RAF activation

Mammals have three RAF isoforms, A, B and C, that are activated downstream of RAS and stimulate the MAPK pathway. Although CRAF (also known as RAF-1) was the first identified and remains perhaps the best studied, BRAF is most similar to the RAF expressed in other organisms. Notably, MAPK (ERK) activation is more compromised in BRAF-deficient cells than in CRAF or ARAF deficient cells (Bonner et al, 1985; Mikula et al, 2001, Huser et al, 2001, Mercer et al, 2002; reviewed in Leicht et al, 2007; Matallanas et al, 2011; Cseh et al, 2014). Consistent with its important role in MAPK pathway activation, mutations in the BRAF gene, but not in those for A- or CRAF, are associated with cancer development (Davies et al, 2002; reviewed in Leicht et al, 2007). ARAF and CRAF may have arisen through gene duplication events, and may play additional roles in MAPK-independent signaling (Hindley and Kolch, 2002; Murakami and Morrison, 2001).<br>Despite divergences in function, all mammalian RAF proteins share three conserved regions (CRs) and each interacts with RAS and MEK proteins, although with different affinities. The N-terminal CR1 contains a RAS-binding domain (RBD) and a cysteine-rich domain (CRD) that mediate interactions with RAS and the phospholipid membrane. CR2 contains inhibitory phosphorylation sites that impact RAS binding and RAF activation, while the C-terminal CR3 contains the bi-lobed kinase domain with its activation loop, and an adjacent upstream "N-terminal acidic motif" -S(S/G)YY in C- and A-RAF,respectively, and SSDD in B-RAF - that is required for RAF activation (Tran et al, 2005; Dhillon et al, 2002; Chong et al, 2001; Cutler et al, 1998; Chong et al, 2003; reviewed in Matallanas et al, 2011).<br><br>Regulation of RAF activity involves multiple phosphorylation and dephosphorylation events, intramolecular conformational changes, homo- and heterodimerization between RAF monomers and changes to protein binding partners, including scaffolding proteins which bring pathway members together (reviewed in Matallanas et al, 2011; Cseh et al, 2014). The details of this regulation are not completely known and differ slightly from one RAF isoform to another. Briefly, in the inactive state, RAF phosphorylation on conserved serine residues in CR2 promote an interaction with 14-3-3 dimers, maintaining the kinase in a closed conformation. Upon RAS activation, these sites are dephosphorylated, allowing the RAF CRD and RBD to bind RAS and phospholipids, facilitating membrane recruitment. RAF activation requires homo- or heterodimerization, which promotes autophosphorylation in the activation loop of the receiving monomer. Of the three isoforms, only BRAF is able to initiate this allosteric activation of other RAF monomers (Hu et al, 2013; Heidorn et al, 2010; Garnett et al, 2005). This activity depends on negative charge in the N-terminal acidic region (NtA; S(S/G)YY or SSDD) adjacent to the kinase domain. In BRAF, this region carries permanent negative charge due to the presence of the two aspartate residues in place of the tyrosine residues of A- and CRAF. In addition, unique to BRAF, one of the serine residues of the NtA is constitutively phosphorylated. In A- and CRAF, residues in this region are subject to phosphorylation by activated MEK downstream of RAF activation, establishing a positive feedback loop and allowing activated A- and CRAF monomers to act as transactivators in turn (Hu et al, 2013; reviewed in Cseh et al, 2014). RAF signaling is terminated through dephosphorylation of the NtA region and phosphorylation of the residues that mediate the inhibitory interaction with 14-3-3, promoting a return to the inactive state (reviewed in Matallanas et al, 2011; Cseh et al, 2014).<br>

哺乳动物具有三种RAS激活因子（RAF）同源异构体，分别为A、B和C型，它们位于RAS下游并被激活，从而刺激MAPK途径。尽管CRAF（亦称RAF-1）是最先被鉴定且可能是研究最为深入的，但BRAF与其它生物体中表达的RAF最为相似。值得注意的是，在BRAF缺陷细胞中，MAPK（ERK）的激活受损程度大于CRAF或ARAF缺陷细胞（Bonner等人，1985；Mikula等人，2001，Huser等人，2001，Mercer等人，2002；参见Leicht等人，2007；Matallanas等人，2011；Cseh等人，2014的综述）。与BRAF在MAPK途径激活中的重要作用相一致，BRAF基因的突变与癌症的发生有关，而A-或CRAF基因的突变则不然（Davies等人，2002；参见Leicht等人，2007的综述）。ARAF和CRAF可能通过基因复制事件产生，并在MAPK非依赖性信号传导中发挥额外的作用（Hindley和Kolch，2002；Murakami和Morrison，2001）。尽管在功能上存在差异，所有哺乳动物的RAF蛋白均共享三个保守区域（CRs），并分别与RAS和MEK蛋白相互作用，尽管亲和力不同。N端保守区域CR1包含一个RAS结合域（RBD）和一个富含半胱氨酸的域（CRD），它们介导与RAS和磷脂膜的相互作用。CR2包含抑制性磷酸化位点，影响RAS结合和RAF激活，而C端保守区域CR3包含双叶状激酶域及其激活环，以及相邻的“上游N端酸性基序”——在C-和A-RAF中分别为-S(S/G)YY，在B-RAF中为SSDD——这是RAF激活所必需的（Tran等人，2005；Dhillon等人，2002；Chong等人，2001；Cutler等人，1998；Chong等人，2003；参见Matallanas等人，2011的综述）。

RAF活性的调控涉及多个磷酸化和去磷酸化事件、分子内构象变化、RAF单体之间的同源二聚化和异源二聚化，以及蛋白质结合伙伴的变化，包括将途径成员聚集在一起的支架蛋白（参见Matallanas等人，2011；Cseh等人，2014的综述）。这种调控的细节尚不完全清楚，并且在不同RAF同源异构体之间略有差异。简而言之，在非活性状态下，CR2中保守丝氨酸残基的磷酸化促进与14-3-3二聚体的相互作用，维持激酶的闭合构象。在RAS激活后，这些位点去磷酸化，使得RAF CRD和RBD能够结合RAS和磷脂，从而促进膜募集。RAF的激活需要同源或异源二聚化，这促进了接受单体激活环中的自磷酸化。在三种同源异构体中，只有BRAF能够启动这种变构激活其它RAF单体的过程（Hu等人，2013；Heidorn等人，2010；Garnett等人，2005）。这种活性依赖于邻近激酶域的N端酸性区域（NtA；S(S/G)YY或SSDD）中的负电荷。在BRAF中，由于存在取代酪氨酸残基的天冬氨酸残基，这一区域携带永久性的负电荷。此外，对于BRAF来说，NtA中的一个丝氨酸残基是组成性磷酸化的。在A-和CRAF中，这一区域的残基受到激活的MEK在RAF激活后的磷酸化，从而建立正反馈回路，使得激活的A-和CRAF单体依次作为转录激活剂（Hu等人，2013；参见Cseh等人，2014的综述）。RAF信号传导通过去磷酸化NtA区域和磷酸化介导与14-3-3抑制性相互作用的残基来终止，促进回到非活性状态（参见Matallanas等人，2011；Cseh等人，2014的综述）。