Negative regulation of Raf activity by binding of 14-3-3 to the amino terminus of Raf in vivo

Mech Dev. 1997 Jun;64(1-2):95-104. doi: 10.1016/s0925-4773(97)00052-x.

Abstract

In the developing eye of Drosophila the protein kinase D-Raf controls the specification of the R7 photoreceptor cells. We show that overexpression 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 formation of supernumerary R7 cells, indicating increased D-Raf activity in vivo. S388 in D-Raf corresponds to S259 in c-Raf; shown to be involved 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-Raf and cause a Ras-independent constitutively increased c-Raf kinase activity. Binding of 14-3-3 zeta to the second binding site at the carboxy 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 negatively regulate Raf kinase activity in vivo.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 14-3-3 Proteins
  • Amino Acid Sequence
  • Animals
  • Binding Sites / genetics
  • Drosophila / genetics
  • Drosophila / growth & development*
  • Drosophila / metabolism*
  • Eye / cytology
  • Eye / growth & development*
  • Eye / metabolism*
  • Microscopy, Electron, Scanning
  • Mutagenesis, Site-Directed
  • Photoreceptor Cells, Invertebrate / cytology
  • Photoreceptor Cells, Invertebrate / growth & development
  • Photoreceptor Cells, Invertebrate / metabolism
  • Protein-Serine-Threonine Kinases / genetics
  • Protein-Serine-Threonine Kinases / metabolism*
  • Proteins / metabolism*
  • Proto-Oncogene Proteins / genetics
  • Proto-Oncogene Proteins / metabolism*
  • Proto-Oncogene Proteins c-raf
  • Signal Transduction
  • Tyrosine 3-Monooxygenase*

Substances

  • 14-3-3 Proteins
  • Proteins
  • Proto-Oncogene Proteins
  • Tyrosine 3-Monooxygenase
  • Protein-Serine-Threonine Kinases
  • Proto-Oncogene Proteins c-raf