NEGATIVE REGULATION OF RAF ACTIVITY BY BINDING OF 14-3-3 TO THE AMINO-TERMINUS OF RAF IN-VIVO

In the developing eye of Drosophila the protein kinase D-Raf controls the specification of the R7 photoreceptor cells. We show that overexpr ession of wild-type D-Raf inhibits the formation of R7 cells in a dose -dependent manner. Conversely, overexpression of mutant D-Raf proteins in which the conserved S388 is replaced by A or by D promotes the for mation of supernumerary R7 cells, indicating increased D-Raf activity in vivo. S388 in D-Raf corresponds to S259 in c-Raf, shown to be invol ved in binding of 14-3-3. We show that analogous substitutions of S259 in c-Raf prevent binding of 14-3-3 zeta to the amino terminus of c-Ra f and cause a Ras-independent constitutively increased c-Raf kinase ac tivity. Binding of 14-3-3 zeta to the second binding site at the carbo xy terminal catalytic domain was unaffected by these mutations. These results suggest that the increased kinase activity of mutant D-Raf is caused by the selective loss of 14-3-3 binding to its amino terminus. Therefore, binding of 14-3-3 to the amino terminus of Raf appears to n egatively regulate Raf kinase activity in vivo. (C) 1997 Elsevier Scie nce Ireland Ltd.