Positive and negative regulation of Raf kinase activity and function by phosphorylation

Chong, Huira; Lee, Jeeyong; Guan, Kun Liang

doi:10.1093/emboj/20.14.3716

Cited by 222 publications

(224 citation statements)

References 42 publications

Supporting

Mentioning

211

Contrasting

Unclassified

Order By: Relevance

“…Exchange of serine for aspartic acid (D) was intended to mimick the phosphorylation state of serine and has been used in similar studies [13]. As shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Novel C‐Raf phosphorylation sites: serine 296 and 301 participate in Raf regulation

et al. 2004

View full text Add to dashboard Cite

The C-Raf kinase is regulated by numerous phosphorylation steps. To quantify the most prominent phosphorylation sites of C-Raf, we performed mass spectrometry analysis of wild-type C-Raf and the constitutively active C-Raf mutant CRaf-Y340D/Y341D. We confirmed phosphorylation of most of the sites reported in the literature with the exception that we did not detect phosphorylation of threonine 268/269 (autophosphorylation sites) and threonine 491/serine 494 (kinase activation loop). Importantly, we detected novel phosphorylation sites at the positions of serine 296 and 301. The degree of phosphorylation in these positions depends on the level of activation of CRaf. Furthermore, we show here, using point mutant forms of C-Raf kinases with serine to alanine and serine to aspartic acid substitution, that serines 296 and 301 contribute to negative regulation of C-Raf.

show abstract

“…Exchange of serine for aspartic acid (D) was intended to mimick the phosphorylation state of serine and has been used in similar studies [13]. As shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Novel C‐Raf phosphorylation sites: serine 296 and 301 participate in Raf regulation

et al. 2004

View full text Add to dashboard Cite

show abstract

“…As N-region and activation segment phosphorylation act synergistically in Raf-1 activation (Chong et al, 2001), it has been suggested that the constitutively charged N-region predisposes B-Raf for oncogenic hits (Davies et al, 2002;Morrison, 2004;Wellbrock et al, 2004a). Indeed, gain-of-function mutations in the human BRAF gene have been identified in about 70% of malignant melanomas, 30% of papillary thyroid and serous ovarian carcinomas, 15% of colorectal cancers and to a lower frequency in a wide range of other tumours (Davies et al, 2002;Garnett and Marais, 2004).…”

Section: Introductionmentioning

confidence: 99%

“…Upon binding to Ras-GTP, 14-3-3 is displaced from phospho-S259 and its re-binding is prevented by S259 dephosphorylation (Dhillon et al, 2002). This induces an open conformation leading to the exposure of the MEK docking site and the phosphorylation of the N-region and activation segment (Chong et al, 2001;Terai and Matsuda, 2005). The N-region plays a critical role in Raf activation (Mason et al, 1999).…”

mentioning

confidence: 99%

Functional analysis of the regulatory requirements of B-Raf and the B-RafV600E oncoprotein

et al. 2006

View full text Add to dashboard Cite

The BRAF V600E mutation is found in approximately 6% of human cancers and mimics the phosphorylation of the kinase domain activation segment. In wild-type B-Raf (B-Raf wt ), activation segment phosphorylation is thought to cooperate with negative charges within the N-region for full activation. In contrast to Raf-1, the N-region of B-Raf is constitutively negatively charged owing to the presence of residues D447/D448 and the phosphorylation of S446. Therefore, it has been suggested that this hallmark predisposes B-Raf for oncogenic activation. In this study, we demonstrate that neutralizing mutations of these residues (in particular S446 and S447), or uncoupling of B-Raf from Ras-guanine 5 0 -triphosphate (GTP), strongly reduce the biological activity of B-Raf in a PC12 cell differentiation assay. We also confirm that S365 is a 14-3-3 binding site, and determine that mutation of this residue rescues the impaired biological activity of B-Raf proteins with a neutralized N-region, suggesting that the N-region opposes a 14-3-3-mediated transition into an inactive conformation. However, in the case of B-Raf V600E , although complete N-region neutralization resulted in a 2.5-fold reduction in kinase activity in vitro, this oncoprotein strongly induced PC12 differentiation or transformation and epithelial-mesenchymal transition of MCF-10A cells regardless of its N-region charge. Furthermore, the biological activity of B-Raf V600E was independent of its ability to bind Ras-GTP. Our analysis identifies important regulatory differences between B-Raf wt and B-Raf V600E and suggests that B-Raf V600E cannot be inhibited by strategies aimed at blocking S446 phosphorylation or Ras activation. Keywords: Ras; 14-3-3 proteins; b-Catenin; E-cadherin; mammary epithelial cells IntroductionThe Ras/Raf/mitogen-activated/extracellular-regulated kinase (MEK)/extracellular signal regulated kinase (ERK) pathway plays a pivotal role in control of proliferation and differentiation and, owing to its role as a gatekeeper of this pathway, Raf appears an attractive therapeutic target (O'Neill and Kolch, 2004;Wilhelm et al., 2004). The Raf-kinase family comprises the A-Raf, B-Raf and Raf-1 isoforms in vertebrates as well as D-Raf and LIN-45 in Drosophila and Caenorhabditis, respectively. B-Raf, the major ERK activator in vertebrates, is required for the maintenance of basal ERK activity and displays the most potent transforming activity (Papin et al., 1998;Brummer et al., 2002;Mercer and Pritchard, 2003). All isoforms share three highly conserved regions (CRs; Figure 1a): the N-terminal CR1 contains the Ras-guanine 5 0 -triphosphate (GTP)-binding domain (RBD), which initiates the interaction with Ras-GTP through a conserved arginine residue (R188 in B-Raf) that is required for the recruitment and activation of Raf at the plasma membrane. Consequently, mutation of this residue prevents Ras/Raf interaction and renders D-Raf, B-Raf and Raf-1 unresponsive to most extracellular signals (Hou et al., 1995;Marais et al., 1997;Brummer et al., 2002). The ...

show abstract

“…These events involve changes in Raf-1 localization, phosphorylation and protein-protein interactions (Weinstein-Oppenheimer et al, 2000;Avruch et al, 2001;Wellbrock et al, 2004a). Studies focusing on Raf-1 phosphorylation document both positive and negative effects of Raf-1 phosphorylation (Avruch et al, 2001;Chong et al, 2001). For example, p21 activated kinase (PAK) and Src mediate Raf-1 phosphorylation on positive reg- ulatory sites and augment Raf-1 activity, whereas cAMPdependent protein kinase (PKA) and AKT mediate Raf-1 phosphorylation on negative regulatory sites and inhibit Raf-1 activity.…”

Section: Introductionmentioning

confidence: 99%

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

Balan

Leicht

Zhu³

et al. 2006

MBoC

View full text Add to dashboard Cite

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...

show abstract

Positive and negative regulation of Raf kinase activity and function by phosphorylation

Cited by 222 publications

References 42 publications

Novel C‐Raf phosphorylation sites: serine 296 and 301 participate in Raf regulation

Novel C‐Raf phosphorylation sites: serine 296 and 301 participate in Raf regulation

Functional analysis of the regulatory requirements of B-Raf and the B-RafV600E oncoprotein

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

Contact Info

Product

Resources

About