A dimeric 14-3-3 protein is an essential cofactor for Raf kinase activity
A dimeric 14-3-3 protein is an essential cofactor for Raf kinase activity
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DOI：
10.1038/27938
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发表时间：
1998-07-02
期刊：
影响因子：
64.8
通讯作者：
Avruch, J
中科院分区：
文献类型：
--
作者：
Tzivion, G;Luo, ZJ;Avruch, J
cRaf-1是一种促分裂原活化蛋白激酶，是GTP结合Ras募集的主要效应子，以激活MAP激酶途径（1）。无活性Raf在细胞溶质中与Hsp 90、Hsp 50（Cdc 37）（2，3）和14-3-3蛋白（4）形成复合物。GTP结合的Ras结合Raf，并且对于响应血清、表皮生长因子、血小板衍生生长因子或胰岛素而发生的Raf的稳定活化是必需的，但不是充分的（5-8）。这些试剂导致丝氨酸/苏氨酸残基上Raf的总体磷酸化增加2 - 3倍（8，9），并且用蛋白质（丝氨酸/苏氨酸）磷酸酶处理cRaf-1可以使其失活，至少部分失活（10）。14-3-3蛋白在Raf激酶活性调节中的作用尚不确定（4，11），本文对此进行了研究。活性Raf可以在体外通过使用合成磷酸肽置换14-3-3而几乎完全失活。通过加入纯化的重组细菌14-3-3可以基本上逆转失活;然而，Raf必须预先在体内被激活，才能在体外被14-3-3重新激活。14-3-3支持Raf活性的能力取决于Raf上丝氨酸残基的磷酸化和14-3-3二聚体的完整性; 14-3-3的突变单体形式虽然能够在体内结合Raf，但不能使Raf在体内活化或在体外置换14-3-3后恢复Raf活性。14-3-3蛋白不需要诱导Raf的二聚化，我们提出，二聚体14-3-3是需要的，以维持Raf在无GTP结合的Ras的情况下处于非活性状态，并稳定在体内活化过程中产生的Raf的活性构象。
cRaf-1 is a mitogen-activated protein kinase that is the main effector recruited by GTP-bound Ras in order to activate the MAP kinase pathway(1). Inactive Raf is found in the cytosol in a complex with Hsp90, Hsp50 (Cdc37)(2,3) and the 14-3-3 proteins(4). GTP-bound Ras binds Raf and is necessary but not sufficient for the stable activation of Raf that occurs in response to serum, epidermal growth factor, platelet-derived growth factor or insulin(5-8). These agents cause a two- to threefold increase in overall phosphorylation of Raf on serine/threonine residues(8,9), and treatment of cRaf-1 with protein (serine/threonine) phosphatases can deactivate it, at least partially(10). The role of 14-3-3 proteins in the regulation of Raf's kinase activity is uncertain(4,11) and is investigated here. Active Raf can be almost completely deactivated in vitro by displacement of 14-3-3 using synthetic phosphopeptides. Deactivation can be substantially reversed by addition of purified recombinant bacterial 14-3-3; however, Raf must have been previously activated in vivo to be reactivated by 14-3-3 in vitro. The ability of 14-3-3 to support Raf activity is dependent on phosphorylation of serine residues on Raf and on the integrity of the 14-3-3 dimer; mutant monomeric forms of 14-3-3, although able to bind Raf in vivo, do not enable Raf to be activated in vivo or restore Raf activity after displacement of 14-3-3 in vitro. The 14-3-3 protein is not required to induce dimerization of Raf, We propose that dimeric 14-3-3 is needed both to maintain Raf in an inactive state in the absence of GTP-bound Ras and to stabilize an active conformation of Raf produced during activation in vivo.