241 human active and 13 inactive phosphatases in total;
194 phosphatases have substrate data;
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336 protein substrates;
83 non-protein substrates;
1215 dephosphorylation interactions;
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299 KEGG pathways;
876 Reactome pathways;
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last scientific update: 11 Mar, 2019
last maintenance update: 01 Sep, 2023
Location-regulated scaffold connecting MEK to RAF Hasvery low protein kinase activity and can phosphorylate MAP2K1 atseveral Ser and Thr residues with very low efficiency (in vitro)Interaction with BRAF enhances KSR2-mediated phosphorylation ofMAP2K1 (in vitro) Blocks MAP3K8 kinase activity and MAP3K8-mediated signaling Acts as a negative regulator of MAP3K3-mediated activation of ERK, JNK and NF-kappa-B pathways,inhibiting MAP3K3-mediated interleukin-8 production
The Ras proteins are GTPases that function as molecular switches for signaling pathways regulating cell proliferation, survival, growth, migration, differentiation or cytoskeletal dynamism. Ras proteins transduce signals from extracellular growth factors by cycling between inactive GDP-bound and active GTP-bound states. The exchange of GTP for GDP on RAS is regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Activated RAS (RAS-GTP) regulates multiple cellular functions through effectors including Raf, phosphatidylinositol 3-kinase (PI3K) and Ral guanine nucleotide-dissociation stimulator (RALGDS).
Activated RAF proteins are restricted substrate kinases whose primary downstream targets are the two MAP2K proteins, MAPK2K1 and MAP2K2 (also known as MEK1 and MEK2) (reviewed in Roskoski, 2010, Roskoski, 2012a). Phosphorylation of the MAPK2K activation loop primes them to phosphorylate the primary effector of the activated MAPK pathway, the two MAPK proteins MAPK3 and MAPK1 (also known as ERK1 and 2). Unlike their upstream counterparts, MAPK3 and MAPK1 catalyze the phosphorylation of hundreds of cytoplasmic and nuclear targets including transcription factors and regulatory molecules (reviewed in Roskoski, 2012b). Activation of MAP2K and MAPK proteins downstream of activated RAF generally occurs in the context of a higher order scaffolding complex that regulates the specificity and localization of the pathway (reviewed in Brown and Sacks, 2009; Matallanas et al, 2011)
In addition to the highly prevalent and activating V600E BRAF mutations, numerous moderately activating and less common mutations have also been identified in human cancers (Forbes et al, 2015)
BRAF is mutated in about 8% of human cancers, with high prevalence in hairy cell leukemia, melanoma, papillary thyroid and ovarian carcinomas, colorectal cancer and a variety of other tumors (Davies et al, 2002; reviewed in Samatar and Poulikakos, 2014). Most BRAF mutations fall in the activation loop region of the kinase or the adjacent glycine rich region. These mutations promote increased kinase activity either by mimicking the effects of activation loop phosphorylations or by promoting the active conformation of the enzyme (Davies et al, 2002; Wan et al, 2004). Roughly 90% of BRAF mutants are represented by the single missense mutation BRAF V600E (Davies et al, 2002; Wan et al, 2004). Other highly active kinase mutants of BRAF include BRAF G469A and BRAF T599dup. G469 is in the glycine rich region of the kinase domain which plays a role in orienting ATP for catalysis, while T599 is one of the two conserved regulatory phosphorylation sites of the activation loop. Each of these mutants has highly enhanced basal kinase activities, phosphorylates MEK and ERK in vitro and in vivo and is transforming when expressed in vivo (Davies et al, 2002; Wan et al, 2004; Eisenhardt et al, 2011). Further functional characterization shows that these highly active mutants are largely resistant to disruption of the BRAF dimer interface, suggesting that they are able to act as monomers (Roring et al, 2012; Brummer et al, 2006; Freeman et al, 2013; Garnett et al, 2005). Activating BRAF mutations occur for the most part independently of RAS activating mutations, and RAS activity levels are generally low in BRAF mutant cells. Moreover, the kinase activity of these mutants is only slightly elevated by coexpression of G12V KRAS, and biological activity of the highly active BRAF mutants is independent of RAS binding (Brummer et al, 2006; Wan et al, 2004; Davies et al, 2002; Garnett et al, 2005). Although BRAF V600E is inhibited by RAF inhibitors such as vemurafenib, resistance frequently develops, in some cases mediated by the expression of a splice variant that lacks the RAS binding domain and shows elevated dimerization compared to the full length V600E mutant (Poulikakos et al, 2011; reviewed in Lito et al, 2013)
Members of the RAS gene family were the first oncogenes to be identified, and mutations in RAS are present in ~20-30% of human cancers (reviewed in Prior et al, 2012). Mutations in the KRAS gene are the most prevalent, and are found with high frequency in colorectal cancer, non-small cell lung cancer and pancreatic cancer, among others. The reasons for the lower prevalence of HRAS and NRAS mutations in human cancers are not fully understood, but may reflect gene-specific functions as well as differential codon usage and spatio-temporal regulation (reviewed in Prior et al, 2012; Stephen et al, 2014; Pylayeva-Gupta et al, 2011). Activating RAS mutations contribute to cellular proliferation, transformation and survival by activating the MAPK signaling pathway, the AKT pathway and the RAL GDS pathway, among others (reviewed in Stephen et al, 2014; Pylayeva-Gupta et al, 2011).Although the frequency and distribution varies between RAS genes and cancer types, the vast majority of activating RAS mutations occur at one of three residues - G12, G13 and Q61. Mutations at these sites favour the RAS:GTP bound form and yield constitutively active versions of the protein (reviewed in Prior et al, 2012)
In addition to the more prevalent point mutations, BRAF and RAF1 are also subject to activation as a result of translocation events that yield truncated or fusion products (Jones et al, 2008; Cin et al, 2011; Palanisamy et al, 2010; Ciampi et al, 2005; Stransky et al, 2014; Hutchinson et al, 2013; Zhang et al, 2013; Lee et al, 2012; Ricarte-Filho et al, 2013; reviewed in Lavoie and Therrien et al, 2015). In general these events put the C-terminal kinase domain of BRAF or RAF1 downstream of an N-terminal sequence provided by a partner protein. This removes the N-terminal region of the RAF protein, relieving the autoinhibition imposed by this region of the protein. In addition, some but not all of the fusion partner proteins have been shown to contain coiled-coil or other dimerization domains. Taken together, the fusion proteins are thought to dimerize constitutively and activate downstream signaling (Jones et al, 2008; Lee et al, 2012; Hutchinson et al, 2013; Ciampi et al, 2005; Cin et al, 2011; Stransky et al, 2014)
While BRAF-specific inhibitors inhibit MAPK/ERK activation in the presence of the BRAF V600E mutant, paradoxical activation of ERK signaling has been observed after treatment of cells with inhibitor in the presence of WT BRAF (Wan et al, 2004; Garnett et al, 2005; Heidorn et al, 2010; Hazivassiliou et al, 2010; Poulikakos et al, 2010). This paradoxical ERK activation is also seen in cells expressing kinase-dead or impaired versions of BRAF such as D594V, which occur with low frequency in some cancers (Wan et al, 2004; Heidorn et al, 2010). Unlike BRAF V600E, which occurs exclusively of activating RAS mutations, kinase-impaired versions of BRAF are coincident with RAS mutations in human cancers, and indeed, paradoxical activation of ERK signaling in the presence of inactive BRAF is enhanced in the presence of oncogenic RAS (Heidorn et al, 2010; reviewed in Holderfield et al, 2014). Although the details remain to be worked out, paradoxical ERK activation in the presence of inactive BRAF appears to rely on enhanced dimerization with and transactivation of CRAF (Heidorn et al, 2010; Hazivassiliou et al, 2010; Poulikakos et al, 2010; Roring et al, 2012; Rajakulendran et al, 2009; Holderfield et al, 2013; Freeman et al, 2013; reviewed in Roskoski, 2010; Samatar and Poulikakos, 2014; Lavoie and Therrien, 2015)