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Vol. 17, Issue 3, 1141-1153, March 2006

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Identification of Novel In Vivo Raf-1 Phosphorylation Sites Mediating Positive Feedback Raf-1 Regulation by Extracellular Signal-regulated Kinase

Vitaly Balan ^* , Deborah T. Leicht ^* , Jun Zhu , Karina Balan ^*, Alexander Kaplun ^*, Vinita Singh-Gupta ^*, Jun Qin , Hong Ruan ^||, Michael J. Comb ^||, and Guri Tzivion ^*

^* Karmanos Cancer Institute and Department of Pathology, Wayne State University, Detroit, MI 48201; Cardiovascular Research Institute, Texas A&M University Health Science Center, Temple, TX 76504; Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030; and ^|| Cell Signaling Technology, Beverly, MA 01915

Submitted December 27, 2004; Accepted January 3, 2006
Monitoring Editor: Carl-Henrik Heldin

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.

These authors contributed equally to this work.

Address correspondence to: Guri Tzivion (tziviong{at}karmanos.org).

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