14-3-3 IS NOT ESSENTIAL FOR RAF-1 FUNCTION - IDENTIFICATION OF RAF-1 PROTEINS THAT ARE BIOLOGICALLY ACTIVATED IN A 14-3-3-INDEPENDENT AND RAS-INDEPENDENT MANNER

Recent reports have demonstrated the in vivo association of Raf-1 with members of the 14-3-3 protein family. To address the significance of the Raf-1-14-3-3 interaction, we investigated the enzymatic activity a nd biological function of Raf-1 in the presence and absence of associa ted 14-3-3. The interaction between these two molecules was disrupted in vivo and in vitro with a combination of molecular and biochemical t echniques. Biochemical studies demonstrated that the enzymatic activit ies of Raf-1 were equivalent in the presence and absence of 14-3-3. Fu rthermore, mixing of purified Raf-1 and 14-3-3 in vitro was not suffic ient to activate Raf-1, With a molecular approach, Cys-165 and Cys-168 as well as Ser-259 were identified as residues of Raf-1 required for the interaction with 14-3-3, Cys-165 and Cys-168 are located within th e conserved cysteine-rich region of the CR1 domain, and Ser-259 is a c onserved site of serine phosphorylation found within the CR2 domain. M utation of either Cys-165 and Cys-168 or Ser-259 prevented the stable interaction of Raf-1 with 14-3-3 in vivo, Consistent with the model in which a site of serine phosphorylation is involved in the Raf-1-14-3- 3 interaction, dephosphorylated Raf-l was unable to associate with 14- 3-3 in vitro. Phosphorylation may represent a general mechanism mediat ing 14-3-3 binding, because dephosphorylation of the Bcr kinase (known to interact with 14-3-3) also eliminated its association with 14-3-3, Finally, mutant Raf-1 proteins unable to stably interact with 14-3-3 exhibited enhanced enzymatic activity in human 293 cells and Xenopus o ocytes and were biologically activated, as demonstrated by their abili ty to induce meiotic maturation of Xenopus oocytes, However, in contra st to wild-type Raf-1, activation of these mutants was independent of Ras, Our results therefore indicate that interaction with 14-3-3 is no t essential for Raf-1 function.