Proteomic screen defines the Polo-box domain interactome and identifies Rock2 as a Plk1 substrate
Proteomic screen defines the Polo-box domain interactome and identifies Rock2 as a Plk1 substrate
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DOI：
10.1038/sj.emboj.7601683
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发表时间：
2007-05-02
期刊：
影响因子：
11.4
通讯作者：
Yaffe, Michael B.
中科院分区：
文献类型：
--
作者：
Lowery, Drew M.;Clauser, Karl R.;Yaffe, Michael B.
Polo样激酶-1（Plk 1）磷酸化许多有丝分裂底物，但Plk 1依赖过程的多样性表明存在其他靶点。Plk 1含有一个专门的磷酸丝氨酸-苏氨酸结合结构域，即波罗盒结构域（PBD），推测其将激酶靶向其底物。使用Plk 1的专用PBD作为亲和捕获剂，我们通过质谱法进行筛选以定义有丝分裂Plk 1-PBD相互作用组。我们鉴定了622种蛋白质，这些蛋白质表现出磷酸化依赖的有丝分裂特异性相互作用，包括参与完善的Plk 1调控过程的蛋白质，以及先前与Plk 1无关的过程，如翻译控制，RNA加工和囊泡运输。在我们的屏幕中确定的许多蛋白质在胞质分裂中发挥重要作用，在哺乳动物细胞中，Plk 1的详细机制作用仍然不清楚。我们继续描述Plk 1-PBD与胞质分裂效应激酶Rhoassociated coiled-coil domain-containing protein kinase 2（Rock 2）的有丝分裂特异性相互作用，证明Rock 2是Plk 1底物，并表明Rock 2在胞质分裂期间与Plk 1共定位。最后，我们表明，Plk 1和RhoA一起发挥作用，最大限度地提高Rock 2激酶活性在体外和细胞内，并牵连Plk 1作为一个中央调节器的多个途径，协同收敛，以调节肌动球蛋白环收缩在卵裂沟侵入。
Polo-like kinase-1 (Plk1) phosphorylates a number of mitotic substrates, but the diversity of Plk1-dependent processes suggests the existence of additional targets. Plk1 contains a specialized phosphoserine-threonine binding domain, the Polo- box domain (PBD), postulated to target the kinase to its substrates. Using the specialized PBD of Plk1 as an affinity capture agent, we performed a screen to define the mitotic Plk1-PBD interactome by mass spectrometry. We identified 622 proteins that showed phosphorylation-dependent mitosis-specific interactions, including proteins involved in well-established Plk1-regulated processes, and in processes not previously linked to Plk1 such as translational control, RNA processing, and vesicle transport. Many proteins identified in our screen play important roles in cytokinesis, where, in mammalian cells, the detailed mechanistic role of Plk1 remains poorly defined. We go on to characterize the mitosis- specific interaction of the Plk1-PBD with the cytokinesis effector kinase Rhoassociated coiled-coil domain-containing protein kinase 2 ( Rock2), demonstrate that Rock2 is a Plk1 substrate, and show that Rock2 colocalizes with Plk1 during cytokinesis. Finally, we show that Plk1 and RhoA function together to maximally enhance Rock2 kinase activity in vitro and within cells, and implicate Plk1 as a central regulator of multiple pathways that synergistically converge to regulate actomyosin ring contraction during cleavage furrow ingression.