Ferrell J E, Bhatt R R
Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, California 94305-5332, USA.
J Biol Chem. 1997 Jul 25;272(30):19008-16. doi: 10.1074/jbc.272.30.19008.
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.
先前在非洲爪蟾卵母细胞提取物系统中对丝裂原活化蛋白（MAP）激酶级联组分反应的研究表明，p42 MAP激酶（MAPK）呈现出一条尖锐的、S形的刺激/反应曲线，而非更为典型的双曲线。对此行为一种合理的解释需要假设MAP激酶激酶（MAPKK）通过一种分布机制对p42 MAPK进行双重磷酸化，即MAPKK在第一次和第二次磷酸化之间与MAPK解离，而非持续性机制，即MAPKK在解离前进行两次磷酸化。在此，我们研究了组成型活性形式的人MAPKK-1（称为MAPKK-1 R4F或MAPKK-1*）在体外磷酸化非洲爪蟾p42 MAPK的机制。我们发现，磷酸化反应过程中形成的单磷酸化MAPK的量超过了存在的MAPKK-1的量，如果磷酸化仅通过持续性机制发生，这是不可能的。单磷酸化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（但并非总是）是酪氨酸磷酸化。我们还发现，脉冲标记的单磷酸化MAPK变成双磷酸化的速率取决于MAPKK-1的浓度，这种行为是分布模型所预测的，但与持续性模型不相符。这些发现表明，MAPKK-1*通过双碰撞、分布机制而非单碰撞、持续性机制磷酸化p42 MAPK，并为理解MAP激酶如何将分级输入转化为开关式输出提供了一个机制基础。
