Identification of novel in vivo Raf-1 phosphorylation sites mediating positive feedback Raf-1 regulation by extracellular signal-regulated kinase.
The Ras-Raf-mitogen-activated protein kinase cascade is a key growth-signaling pathway, which uncontrolled activation results in transformation. Although the exact mechanisms underlying Raf-1 regulation remain incompletely understood, phosphorylation has been proposed to play a critical role in this regulation. We report here three novel epidermal growth factor-induced in vivo Raf-1 phosphorylation sites that mediate positive feedback Raf-1 regulation. Using mass spectrometry, we identified Raf-1 phosphorylation on three SP motif sites: S289/S296/S301 and confirmed their identity using two-dimensional-phosphopeptide mapping and phosphospecific antibodies. These sites were phosphorylated by extracellular signal-regulated kinase (ERK)-1 in vitro, and their phosphorylation in vivo was dependent on endogenous ERK activity. Functionally, ERK-1 expression sustains Raf-1 activation in a manner dependent on Raf-1 phosphorylation on the identified sites, and S289/296/301A substitution markedly decreases the in vivo activity of Raf-1 S259A. Importantly, the ERK-phosphorylated Raf-1 pool has 4 times higher specific kinase activity than total Raf-1, and its phosphopeptide composition is similar to that of the general Raf-1 population, suggesting that the preexisting, phosphorylated Raf-1, representing the activatable Raf-1 pool, is the Raf-1 subpopulation targeted by ERK. Our study describes the identification of new in vivo Raf-1 phosphorylation sites targeted by ERK and provides a novel mechanism for a positive feedback Raf-1 regulation.
Balan
Vitaly
V
Karmanos Cancer Institute, Department of Pathology, Wayne State University, Detroit, MI 48201, USA.
Leicht
Deborah T
DT
Zhu
Jun
J
Balan
Karina
K
Kaplun
Alexander
A
Singh-Gupta
Vinita
V
Qin
Jun
J
Ruan
Hong
H
Comb
Michael J
MJ
Tzivion
Guri
G
D000595:Amino Acid Sequence; D000818:Animals; D045163:Antibodies, Phospho-Specific / Q000378:metabolism; D019556:COS Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D004815:Epidermal Growth Factor / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D025461:Feedback, Physiological; D015870:Gene Expression; D020930:MAP Kinase Kinase Kinases / Q000037:antagonists & inhibitors; D008969:Molecular Sequence Data; D010766:Phosphorylation / Q000187:drug effects; D019908:Proto-Oncogene Proteins c-raf / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000378:metabolism
D045163:Antibodies, Phospho-Specific; D012694:Serine; D004815:Epidermal Growth Factor; D019908:Proto-Oncogene Proteins c-raf; D048049:Extracellular Signal-Regulated MAP Kinases; D020930:MAP Kinase Kinase Kinases