Plk1-Dependent Recruitment of γ-Tubulin Complexes to Mitotic Centrosomes Involves Multiple PCM Components

Abstract

The nucleation of microtubules requires protein complexes containing γ-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 γ-tubulin targeting factor GCP-WD/NEDD1, which has an essential role in spindle formation. The recruitment of additional γ-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 γ-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 γ-tubulin. Moreover, Plk1 also controls the recruitment of other PCM proteins implicated in centrosomal γ-tubulin attachment (Cep192/hSPD2, pericentrin, Cep215/Cdk5Rap2). Our results support a model in which Plk1-dependent recruitment of γ-tubulin to mitotic centrosomes is regulated upstream of GCP-WD, involves multiple PCM proteins and therefore potentially multiple Plk1 substrates.

Plk1 inhibition leads to a failure of mitotic division during the first mitotic division in pig embryos

“…It was also localized at the midbody in telophase in HeLa cells [7]. Haren et al found that recruitment of γ-tubulin and AURKA (Aurora Kinase A) to the mitotic centrosome were Plk1 dependent before spindle formation in HeLa cells [22]. Petronczki et al also found that Plk1 was localized to the centromere at the metaphase stage and involved in the stability of spindle assembly checkpoint (SAC)-dependent kinetochore-microtubule interactions.…”

The Centrosome and Its Duplication Cycle

“…Spd-2 mutant flies have reduced CNN and gtubulin in several cell types (Dix and Raff 2007;Giansanti et al 2008), and injection of antibodies against Spd-2 into Drosophila embryos prevents CNN recruitment in the vicinity of the centriole ). CDKRAP2 and Cep192, human counterparts of CNN and Spd-2, also increase on centrosome maturation as does pericentrin, and the proteins show interdependency for the recruitment of g-tubulin (Haren et al 2009;Lee and Rhee 2011). In part, this reflects the physical association of g-tubulin with the amino-terminal part, and pericentrin with the carboxy-terminal part of CDKRAP2 (Fong et al 2008;Choi et al 2010;Wang et al 2010).…”

“…Polo kinase or Plk1 (Polo-like kinase 1) is the major kinase required for the dramatic increase of PCM that occurs before mitotic entry (Blagden and Glover 2003;Glover 2005). Its kinase activity is needed for the normal localization of Spd-2/Cep192, CNN/CDK5RAP2, pericentrin, and Nedd1 (Haren et al 2009;Zhang et al 2009;Hatch et al 2010;Lee and Rhee 2011;Fu and Glover 2012). Interestingly, Drosophila Polo is restricted to zone II (i.e., the vicinity of the microtubule wall).…”

Let's huddle to prevent a muddle: centrosome declustering as an attractive anticancer strategy

“…43 Nevertheless, Plk1 activity slowly rises from S through G2 and peaks in mitosis. Plk1 not only mediates centriole maturation 42,44,45 but also primes centrioles for disengagement 34 by phosphorylating cohesin and accelerating its cleavage by separase. 46 In addition, Plk1 may regulate centrosomal localization of shugoshin and mediate centriole disengagement by modifying shugoshin's activity in preserving centriole cohesion.…”