Kinase suppressor of Ras transphosphorylates c-Raf-1 Academic Article uri icon

Overview

MeSH Major

  • Protein Kinases
  • Proto-Oncogene Proteins c-raf

abstract

  • Whether kinase suppressor of Ras1 (KSR1) is an active kinase that phosphorylates c-Raf-1 or a scaffold that coordinates signaling along the Ras/ERK1 signaling module is actively debated. In this study, we generated a monoclonal antibody against a c-Raf-1 peptide containing phosphorylated Thr(269), the putative target for KSR1 kinase activity. We show that this antibody detects Thr(269)-phosphorylated c-Raf-1 in A431 cells upon epidermal growth factor (EGF) stimulation, preceding MEK1 activation. Furthermore, this antibody detects in vitro phosphorylation of FLAG-c-Raf-1 and kinase-dead FLAG-c-Raf-1(K375M) by immunopurified KSR1, but fails to detect phosphorylation of FLAG-c-Raf-1(K375M/T269V), engineered with a Thr(269) to valine substitution. To provide unequivocal evidence that KSR1 is a legitimate kinase, we purified KSR1 to homogeneity, confirmed by mass spectrometry, renatured it in-gel, and demonstrated that it phosphorylates BSA-conjugated c-Raf-1 peptide at Thr(269). These studies add to emerging data validating KSR1 as a kinase that phosphorylates c-Raf-1.

publication date

  • December 18, 2009

Research

keywords

  • Academic Article

Identity

Language

  • eng

PubMed Central ID

  • PMC2800940

Digital Object Identifier (DOI)

  • 10.1016/j.bbrc.2009.09.048

PubMed ID

  • 19766101

Additional Document Info

start page

  • 434

end page

  • 40

volume

  • 390

number

  • 3