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Vol. 16, Issue 10, 4733-4744, October 2005

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Identification of Raf-1 S471 as a Novel Phosphorylation Site Critical for Raf-1 and B-Raf Kinase Activities and for MEK Binding

Jun Zhu ^* , Vitaly Balan ^*  , Agnieszka Bronisz ^*, Karina Balan ^* , Hengrui Sun ^*, Deborah T. Leicht , Zhijun Luo , Jun Qin ^||, Joseph Avruch ^¶, and Guri Tzivion ^* 

^* Cardiovascular Research Institute, The Texas A&M University Health Science Center, Temple, TX 76504; Karmanos Cancer Institute and Department of Pathology, Wayne State University, Detroit, MI 48201; Diabetes and Metabolism Research Unit, Evans Department of Medicine and Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118; ^|| Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030; and ^¶ Diabetes Unit, Department of Molecular Biology, Massachusetts General Hospital and Department of Medicine, Harvard Medical School, Boston, MA 02114

Submitted February 2, 2005; Revised July 2, 2005; Accepted July 29, 2005
Monitoring Editor: M. Bishr Omary

The Ras-Raf-MAPK cascade is a key growth-signaling pathway and its uncontrolled activation results in cell transformation. Although the general features of the signal transmission along the cascade are reasonably defined, the mechanisms underlying Raf activation remain incompletely understood. Here, we show that Raf-1 dephosphorylation, primarily at epidermal growth factor (EGF)-induced sites, abolishes Raf-1 kinase activity. Using mass spectrometry, we identified five novel in vivo Raf-1 phosphorylation sites, one of which, S471, is located in subdomain VIB of Raf-1 kinase domain. Mutational analyses demonstrated that Raf-1 S471 is critical for Raf-1 kinase activity and for its interaction with mitogen-activated protein kinase kinase (MEK). Similarly, mutation of the corresponding B-Raf site, S578, resulted in an inactive kinase, suggesting that the same Raf-1 and B-Raf phosphorylation is needed for Raf kinase activation. Importantly, the naturally occurring, cancer-associated B-Raf activating mutation V599E suppressed the S578A mutation, suggesting that introducing a charged residue at this region eliminates the need for an activating phosphorylation. Our results demonstrate an essential role of specific EGF-induced Raf-1 phosphorylation sites in Raf-1 activation, identify Raf-1 S471 as a novel phosphorylation site critical for Raf-1 and B-Raf kinase activities, and point to the possibility that the V599E mutation activates B-Raf by mimicking a phosphorylation at the S578 site.

These authors contributed equally to this work.

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

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