Mitogen-induced rapid phosphorylation of serine 795 of the retinoblastoma gene product in vascular smooth muscle cells involves ERK activation
Mitogen-induced rapid phosphorylation of serine 795 of the retinoblastoma gene product in vascular smooth muscle cells involves ERK activation
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DOI：
10.1074/jbc.m311622200
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发表时间：
2004-06-04
影响因子：
4.8
通讯作者：
Raymond, JR
中科院分区：
文献类型：
--
作者：
Garnovskaya, MN;Mukhin, YV;Raymond, JR
我们研究了有丝分裂原激活的MEK（有丝分裂原和细胞外信号调节蛋白激酶激酶）和血管平滑肌细胞中视网膜母细胞瘤（以下简称Rb）编码的基因产物磷酸化之间的关系。用100 nM血管紧张素II或1 μ M 5-羟色胺短暂处理细胞导致Rb的丝氨酸磷酸化，其幅度与用10%胎牛血清处理细胞20 h诱导的幅度相等（约为基础值的3倍）。没有检测到Rb的两个近亲p107和p130的快速磷酸化。磷酸化状态特异性抗血清表明，快速磷酸化发生在Ser（795）上，而不是Ser（249）、Thr（252）、Thr（373）、Ser（780）、Ser（807）或Ser（811）上。Rb Ser（795）的磷酸化在10分钟达到峰值，落后于MEK和ERK（细胞外信号调节蛋白激酶）的磷酸化。Rb Ser（795）磷酸化可被MEK抑制剂PD 98059阻断，并被Ras -> Raf -> MEK -> ERK通路抑制剂芹菜素显著减弱。该作用似乎也由CDK 4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CDK 4，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Rb Ser（795）被有丝分裂原快速、新颖和选择性磷酸化，并证明了Ras -> Raf -> MEK -> ERK途径的作用与Rb的磷酸化状态之间的意想不到的快速联系。
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 muM 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 (approximate to3 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.