of Raf-1 that had been pre-activated by coexpression of Ras or the Lck tyrosine kinase, In contrast, conditioned media from PKC epsilon transformed cells induced a robust activation of Raf-1. This activatio n could be partially reproduced by recombinant TGF beta, a growth fact ors secreted by PKC epsilon transformed Rat6 cells. In conclusion, our results suggest that PKC epsilon stimulates Raf-1 indirectly by induc ing the production of autocrine growth factors.