MEK-1 activates C-Raf through a Ras-independent mechanism

Abstract

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 32P-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 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 downregulation of RKIP and MST2.

Allosteric Activation of Functionally Asymmetric RAF Kinase Dimers

“…This model (Fig. 6) is supported by our experiments showing that MEK controls NtA phosphorylation of CRAF and a recent report that MEK can activate CRAF (Leicht et al, 2013). Whether MEK can directly phosphorylate the NtA, or whether this is the property of an intermediary kinase, is not known.…”

Structure of the BRAF-MEK Complex Reveals a Kinase Activity Independent Role for BRAF in MAPK Signaling

“…This agrees well with our observations that cell lines with low basal pMEK levels are enriched with high BRAF-MEK1 complexes and less so with CRAF-MEK1. As the relative stoichiometry of BRAF and CRAF can affect MAPK signaling output and since the MEK interaction with CRAF drives CRAF activation (Hu et al, 2013;Leicht et al, 2013), our data suggest that the presence of a BRAF-MEK1 complex modulates flux through the MAPK pathway. Accordingly, mutations in the BRAF P-loop , which exhibit weakened interaction with MEK1 and enhanced dimerization with CRAF, likely position BRAF P-loop to function as an ''activator'' of CRAF through a dimerization-dependent manner.…”

Families of microRNAs Expressed in Clusters Regulate Cell Signaling in Cervical Cancer

“…Proliferation and apoptosis are regulated through Raf-1 and MST2 activation. Raf-1 is activated by Ras inducing MEK1, ERK1, and ERK2 phosphorylation [ 14 ], while MST2 triggers apoptosis through PUMA and caspases activation. MST2–Raf-1 interaction inhibits MST2 activation while MST2-RASSF1A interaction activates MST2.…”

“…MST2–Raf-1 interaction inhibits MST2 activation while MST2-RASSF1A interaction activates MST2. RASSF1A and Raf-1 compete for binding sites within MST2 [ 14 , 15 ]. Apoptosis stimulus induces MST2-RASSF1A interaction, activating MST2.…”