Kinase suppressor of Ras transphosphorylates c-Raf-1.
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.
Zafrullah
Mohammad
M
Laboratory of Signal Transduction, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA.
Yin
Xianglei
X
Haimovitz-Friedman
Adriana
A
Fuks
Zvi
Z
Kolesnick
Richard
R
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D045163:Antibodies, Phospho-Specific / Q000276:immunology; D019556:COS Cells; D002522:Chlorocebus aethiops; D004815:Epidermal Growth Factor / Q000494:pharmacology; D006801:Humans; D051379:Mice; D010766:Phosphorylation / Q000235:genetics; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000378:metabolism*; D013912:Threonine / Q000378:metabolism
D000911:Antibodies, Monoclonal; D045163:Antibodies, Phospho-Specific; D013912:Threonine; D004815:Epidermal Growth Factor; D011494:Protein Kinases; C097199:KSR-1 protein kinase; D019908:Proto-Oncogene Proteins c-raf