Haren Laurence, Stearns Tim, Lüders Jens
Institut de Sciences et Technologies du Médicament de Toulouse, Centre National de la Recherche Scientifique/Pierre Fabre, Toulouse, France.
PLoS One. 2009 Jun 19;4(6):e5976. doi: 10.1371/journal.pone.0005976.
The nucleation of microtubules requires protein complexes containing gamma-tubulin, which are present in the cytoplasm and associate with the centrosome and with the mitotic spindle. We have previously shown that these interactions require the gamma-tubulin targeting factor GCP-WD/NEDD1, which has an essential role in spindle formation. The recruitment of additional gamma-tubulin to the centrosomes occurs during centrosome maturation at the G2/M transition and is regulated by the mitotic kinase Plk1. However, the molecular details of this important pathway are unknown and a Plk1 substrate that controls gamma-tubulin recruitment has not been identified. Here we show that Plk1 associates with GCP-WD in mitosis and Plk1 activity contributes to phosphorylation of GCP-WD. Plk1 depletion or inhibition prevents accumulation of GCP-WD at mitotic centrosomes, but GCP-WD mutants that are defective in Plk1-binding and -phosphorylation still accumulate at mitotic centrosomes and recruit gamma-tubulin. Moreover, Plk1 also controls the recruitment of other PCM proteins implicated in centrosomal gamma-tubulin attachment (Cep192/hSPD2, pericentrin, Cep215/Cdk5Rap2). Our results support a model in which Plk1-dependent recruitment of gamma-tubulin to mitotic centrosomes is regulated upstream of GCP-WD, involves multiple PCM proteins and therefore potentially multiple Plk1 substrates.
微管的成核需要含有γ-微管蛋白的蛋白质复合物，这些复合物存在于细胞质中，并与中心体和有丝分裂纺锤体相关联。我们之前已经表明，这些相互作用需要γ-微管蛋白靶向因子GCP-WD/NEDD1，它在纺锤体形成中起关键作用。在G2/M期转换时中心体成熟过程中，额外的γ-微管蛋白会被招募到中心体，这一过程由有丝分裂激酶Plk1调控。然而，这条重要途径的分子细节尚不清楚，且尚未鉴定出控制γ-微管蛋白招募的Plk1底物。在这里，我们表明Plk1在有丝分裂过程中与GCP-WD结合，且Plk1的活性有助于GCP-WD的磷酸化。Plk1的缺失或抑制会阻止GCP-WD在有丝分裂中心体的积累，但在Plk1结合和磷酸化方面存在缺陷的GCP-WD突变体仍会在有丝分裂中心体积累并招募γ-微管蛋白。此外，Plk1还控制着其他参与中心体γ-微管蛋白附着的PCM蛋白（Cep192/hSPD2、中心粒周蛋白、Cep215/Cdk5Rap2）的招募。我们的结果支持一种模型，即Plk1依赖的γ-微管蛋白向有丝分裂中心体的招募在GCP-WD的上游受到调控，涉及多种PCM蛋白，因此可能涉及多个Plk1底物。
