The Ras-Raf-MAPK pathway regulates diverse physiological processes by transmitting signals from membrane based receptors to various nuclear, cytoplasmic and membrane-bound targets, coordinating a large variety of cellular responses. Function of Raf family kinases has been shown to play a role during organism development, cell cycle regulation, cell proliferation and differentiation, cell survival and apoptosis and many other cellular and physiological processes. Aberrations along the Ras-Raf-MAPK pathway play an integral role in various biological processes concerning human health and disease. Overexpression or activation of the pathway components is a common indicator in proliferative diseases such as cancer and contributes to tumor initiation, progression and metastasis. In this review, we focus on the physiological roles of Raf kinases in normal and disease conditions, specifically cancer, and the current thoughts on Raf regulation.

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 twodimensional-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 ERKphosphorylated 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. INTRODUCTIONRaf-1 is part of the Ras-Raf-mitogen-activated protein kinase kinase (MEK)-mitogen-activated protein kinase (MAPK) pathway, among the first mammalian signaling cascades to be elucidated (Avruch et al., 2001;Chang and Karin, 2001). The Ras-Raf-MAPK pathway is responsible for transmitting signals from membrane-bound receptors to intracellular and to nuclear targets, coordinating cellular response to a variety of environmental factors (Liebmann, 2001;Pearson et al., 2001). Aberrations at the receptor level and along the Ras-Raf-MAPK pathway are associated with a variety of diseases, especially cancer, with mutations in Ras detected in Ͼ30% of all human cancers and reaching frequencies of up to 50 -90% in specific carcinomas (Porter and Vaillancourt, 1998;Lyons et al., 2001;Herrera and Sebolt-Leopold, 2002). Recently, also activating mutations in the B-Raf gene have been identified in various cancers, most predominantly melanomas (Davies et al., 2002;Mercer and Pritchard, 2003). In addition, both Ras and Raf are protooncogenes that occur naturally as viral-transmitted oncoproteins (Rapp et al., 1983). The initial elucidation of the Ras-Raf-MAPK pathway resulted from complimentary molecular and biochemical studies in mammalian cells and genetic studies in yeast, Drosophila and Caenorhabditis elegans (Avruch et al., 2001). Although the general features of the pathway and its activation mode are known, the exact regulatory mechanisms, at the molecular level, remain incompletely underst...

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.