Raf-1 or c-Raf-1 is part of the Ras-Raf-MEK-MAPK pathway, which was among the first mammalian signaling cascades to be elucidated (Avruch et al., 1994
). The Ras-Raf-MAPK pathway transmits signals from membrane-bound receptors to intracellular and nuclear targets, coordinating cellular response to a variety of environmental factors (Avruch et al., 2001
; Chang and Karin, 2001
). Aberrations at the receptor level and along the Ras-Raf-MAPK pathway are associated with a variety of diseases, especially cancer, with Ras mutations detected in >30% of all human cancers (Porter and Vaillancourt, 1998
; Lyons et al., 2001
; Herrera and Sebolt-Leopold, 2002
). Recently, also B-Raf activating mutations were found in various cancers, most predominantly, melanomas (Davies et al., 2002
; Mercer and Pritchard, 2003
; Garnett and Marais, 2004
). The 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 are known, the exact mechanisms at the molecular level remain incompletely understood. Initially, a simple linear activation process was proposed, i.e., Ras activates Raf, Raf activates mitogen-activated protein kinase kinase (MEK), and MEK activates extracellular signal-regulated kinase (ERK). However, it is clear now that this pathway is more complex than initially thought and that other processes and accessory proteins are involved in its regulation, with Raf being the most complexly and strictly regulated member along the pathway (Kerkhoff and Rapp, 2001
; Dhillon and Kolch, 2002
; Wellbrock et al., 2004a
The mammalian Raf family of serine/threonine kinases consists of three highly conserved members, i.e., A-Raf, B-Raf, and Raf-1. Whereas Raf-1 is ubiquitously expressed, A- and B-Raf display a more tissue-specific expression. Raf-1, being the first Raf member to be identified is the most studied Raf isoform (Hagemann and Rapp, 1999
). The first step in Raf-1 activation by receptor tyrosine kinases, such as the epidermal growth factor (EGF) receptor, involves Ras activation (i.e., Ras loading with GTP). The exchange of Ras from a GDP- to a GTP-bound form results in a conformational change allowing its high-affinity interaction with Raf-1. The association of Raf-1 with Ras is insufficient by itself for Raf-1 activation, and other modulations take place at the membrane, producing a stable active Raf-1. This complex process involves changes in Raf-1 localization, posttranslational modifications (e.g., phosphorylation), dimerization, and protein–protein interactions (Luo et al., 1996
; Wellbrock et al., 2004a
). The main unresolved question in the Ras-mediated Raf-1 activation relates to the events at the membrane resulting in stable Raf-1 activation.
Phosphorylation is the major modification suggested to take place at the membrane. This idea is supported by studies demonstrating increased Raf-1 phosphorylation after mitogenic stimulation and studies showing that phosphatase treatment results in Raf-1 inactivation (Morrison et al., 1993
; Dent et al., 1995
; Marais et al., 1995
). Several Raf-1 phosphorylation sites have been identified, including basal and mitogen-activated sites. The major basal in vivo Raf-1 phosphorylation sites are located at S43, S259, S621, and an as-of-yet-unidentified site (). The S43 site, a postulated inhibitory phosphorylation site, is targeted by protein kinase A (PKA); however, the functional significance of this site remains controversial. The kinases responsible for the constitutive phosphorylation of S259 and S621 have not been fully defined; however, several candidate kinases, including PKA, AKT, and AMPK have been proposed (Wellbrock et al., 2004a
). S259 provides a binding point for the regulatory adapter protein 14-3-3 and serves as a negative regulatory site (Tzivion et al., 1998
; Tzivion et al., 2001
; Tzivion and Avruch, 2002
). Phosphorylation of the site by AKT or PKA negatively regulates Raf-1, whereas its dephosphorylation by protein phosphatase 2A has been reported to be part of the Raf-1 activation mechanism. Conversely, Raf-1 phosphorylation at the S621 supports Raf-1 kinase activity by providing a second binding point for 14-3-3, which binding at this site is critical for sustaining Raf-1 kinase activity (Tzivion et al., 1998
; Yip-Schneider et al., 2000
). It is not known yet whether phosphorylation on S259 and S621 occur simultaneously or whether they represent two separate Raf-1 subgroups.
Figure 1. Raf-1 phosphorylation sites. (A) Schematic diagram of Raf-1 showing known phosphorylation sites and potential kinases reported to phosphorylate these sites. The locations of the kinase domain, the Ras binding domain (RBD), the cysteine-rich domain (CRD), (more ...)
Beside the major sites, Raf-1 phosphorylation at several minor sites also has been reported. These include T268/269, S338/339, Y340/341, and S497/499. T268 is a proposed Raf-1 autophosphorylation site (Morrison et al., 1993
), and T269 was reported to be phosphorylated by KSR (Zhang et al., 1997
). Although the role of these phosphorylations in Raf-1 regulation remains unresolved, it seems that phosphorylation at these sites does not notably affect Raf-1 activation.
The Rac/CDC42-activated kinases PAK-2 and PAK-3 can phosphorylate Raf-1 on S338/339 and support Raf-1 activation by Ras and growth factors (King et al., 1998
). However, phosphorylation at these sites does not activate Raf-1 per se, suggesting that other modifications may be required for stable Raf-1 activation (Wellbrock et al., 2004a
). Similarly, Raf-1 phosphorylation at Y340/341, mediated by the tyrosine kinase Src, results in an only partial Raf-1 activation. S497 and S499 can be phosphorylated by protein kinase C; however, this phosphorylation does not result in Raf-1 activation, and phosphorylation at these sites is not required for Raf-1 activation by phorbol 12-myristate 13-acetate (PMA), growth factors, or Ras (Marais et al., 1998
In addition to these established sites, several novel feedback in vivo Raf-1 phosphorylation sites targeted by ERK have been reported recently. These include S29, S289, S296, S301, and S642 (Dougherty et al., 2005
; Hekman et al., 2005
). The role of these phosphorylations in Raf-1 regulation remains, however, controversial, because both negative (Alessi et al., 1995
; Dougherty et al., 2005
; Hekman et al., 2005
) and positive (Alessandrini et al., 1996
; Zimmermann et al., 1997
; Laird et al., 1999
; Balan et al.
, 2005) MEK/ERK effects on Raf-1 have been observed.
The mechanism/s responsible for Raf-1 inactivation is much less known, and it is proposed that Raf-1 dephosphorylation should play a role in this process (Avruch et al., 2001
Here, we demonstrate the necessity of EGF-induced Raf-1 phosphorylation in the EGF-induced Raf-1 activation and by using mass spectrometry, identify five novel in vivo Raf-1 phosphorylation sites. One of these sites, S471, located in subdomain VIB of the Raf-1 kinase domain, seems critical for Raf-1 kinase activity and for Raf-1 binding to MEK. Accordingly, mutation of the corresponding B-Raf site, S578, results in B-Raf inactivation, and, more importantly, is suppressed by the activating B-Raf V599E mutation, suggesting that introducing a charged residue at this region eliminates the need for an activating phosphorylation. Our results demonstrate a vital role of the EGF-induced Raf-1 phosphorylation in Raf-1 activation, identify Raf-1 S471 and B-Raf S578 as critical sites for Raf-1 and B-Raf kinase activities, and point to the possibility that the V599E mutation activates B-Raf by imitating phosphorylation at the S578 site.