Selective activation of MEK1 but not MEK2 by A-Raf from epidermal growth factor-stimulated Hela cells

J Biol Chem. 1996 Feb 9;271(6):3265-71. doi: 10.1074/jbc.271.6.3265.


Activation of the mitogen-activated protein kinase cascade is a critical event in mitogenic growth factor signal transduction. Mitogen-activated protein kinase is directly activated by a dual specific kinase, MEK, which itself is activated by serine phosphorylation. The c-Raf kinase has been implicated in mediating the signal transduction from mitogenic growth factor receptors to MEK activation. Recently, the B-Raf kinase was shown to be capable of phosphorylating and activating MEK as a result of growth factor stimulation. In this report, we used the yeast two-hybrid screening to isolate MEK interacting proteins. All three members of the Raf family kinases were identified as positive clones when the mutant MEK1S218/222A, in which the two phosphorylation serine residues were substituted by alanines, was used as a bait, whereas no positive clones were isolated when the wild type MEK1 was used as a bait in a similar screening. These results suggest that elimination of the phosphorylation sites of a target protein (MEK1 in our study) may stabilize the interaction between the kinase (Raf) and its substrate (MEK1), possibly due the formation of a nonproductive complex. These observations seem to suggest a general strategy using mutants to identify the upstream kinase of a phosphoprotein or the downstream targets of a kinase. Although c-Raf and B-Raf have been implicated in growth factor-induced MEK activation, little is known about A-Raf. We observed that stimulation of Hela cells with epidermal growth factor resulted in a rapid and transient activation of A-Raf, which is then capable of phosphorylating and activating MEK1. Interestingly, A-Raf does not activate MEK2, although c-Raf can activate both MEK1 and MEK2. Our data demonstrated that A-Raf is, indeed, a MEK1 activator and may play a role in growth factor signaling.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Cloning, Molecular
  • Enzyme Activation
  • Epidermal Growth Factor / pharmacology*
  • HeLa Cells
  • Humans
  • MAP Kinase Kinase 1
  • MAP Kinase Kinase 2
  • Mitogen-Activated Protein Kinase Kinases*
  • Mutagenesis, Site-Directed
  • Phosphorylation
  • Point Mutation
  • Protein-Serine-Threonine Kinases / metabolism*
  • Protein-Tyrosine Kinases / metabolism*
  • Proto-Oncogene Proteins / metabolism*
  • Proto-Oncogene Proteins c-raf
  • Recombinant Proteins / metabolism
  • Saccharomyces cerevisiae / growth & development
  • Saccharomyces cerevisiae / metabolism
  • Serine
  • Substrate Specificity


  • Proto-Oncogene Proteins
  • Recombinant Proteins
  • Serine
  • Epidermal Growth Factor
  • MAP2K2 protein, human
  • Protein-Tyrosine Kinases
  • Protein-Serine-Threonine Kinases
  • Proto-Oncogene Proteins c-raf
  • MAP Kinase Kinase 1
  • MAP Kinase Kinase 2
  • MAP2K1 protein, human
  • Mitogen-Activated Protein Kinase Kinases