Mechanistic studies of the dual phosphorylation of mitogen-activated protein kinase
Mechanistic studies of the dual phosphorylation of mitogen-activated protein kinase
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DOI：
10.1074/jbc.272.30.19008
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发表时间：
1997-07-25
影响因子：
4.8
通讯作者：
Bhatt, RR
中科院分区：
文献类型：
--
作者：
Ferrell, JE;Bhatt, RR
以前关于非洲爪哇卵母细胞提取系统中有丝分裂原激活蛋白(MAP)激酶级联组分的反应的工作表明，P42 MAP激酶(MAPK)呈现出一条陡峭的S型刺激/反应曲线，而不是更典型的双曲线。对这种行为的一个合理的解释需要假设MAPK(MAPKK)通过一种分配机制实现其对P42 MAPK的双重磷酸化，其中MAPKK在第一次和第二次磷酸化之间从MAPK解离，而不是一种过程机制，其中MAPKK在解离之前执行两种磷酸化。本文研究了一种活性形式的人MAPKK-1(记为MAPKK-1 R4F或MAPKK-1*)在体外磷酸化非洲爪哇P42 MAPK的机制。我们发现，在磷酸化反应中形成的单磷酸化MAPK的量超过了MAPKK-1*的量，这是不可能的。如果磷酸化完全以过程机制发生，单磷酸化的MAPK主要在酪氨酸上被磷酸化，但一小部分在苏氨酸上被磷酸化，这表明第一次磷酸化通常是，但不是一成不变的，酪氨酸磷酸化。我们还发现，标记单磷酸化的MAPK变成双磷酸化的脉冲速率随着MAPKK-1的浓度的增加而不同。这些发现表明，MAPKK-1*通过两次碰撞的分配机制而不是单碰撞的过程机制来磷酸化p42 MAPK，并为理解MAP激酶如何将分级输入转换为开关样输出提供了机制基础。
Previous work on the responses of mitogen-activated protein (MAP) kinase cascade components in a Xenopus oocyte extract system demonstrated that p42 MAP kinase (MAPK) exhibits a sharp, sigmoidal stimulus/response curve, rather than a more typical hyperbolic curve, One plausible explanation for this behavior requires the assumption that MAP kinase kinase (MAPKK) carries out its dual phosphorylation of p42 MAPK by a distributive mechanism, where MAPKK dissociates from MAPK between the first and second phosphorylations, rather than a processive mechanism, where MAPKK carries out both phosphorylations before dissociating, Here we have investigated the mechanism through which a constitutively active form of human MAPKK-1 (denoted MAPKK-1 R4F or MAPKK-1*) phosphorylates Xenopus p42 MAPK in vitro, We found that the amount of monophosphorylated MAPK formed during the phosphorylation reaction exceeded the amount of MAPKK-1* present, which would not be possible if the phosphorylation occurred exclusively by a processive mechanism, The monophosphorylated MAPK was phosphorylated predominantly on tyrosine, but a small proportion was phosphorylated on threonine, indicating that the first phosphorylation is usually, but not invariably, the tyrosine phosphorylation, We also found that the rate at which pulse labeled monophosphorylated MAPK became bisphosphorylated depended on the MAPKK-1* concentration, behavior that is predicted by the distributive model but incompatible with the processive model, These findings indicate that MAPKK-1* phosphorylates p42 MAPK by a two-collision, distributive mechanism rather than a single-collision, processive mechanism, and provide a mechanistic basis for understanding how MAP kinase can convert graded inputs into switch-like outputs.