Plk1 self-organization and priming phosphorylation of HsCYK-4 at the spindle midzone regulate the onset of division in human cells.
Plk1 self-organization and priming phosphorylation of HsCYK-4 at the spindle midzone regulate the onset of division in human cells.
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DOI：
10.1371/journal.pbio.1000111
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发表时间：
2009-05-05
期刊：
影响因子：
9.8
通讯作者：
Jallepalli PV
中科院分区：
文献类型：
--
作者：
Burkard ME;Maciejowski J;Rodriguez-Bravo V;Repka M;Lowery DM;Clauser KR;Zhang C;Shokat KM;Carr SA;Yaffe MB;Jallepalli PV
自我调节的Polo-like kinase1移动到有丝分裂纺锤体的中区，启动了局部信号级联，激活了细胞赤道的细胞分裂机制。动物细胞启动胞质分裂与后期开始平行，当肌动球蛋白环通过小GTP酶RhoA的局部激活组装和收缩时，产生分裂沟。沟的形成依赖于后期纺锤体微管(MTS)提供的位置线索，但这种线索是如何产生的仍不清楚。利用化学遗传学来实现时间和空间控制，我们证明了Polo-like kinase1(Plk1)的自组织递送到中带及其对MT结合底物的局部磷酸化是产生这种沟纹诱导信号的关键。当Plk1激活但不能针对这一赤道地标时，皮质RhoA募集和沟槽诱导都无法发生，从而概括了后期特异性Plk1抑制的效果。利用串联质谱仪和磷酸特异性抗体，我们发现Plk1结合并直接磷酸化中央纺锤体的HsCyK-4亚单位(也称为mGcracGAP)。在丝氨酸157处，这种修饰为Rho GTP交换因子ect2的串联BRCT重复序列创建了一个主要的对接位置。只表达非磷酸化形式的HsCyk-4的细胞不能在中带定位Ect2，并且在卵裂沟的形成中受到严重破坏，这意味着HsCyk-4是RhoA上游的Plk1‘S限速靶点。相反，将Plk1的抗抑制剂等位基因与HsCyk-4捆绑在一起，可以在细胞内所有其他Plk1分子受到全局抑制的情况下形成皱纹。我们的发现阐明了控制人类细胞胞质分裂启动的两个关键机制，并说明了化学遗传学在时间和空间上探索这种调控的力量。在有丝分裂期间，复制的染色体的分离和随后的细胞质分裂(细胞分裂)是紧密耦合的过程。胞质分裂不仅必须发生在染色体分离之后，而且还必须发生在染色体之间的物理空间中，以便每个子细胞继承适当的遗传物质。然而，负责这种细胞编排的机制却鲜为人知。我们使用化学遗传学来剖析细胞分裂的关键调节因子--Polo-like kinase1(Plk1)在人类细胞中的作用。我们发现，与以前的模型相反，Plk1寻找位于分离的染色体之间的微管(所谓的中区微管)的能力为细胞提供了分裂的肯定命令。一旦在这个里程碑组装，Plk1就会磷酸化HsCyK-4，这是中央纺锤体复合体的一个组成部分(之所以这样命名，是因为它在纺锤体中区组装)，并使HsCyK-4与另一种细胞分裂调节因子Ect2结合。然后，结合的Ect2与组装基于肌动蛋白和肌球蛋白的收缩环的机械联系，导致细胞分裂成两个子体。因此，我们的工作揭示了Plk1如何在时间和空间上协调人类细胞分裂的新见解。
Self-regulated movement of Polo-like kinase 1 to the midzone of the mitotic spindle initiates a local signaling cascade that activates the cell division machinery at the cell's equator. Animal cells initiate cytokinesis in parallel with anaphase onset, when an actomyosin ring assembles and constricts through localized activation of the small GTPase RhoA, giving rise to a cleavage furrow. Furrow formation relies on positional cues provided by anaphase spindle microtubules (MTs), but how such cues are generated remains unclear. Using chemical genetics to achieve both temporal and spatial control, we show that the self-organized delivery of Polo-like kinase 1 (Plk1) to the midzone and its local phosphorylation of a MT-bound substrate are critical for generating this furrow-inducing signal. When Plk1 was active but unable to target itself to this equatorial landmark, both cortical RhoA recruitment and furrow induction failed to occur, thus recapitulating the effects of anaphase-specific Plk1 inhibition. Using tandem mass spectrometry and phosphospecific antibodies, we found that Plk1 binds and directly phosphorylates the HsCYK-4 subunit of centralspindlin (also known as MgcRacGAP) at the midzone. At serine 157, this modification creates a major docking site for the tandem BRCT repeats of the Rho GTP exchange factor Ect2. Cells expressing only a nonphosphorylatable form of HsCYK-4 failed to localize Ect2 at the midzone and were severely impaired in cleavage furrow formation, implying that HsCYK-4 is Plk1's rate-limiting target upstream of RhoA. Conversely, tethering an inhibitor-resistant allele of Plk1 to HsCYK-4 allowed furrows to form despite global inhibition of all other Plk1 molecules in the cell. Our findings illuminate two key mechanisms governing the initiation of cytokinesis in human cells and illustrate the power of chemical genetics to probe such regulation both in time and space. During mitosis, the separation of duplicated chromosomes and subsequent cytokinesis (cell division) are tightly coupled processes. Cytokinesis must occur not only after chromosomes have separated but also in the physical space between the chromosomes, so that each daughter cell inherits the appropriate genetic material. The mechanisms responsible for this cellular choreography are poorly understood, however. We used chemical genetics to dissect the role of a key regulator of cell division, Polo-like kinase 1 (Plk1) in human cells. We show that, contrary to previous models, the ability of Plk1 to seek out microtubules that lie between the separated chromosomes (so-called midzone microtubules) provides the cell with an affirmative command to divide. Once assembled at this landmark, Plk1 phosphorylates HsCYK-4, a component of the centralspindlin complex (so named because it assembles at the spindle midzone) and enables binding between HsCYK-4 and Ect2, another regulator of cell division. Bound Ect2 then communicates with the machinery that assembles the actin- and myosin-based contractile ring, leading to division of the cell into two daughters. Our work therefore reveals new insights into how Plk1 temporally and spatially orchestrates division of human cells.
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DOI：
10.1083/jcb.129.6.1617
发表时间：
1995-06
期刊：
The Journal of cell biology
影响因子：
--
作者：
Golsteyn RM;Mundt KE;Fry AM;Nigg EA
通讯作者：
Nigg EA
影响因子：
3.7
作者：
Brennan IM;Peters U;Kapoor TM;Straight AF
通讯作者：
Straight AF
DOI：
10.1083/jcb.143.6.1603
发表时间：
1998-12-14
期刊：
The Journal of cell biology
影响因子：
--
作者：
Bähler J;Steever AB;Wheatley S;Wang Yl;Pringle JR;Gould KL;McCollum D
通讯作者：
McCollum D
DOI：
10.1073/pnas.0701140104
发表时间：
2007-03-13
影响因子：
11.1
作者：
Burkard, Mark E.;Randall, Catherine L.;Jallepalli, Prasad V.
通讯作者：
Jallepalli, Prasad V.
影响因子：
3.7
作者：
D'Avino PP;Archambault V;Przewloka MR;Zhang W;Lilley KS;Laue E;Glover DM
通讯作者：
Glover DM