Garnovskaya Maria N, Mukhin Yurii V, Vlasova Tamara M, Grewal Jasjit S, Ullian Michael E, Tholanikunnel Baby G, Raymond John R
Medical and Research Services of the Ralph H. Johnson Veterans Affairs Medical Center and Department of Medicine (Nephrology Division) of the Medical University of South Carolina, Charleston, South Carolina 29425, USA.
J Biol Chem. 2004 Jun 4;279(23):24899-905. doi: 10.1074/jbc.M311622200. Epub 2004 Apr 6.
We examined the relationship between mitogen-activated MEK (mitogen and extracellular signal-regulated protein kinase kinase) and phosphorylation of the gene product encoded by retinoblastoma (hereafter referred to as Rb) in vascular smooth muscle cells. Brief treatment of the cells with 100 nm angiotensin II or 1 microm serotonin resulted in serine phosphorylation of Rb that was equal in magnitude to that induced by treating cells for 20 h with 10% fetal bovine serum ( approximately 3 x basal). There was no detectable rapid phosphorylation of two close cousins of Rb, p107 and p130. Phosphorylation state-specific antisera demonstrated that the rapid phosphorylation occurred on Ser(795), but not on Ser(249), Thr(252), Thr(373), Ser(780), Ser(807), or Ser(811). Phosphorylation of Rb Ser(795) peaked at 10 min, lagging behind phosphorylation of MEK and ERK (extracellular signal-regulated protein kinase). Rb Ser(795) phosphorylation could be blocked by PD98059, a MEK inhibitor, and greatly attenuated by apigenin, an inhibitor of the Ras --> Raf --> MEK --> ERK pathway. The effect also appears to be mediated by CDK4. Immunoprecipitation/immunoblot studies revealed that serotonin and angiotensin II induced complex formation between CDK4, cyclin D1, and phosphorylated ERK. These studies show a rapid, novel, and selective phosphorylation of Rb Ser(795) by mitogens and demonstrate an unexpected rapid linkage between the actions of the Ras --> Raf --> MEK --> ERK pathway and the phosphorylation state of Rb.
我们研究了丝裂原活化的MEK（丝裂原和细胞外信号调节蛋白激酶激酶）与血管平滑肌细胞中视网膜母细胞瘤基因产物（以下简称Rb）磷酸化之间的关系。用100 nM血管紧张素II或1 μM血清素对细胞进行短暂处理，可导致Rb的丝氨酸磷酸化，其程度与用10%胎牛血清处理细胞20小时所诱导的磷酸化程度相当（约为基础水平的3倍）。未检测到Rb的两个近亲p107和p130有快速磷酸化现象。磷酸化状态特异性抗血清表明，快速磷酸化发生在Ser(795)，而非Ser(249)、Thr(252)、Thr(373)、Ser(780)、Ser(807)或Ser(811)。Rb Ser(795)的磷酸化在10分钟时达到峰值，滞后于MEK和细胞外信号调节蛋白激酶（ERK）的磷酸化。Rb Ser(795)的磷酸化可被MEK抑制剂PD98059阻断，并被Ras→Raf→MEK→ERK途径的抑制剂芹菜素大大减弱。这种作用似乎也由细胞周期蛋白依赖性激酶4（CDK4）介导。免疫沉淀/免疫印迹研究表明，血清素和血管紧张素II诱导了CDK4、细胞周期蛋白D1和磷酸化ERK之间的复合物形成。这些研究显示了丝裂原对Rb Ser(795)的快速、新颖且选择性的磷酸化，并证明了Ras→Raf→MEK→ERK途径的作用与Rb的磷酸化状态之间存在意想不到的快速联系。
