MEK-1 activates C-Raf through a Ras-independent mechanism.
C-Raf is a member of the Ras-Raf-MEK-ERK mitogen-activated protein kinase (MAPK) signaling pathway that plays key roles in diverse physiological processes and is upregulated in many human cancers. C-Raf activation involves binding to Ras, increased phosphorylation and interactions with co-factors. Here, we describe a Ras-independent in vivo pathway for C-Raf activation by its downstream target MEK. Using (32)P-metabolic labeling and 2D-phosphopeptide mapping experiments, we show that MEK increases C-Raf phosphorylation by up-to 10-fold. This increase was associated with C-Raf kinase activation, matching the activity seen with growth factor stimulation. Consequently, coexpression of wildtype C-Raf and MEK was sufficient for full and constitutive activation of ERK. Notably, the ability of MEK to activate C-Raf was completely Ras independent, since mutants impaired in Ras binding that are irresponsive to growth factors or Ras were fully activated by MEK. The ability of MEK to activate C-Raf was only partially dependent on MEK kinase activity but required MEK binding to C-Raf, suggesting that the binding results in a conformational change that increases C-Raf susceptibility to phosphorylation and activation or in the stabilization of the phosphorylated-active form. These findings propose a novel Ras-independent mechanism for activating the C-Raf and the MAPK pathway without the need for mutations in the pathway. This mechanism could be of significance in pathological conditions or cancers overexpressing C-Raf and MEK or in conditions where C-Raf-MEK interaction is enhanced due to the down-regulation of RKIP and MST2.
Department of Pathology, Wayne State University, Detroit, MI 48201, USA.
Deborah T
DT
Leicht
Vitaly
V
Balan
Jun
J
Zhu
Alexander
A
Kaplun
Agnieszka
A
Bronisz
Ajay
A
Rana
Guri
G
Tzivion
D000818:Animals; D019556:COS Cells; D002455:Cell Division; D002522:Chlorocebus aethiops; D015536:Down-Regulation; D006801:Humans; D048369:MAP Kinase Kinase 1 / Q000378:metabolism*; D020935:MAP Kinase Signaling System; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D020929:Mitogen-Activated Protein Kinase Kinases / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism
D019908:Proto-Oncogene Proteins c-raf; D048052:Mitogen-Activated Protein Kinase 3; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human; D020929:Mitogen-Activated Protein Kinase Kinases; D016283:Proto-Oncogene Proteins p21(ras)