Regulation of Raf-1 activation and signalling by dephosphorylation

Dhillon, Amardeep S.; Meikle, Sharon; Yazıcı, Zihni Açar; Eulitz, Manfred; Kölch, Walter

doi:10.1093/emboj/21.1.64

Cited by 258 publications

(241 citation statements)

References 49 publications

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“…The measurement of the in vitro kinase activity of B-Raf often does not reflect the biological activity in the cell (19,(45)(46)(47). Thus, we monitored its phosphorylation status at serine 365, because B-Raf and Raf-1 require dephosphorylation at this site for activation (47,48), and dephosphorylation of this site in Raf-1 correlates with an increase in its activity (49). In 2B4 cells, B-Raf is phosphorylated at this inhibitory residue in nonstimulated cells but becomes rapidly dephosphorylated within the first 3 min of stimulation, which also coincides with the rapid onset of Erk activation (Fig.…”

Section: Kidins220 Binds To the Tcr In Unstimulated T Cellsmentioning

confidence: 99%

Kidins220/ARMS Associates with B-Raf and the TCR, Promoting Sustained Erk Signaling in T Cells

Deswal

Meyer

Fiala

et al. 2013

The Journal of Immunology

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The activation kinetics of MAPK Erk are critical for T cell development and activation. In particular, sustained Erk signaling is required for T cell activation and effector functions, such as IL-2 production. Although Raf-1 triggers transient Erk activation, B-Raf is implicated in sustained Erk signaling after TCR stimulation. In this study, we show that B-Raf is dephosphorylated on its inhibitory serine 365 upon TCR triggering. However, it is unknown how B-Raf activation is coupled to the TCR. Using mass spectrometry, we identified protein kinase D–interacting substrate of 220 kDa (Kidins220)/ankyrin repeat-rich membrane spanning protein, mammalian target of rapamycin, Rictor, Dock2, and GM130 as novel B-Raf interaction partners. We focused on Kidins220, a protein that has been studied in neuronal cells and found that it associated with the pre-TCR, αβTCR, and γδTCR. Upon prolonged TCR stimulation, the Kidins220–TCR interaction was reduced, as demonstrated by immunoprecipitation and proximity ligation assays. We show that Kidins220 is required for TCR-induced sustained, but not transient, Erk activation. Consequently, induction of the immediate early gene products and transcription factors c-Fos and Erg-1 was blocked, and upregulation of the activation markers CD69, IL-2, and IFN-γ was reduced. Further, Kidins220 was required for optimal calcium signaling. In conclusion, we describe Kidins220 as a novel TCR-interacting protein that couples B-Raf to the TCR. Kidins220 is mandatory for sustained Erk signaling; thus, it is crucial for TCR-mediated T cell activation.

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Section: Kidins220 Binds To the Tcr In Unstimulated T Cellsmentioning

confidence: 99%

Kidins220/ARMS Associates with B-Raf and the TCR, Promoting Sustained Erk Signaling in T Cells

Deswal

Meyer

Fiala

et al. 2013

The Journal of Immunology

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“…Removing the inhibitory signal on Ser-259 is also able to induce high basal activity of the full-length c-Raf kinase. Phosphorylation on Ser-259 creates a 14-3-3-binding site and is thought to maintain c-Raf in an auto-inhibited conformation as well as mediate subcellular localization (27). Mutation of Ser-259 to alanine results in high basal activity that is suppressed by alanine mutations of the activating phosphorylation sites and increased when these sites are substituted by acidic residues.…”

Section: Ras Regulates the Interaction Between The N Terminus And C Tmentioning

confidence: 99%

Regulation of Raf through Phosphorylation and N Terminus-C Terminus Interaction

Chong¹,

Guan

2003

Journal of Biological Chemistry

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The Raf family of serine/threonine kinases is highly conserved in structure. There are three conserved regions (CR) 1 in Raf kinases, CR1, CR2, and CR3 (1). CR1 contains the Ras binding domain (RBD) and the cysteine-rich domain (CRD), and both bind the activated small GTPase Ras (2, 3). CR2 is serine/threonine-rich, and CR3 is the catalytic kinase domain and contains several activating phosphorylation sites. Activated Ras binds to CR1 and recruits Raf to the plasma membrane, which is necessary for Raf activation. Phosphorylation of activating sites and dephosphorylation of inhibitory sites must be coordinated for full Raf activation. Once Raf is activated, it can phosphorylate and activate MEK, which is then able to phosphorylate and activate MAPK/ERK, which phosphorylates cytosolic and nuclear proteins.Raf was first identified as a retroviral oncogene v-Raf, which has a deletion of the 5Ј end of the normal cellular gene encoding CR1 and CR2 (4). This deletion correlates to an increase in transforming activity independent of Ras, which leads to the hypothesis that the N-terminal domain of Raf proteins has a regulatory function (5-9). In defining the minimal region required for cell transformation, deletion of both CR1 and CR2 but an intact kinase domain was required for high transforming activity (6, 9). Deletion of the N-terminal region renders Raf kinase activity independent of Ras.The N-terminal domain serves the following two functions. 1) It binds to the activated form of the small GTPase Ras required for membrane recruitment. 2) It binds and suppresses the C-terminal kinase. Expression of the N terminus of c-Raf can inhibit Ras activation of the MAPK pathway in germinal vesicle breakdown (GVBD) in Xenopus oocytes by binding and sequestering activated Ras from endogenous c-Raf (10). The N-terminal half was also shown to co-immunoprecipitate and inhibit the C-terminal half of Raf. The mechanism of Raf activation may be similar to that of p21-activated kinase (PAK) (11). The Rac and Cdc42, members of the Rho family of small GTPases, directly bind the N terminus of PAK and relieve an auto-inhibited conformation held together by intramolecular interactions. The new conformation allows phosphorylation of activating sites, which are thought to help maintain an open conformation of PAK.Two different mechanisms have given rise to oncogenic forms of Raf kinases. v-Raf has a deletion of the entire 5Ј end of the cellular gene that encodes for the N-terminal regulatory domain. v-Raf has high transforming activity because of a loss of the auto-inhibitory domain. Recently, mutations in B-RAF were identified in 60% of human melanomas and identified B-RAF as an oncogene (12, 13). A common oncogenic mutation of B-RAF has a Val to Glu mutation in the activation loop of the kinase domain and results in high kinase activity. This mutation, V599E, lies between two conserved phosphorylation sites, Thr-598 and Ser-601, in the activation loop of B-Raf, previously identified by our lab (14,15). We have shown that acidic residue sub...

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“…This dimer is proposed to tether the regulatory and catalytic lobes together and thereby stabilize a closed, inactive conformation of Raf-1. 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).…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2006

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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 ...

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Regulation of Raf-1 activation and signalling by dephosphorylation

Cited by 258 publications

References 49 publications

Kidins220/ARMS Associates with B-Raf and the TCR, Promoting Sustained Erk Signaling in T Cells

Kidins220/ARMS Associates with B-Raf and the TCR, Promoting Sustained Erk Signaling in T Cells

Regulation of Raf through Phosphorylation and N Terminus-C Terminus Interaction

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

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