Phosphorylation of Raf-1 by p21-activated kinase 1 and Src regulates Raf-1 autoinhibition

J Biol Chem. 2003 Mar 28;278(13):11221-6. doi: 10.1074/jbc.M210318200. Epub 2003 Jan 27.

Abstract

Exposure of cells to mitogens or growth factors stimulates Raf-1 activity through a complex mechanism that involves binding to active Ras, phosphorylation on multiple residues, and protein-protein interactions. Recently it was shown that the amino terminus of Raf-1 contains an autoregulatory domain that can inhibit its activity in Xenopus oocytes. In the present work we show that expression of the Raf-1 autoinhibitory domain blocks extracellular signal-regulated kinase 2 activation by the Raf-1 catalytic domain in mammalian cells. We also show that phosphorylation of Raf-1 on serine 338 by PAK1 and tyrosines 340 and 341 by Src relieves autoinhibition and that this occurs through a specific decrease in the binding of the Raf-1 regulatory domain to its catalytic domain. In addition, we demonstrate that phosphorylation of threonine 491 and serine 494, two phosphorylation sites in the catalytic domain that are required for Raf-1 activation, is unlikely to regulate autoinhibition. These results demonstrate that the autoinhibitory domain of Raf-1 is functional in mammalian cells and that its interaction with the Raf-1 catalytic domain is regulated by phosphorylation of serine 338 and tyrosines 340 and 341.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Catalytic Domain
  • Humans
  • Phosphorylation
  • Precipitin Tests
  • Protein-Serine-Threonine Kinases / metabolism*
  • Proto-Oncogene Proteins c-raf / antagonists & inhibitors*
  • Proto-Oncogene Proteins c-raf / chemistry
  • Proto-Oncogene Proteins c-raf / metabolism*
  • Serine / metabolism
  • Transfection
  • Xenopus
  • p21-Activated Kinases

Substances

  • Serine
  • PAK1 protein, human
  • Protein-Serine-Threonine Kinases
  • Proto-Oncogene Proteins c-raf
  • p21-Activated Kinases