REGULATION OF RAF-1 KINASE-ACTIVITY BY THE 14-3-3-FAMILY OF PROTEINS

We have identified the beta (beta) isoform of the 14-3-3 family of pro teins as an activator of the Raf-1 protein kinase. 14-3-3 was isolated in a yeast two-hybrid screen for Raf-1 kinase domain binding proteins . Purified bovine brain 14-3-3 interacted specifically with both c-Raf -1 and the isolated Raf-1 kinase domain, Association was sensitive to the activation status of Raf-1; 14-3-3 bound to unactivated Raf-1, but not Raf-1 activated by protein kinase C alpha or Ras and Lck, The sig nificance of these interactions under physiological conditions was dem onstrated by co-immunoprecipitation of Raf-1 and 14-3-3 from extracts of quiescent, but not mitogen-stimulated, NIH 3T3 cells, 14-3-3 was no t a preferred Raf-l substrate in vitro and did not significantly affec t Raf-l kinase activity in a purified system, However, in cell-free ex tracts 14-3-3 acted as a Ras-independent activator of both c-Raf-1 and the Raf-1 kinase domain, The same results were obtained in vivo using transfection assays; 14-3-3 enhanced both c-Raf-1- and Raf-1 kinase d omain-stimulated expression of AP-1- and NF-kappa B-dependent reporter genes and accelerated Raf-1 kinase domain-triggered differentiation o f PC12 cells. We conclude that 14-3-3 is a latent co-activator bound t o unactivated Raf-1 in quiescent cells and mediates mitogen-triggered but Ras-independent regulatory effects aimed directly at the kinase do main.