Noncatalytic Function of ERK1/2 Can Promote Raf/MEK/ERK-mediated Growth Arrest Signaling
Noncatalytic Function of ERK1/2 Can Promote Raf/MEK/ERK-mediated Growth Arrest Signaling
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DOI：
10.1074/jbc.m109.012591
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发表时间：
2009-11-27
影响因子：
4.8
通讯作者：
Park, Jong-In
中科院分区：
文献类型：
--
作者：
Hong, Seung-Keun;Yoon, Seunghee;Park, Jong-In
激酶活性被认为是MAPK的关键生化特性。在这里，我们报告说，ERK 1/2也利用其非催化功能介导某些信号转导。Raf/MEK/ERK通路的持续激活诱导生长停滞，伴随着细胞周期调节因子的变化（视网膜母细胞瘤磷酸化降低、E2 F1下调和/或p21（CIP 1）上调）以及人肿瘤细胞系LNCaP、U251和TT中c-Myc或RET的形态学和表达的细胞类型特异性变化。通过RNA干扰去除ERK 1/2消除了所有这些作用。然而，活性位点失活的ERK突变体（ERK 1-K71 R，ERK 2-K52 R和ERK 2-D147 A），竞争性抑制内源性ERK 1/2的激活，不能阻断Raf/MEK诱导的生长停滞以及细胞周期调节因子的变化，尽管它们有效地阻断了ERK 1/2催化活性读数，p90（RSK）和ELK 1的磷酸化以及细胞类型特异性变化。因为这表明了潜在的非催化性ERK 1/2功能，我们产生了稳定的肿瘤细胞系，其中ERK 1和ERK 2都被显著敲低，我们进一步研究了使用大鼠来源的激酶缺陷型ERK突变体（ERK 2-K52 R和ERK 2-T183 A/Y185 F）的可能性，这些突变体不被人小发夹RNA靶向。事实上，ERK 2-K52 R选择性地恢复ERK 1/2缺失细胞中Raf-induced生长抑制信号传导，如通过恢复细胞经历生长停滞和控制细胞周期调节剂而不影响c-Myc和形态的能力所证明的。然而，ERK 2-T183 A/Y185 F的效果较差，表明需要TEY位点磷酸化。我们的研究表明，ERK 1/2的功能以外的“经典”激酶活性也参与了途径介导的生长停滞信号。
Kinase activity is known as the key biochemical property of MAPKs. Here, we report that ERK1/2 also utilizes its noncatalytic function to mediate certain signal transductions. Sustained activation of the Raf/MEK/ERK pathway induces growth arrest, accompanied by changes in cell cycle regulators (decreased retinoblastoma phosphorylation, E2F1 down-regulation, and/or p21(CIP1) up-regulation) and cell type-specific changes in morphology and expression of c-Myc or RET in the human tumor lines LNCaP, U251, and TT. Ablation of ERK1/2 by RNA interference abrogated all these effects. However, active site-disabled ERK mutants (ERK1-K71R, ERK2-K52R, and ERK2-D147A), which competitively inhibit activation of endogenous ERK1/2, could not block Raf/MEK-induced growth arrest as well as changes in the cell cycle regulators, although they effectively blocked phosphorylation of the ERK1/2 catalytic activity readouts, p90(RSK) and ELK1, as well as the cell type-specific changes. Because this indicated a potential noncatalytic ERK1/2 function, we generated stable lines of the tumor cells in which both ERK1 and ERK2 were significantly knocked down, and we further investigated the possibility using rat-derived kinase-deficient ERK mutants (ERK2-K52R and ERK2-T183A/Y185F) that were not targeted by human small hairpin RNA. Indeed, ERK2-K52R selectively restored Raf-induced growth inhibitory signaling in ERK1/2-depleted cells, as manifested by regained cellular ability to undergo growth arrest and to control the cell cycle regulators without affecting c-Myc and morphology. However, ERK2-T183A/Y185F was less effective, indicating the requirement of TEY site phosphorylation. Our study suggests that functions of ERK1/2 other than its "canonical" kinase activity are also involved in the pathway-mediated growth arrest signaling.