LRRK1 regulates spindle orientation by phosphorylating CDK5RAP2.
LRRK1 regulates spindle orientation by phosphorylating CDK5RAP2.
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LRRK1 通过磷酸化 CDK5RAP2 来调节纺锤体方向。
DOI：
10.1080/15384101.2015.1093446
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发表时间：
2015
期刊：
影响因子：
4.3
通讯作者：
K
中科院分区：
文献类型：
--
作者：
Hanafusa H;Matsumoto;K
有丝分裂纺锤体的精确定向决定了正确的细胞分裂轴，对组织发育和体内平衡至关重要。已知纺锤体错误取向是某些哺乳动物疾病的基础，例如肿瘤发生和多囊肾病。Polo样激酶1（PLK 1）和亮氨酸重复序列激酶1（LRRK 1）是最近通过基于RNAi的筛选被鉴定为纺锤体定向所需的候选激酶。1 PLK 1是一种有丝分裂激酶，调节不同的有丝分裂事件，包括中心体成熟。然而，它在主轴方向的确切作用还没有得到很好的理解。LRRK 1与家族性帕金森病基因产物Park 8/LRRK 2相关，并且含有复合蛋白（ROC）的Ras结构域和MAPKKK样激酶结构域。我们以前曾报道，LRRK 1参与表皮生长因子（EGF）受体的细胞间运输。2然而，LRRK 1在有丝分裂中的作用仍然未知。最近，我们表征了PLK 1和LRRK 1在纺锤体取向中的作用。我们发现PLK 1通过Polo-box结构域与LRRK 1相互作用，并在Ser-1790磷酸化LRRK 1，这是细胞周期蛋白依赖性激酶1（CDK 1）介导的中心体LRRK 1激活所必需的事件。CDK 1在Thr-1400处磷酸化LRRK 1，其位于LRRK 1的活化环中，从而开启LRRK 1激酶活性。因此，LRRK 1通过2种有丝分裂激酶PLK 1和CDK 1的顺序磷酸化而被激活。重要的是，这种LRRK 1的PLK 1依赖性激活是有丝分裂纺锤体方向调节所必需的。我们发现，LRRK 1调节纺锤体的方向依赖于其激酶活性的方式。此外，由PLK 1失活引起的纺锤体错误取向被磷酸化模拟突变体LRRK 1（S1790 D）的表达所拯救，但不能被磷酸化的突变体LRRK 1（S1790 A）拯救，这表明LRRK 1 Ser-1790的PLK 1磷酸化调节有丝分裂纺锤体的取向。因此，LRRK 1调节PLK 1下游的纺锤体取向。有丝分裂纺锤体的正确定向依赖于星形微管（MT）与细胞皮层的相互作用，有丝分裂中心体的MT成核活性是星形MT形成所必需的。g-微管蛋白环复合物（gTuRC）是一种含有g-微管蛋白的大的多蛋白复合物，已知其对于MT成核是必需的。原则上，中心体的MT成核可以在一个或多个步骤中调节，包括gTuRC的组装、其在中心体的定位以及其随后的成核活性。4由于LRRK 1缺失导致有丝分裂中心体的MT成核减少，我们检查了LRRK 1在这些步骤中的作用。作为LRRK 1的一个结合伴侣，我们鉴定了中心体蛋白CDK 5 RAP 2，一种果蝇中心体蛋白（Cnn）的人类同源物。CDK 5 RAP 2是中心体MT成核活性所必需的，并且在其2个结构域CM 1和CM 2中与果蝇Cnn具有同源性：CM 1参与与g-微管蛋白的缔合，而CM 2对中心体定位很重要。有趣的是，LRRK 1与CDK 5 RAP 2的N-末端区域（包括CM 1结构域）结合，并在靠近CM 1结构域的Ser-140处磷酸化CDK 5 RAP 2。由于CDK 5 RAP 2的CM 1结构域直接与gTuRC结合，因此认为CDK 5 RAP 2可促进gTuRC募集至有丝分裂中心体。然而，尽管LRRK 1的敲低导致CDK 5 RAP 2的CM 1结构域与g-微管蛋白之间的相互作用减少，但我们发现它不影响g-微管蛋白。
Precise orientation of the mitotic spindle determines the correct cell division axis and is essential for tissue development and homeostasis. It is known that spindle misorientation underlies some mammalian diseases, such as tumourigenesis and polycystic kidney disease. Two kinases, Polo-like kinase 1 (PLK1) and leucinerich repeat kinase 1 (LRRK1), were recently identified as candidate kinases required for spindle orientation by an RNAi-based screen. 1 PLK1 is a mitotic kinase that regulates diverse mitotic events, including centrosome maturation. However, its precise role in spindle orientation has not been well understood. LRRK1 is related to the familial Parkinsonism gene product Park8/LRRK2, and contains a Ras of complex proteins (ROC) GTPase domain and a MAPKKK-like kinase domain. We have previously reported that LRRK1 participates in the intercellular trafficking of the epidermal growth factor (EGF) receptor. 2 However, the role of LRRK1 in mitosis has remained unknown. Recently, we characterized the roles of PLK1 and LRRK1 in spindle orientation. 3 We found that PLK1 interacts with LRRK1 through its Polo-box domain and phosphorylates LRRK1 at Ser-1790, an event that is required for cyclin-dependent kinase 1 (CDK1)-mediated activation of LRRK1 at centrosomes. CDK1 phosphorylates LRRK1 at Thr-1400, which is located in the activation loop of LRRK1, turning on LRRK1 kinase activity. Thus, LRRK1 is activated by the sequential phosphorylation of 2 mitotic kinases, PLK1 and CDK1. Importantly, this PLK1-dependent activation of LRRK1 is required for the regulation of mitotic spindle orientation. We showed that LRRK1 regulates spindle orientation in a manner dependent on its kinase activity. Furthermore, the spindle misorientation caused by PLK1 inactivation is rescued by expression of the phospho-mimicking mutant LRRK1 (S1790D), but not the non-phosphorylatable mutant LRRK1 (S1790A), suggesting that PLK1 phosphorylation of LRRK1 Ser-1790 regulates the orientation of the mitotic spindle. Thus, LRRK1 regulates spindle orientation downstream of PLK1. Correct mitotic spindle orientation depends on the interaction of astral microtubules (MTs) with the cell cortex and MT nucleation activity of the mitotic centrosome is indispensable for astral MT formation. g-tubulin ring complex (gTuRC), which is a large multi-protein complex containing g-tubulin, is known to be essential for MT nucleation. In principle, MT nucleation of centrosomes could be regulated at one or more steps including the assembly of gTuRC, its localization at centrosomes, and its subsequent nucleating activity. 4 Since LRRK1 depletion causes a reduction in MT nucleation of mitotic centrosomes, we examined the role of LRRK1 in each of these steps. As one binding partner of LRRK1, we identified the centrosomal protein CDK5RAP2, a human homolog of Drosophila Centrosomin (Cnn). CDK5RAP2 is required for centrosome MT nucleation activity and shares homology with Drosophila Cnn in 2 of its domains, CM1 and CM2: CM1 is involved in the association with g-tubulin, while CM2 is important for centrosomal localization. 5 Interestingly, LRRK1 associates with the N-terminal region of CDK5RAP2, which includes the CM1 domain, and phosphorylates CDK5RAP2 at Ser-140, which is located close to the CM1 domain. Since the CM1 domain of CDK5RAP2 directly binds to gTuRC, it has been thought that CDK5RAP2 may facilitate the recruitment of gTuRC to mitotic centrosomes. However, although knockdown of LRRK1 results in a reduced interaction between the CM1 domain of CDK5RAP2 and g-tubulin, we found that it does not affect g-tubulin …