The autoregulated instability of Polo-like kinase 4 limits centrosome duplication to once per cell cycle (original) (raw)
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Autophosphorylation of Polo-like Kinase 4 and Its Role in Centriole Duplication
Molecular Biology of the Cell, 2009
Centrosome duplication occurs once every cell cycle in a strictly controlled manner. Polo-like kinase 4 (PLK4) is a key regulator of this process whose kinase activity is essential for centriole duplication. Here, we show that PLK4 autophosphorylation of serine S305 is a consequence of kinase activation and enables the active fraction to be identified in the cell. Active PLK4 is detectable on the replicating mother centriole in G1/S, with the proportion of active kinase increasing through interphase to reach a maximum in mitosis. Activation of PLK4 at the replicating daughter centriole is delayed until G2, but a level equivalent to the replicating mother centriole is achieved in M phase. Active PLK4 is regulated by the proteasome, because either proteasome inhibition or mutation of the degron motif of PLK4 results in the accumulation of S305-phosphorylated PLK4. Autophosphorylation probably plays a role in the process of centriole duplication, because mimicking S305 phosphorylation enhances the ability of overexpressed PLK4 to induce centriole amplification. Importantly, we show that S305-phosphorylated PLK4 is specifically sequestered at the centrosome contrary to the nonphosphorylated form. These data suggest that PLK4 activity is restricted to the centrosome to prevent aberrant centriole assembly and sustained kinase activity is required for centriole duplication.
Nature communications, 2017
Centrosome number is tightly controlled during the cell cycle to ensure proper spindle assembly and cell division. However, the underlying mechanism that controls centrosome number remains largely unclear. We show herein that the DNA replication licensing factor Cdc6 is recruited to the proximal side of the centrioles via cyclin A to negatively regulate centrosome duplication by binding and inhibiting the cartwheel protein Sas-6 from forming a stable complex with another centriole duplication core protein, STIL. We further demonstrate that Cdc6 colocalizes with Plk4 at the centrosome, and interacts with Plk4 during S phase. Plk4 disrupts the interaction between Sas-6 and Cdc6, and suppresses the inhibitory role of Cdc6 on Sas-6 by phosphorylating Cdc6. Overexpressing wild-type Cdc6 or Plk4-unphosphorylatable Cdc6 mutant 2A reduces centrosome over-duplication caused by Plk4 overexpression or hydroxyurea treatment. Taken together, our data demonstrate that Cdc6 and Plk4 antagonistical...
Oncogene, 2006
Cyclin-dependent kinase 2 (CDK2) has been proposed to function as a master regulator of centrosome duplication. Using mouse embryonic fibroblasts (MEFs) in which Cdk2 has been genetically deleted, we show here that CDK2 is not required for normal centrosome duplication, maturation and bipolar mitotic spindle formation. In contrast, Cdk2 deficiency completely abrogates aberrant centrosome duplication induced by a viral oncogene. Mechanistically, centrosome overduplication in MEFs wild-type for Cdk2 involves the formation of supernumerary immature centrosomes. These results indicate that normal and abnormal centrosome duplication have significantly different requirements for CDK2 activity and point to a role of CDK2 in licensing centrosomes for aberrant duplication. Furthermore, our findings suggest that CDK2 may be a suitable therapeutic target to inhibit centrosome-mediated chromosomal instability in tumor cells.
Cyclin-dependent kinase control of centrosome duplication
Proceedings of the National Academy of Sciences, 1999
Centrosomes nucleate microtubules and duplicate once per cell cycle. This duplication and subsequent segregation in mitosis results in maintenance of the one centrosome͞cell ratio. Centrosome duplication occurs during the G 1 ͞S transition in somatic cells and must be coupled to the events of the nuclear cell cycle; failure to coordinate duplication and mitosis results in abnormal numbers of centrosomes and aberrant mitoses. Using both in vivo and in vitro assays, we show that centrosome duplication in Xenopus laevis embryos requires cyclin͞cdk2 kinase activity. Injection of the cdk (cyclin-dependent kinase) inhibitor p21 into one blastomere of a dividing embryo blocks centrosome duplication in that blastomere; the related cdk inhibitor p27 has a similar effect. An in vitro system using Xenopus extracts carries out separation of the paired centrioles within the centrosome. This centriole separation activity is dependent on cyclin͞cdk2 activity; depletion of either cdk2 or of the two activating cyclins, cyclin A and cyclin E, eliminates centriole separation activity. In addition, centriole separation is inhibited by the mitotic state, suggesting a mechanism of linking the cell cycle to periodic duplication of the centrosome.
2010
The two mitotic centrosomes direct spindle bipolarity to maintain euploidy. Centrosome amplification-the acquisition of >3 centrosomes-generates multipolar mitoses, aneuploidy, and chromosome instability to promote cancer biogenesis. While much evidence suggests that Cdk2 is the major conductor of the centrosome cycle and that it mediates centrosome amplification induced by various altered tumor suppressors, the role played by Cdk4 in a normal or deregulated centrosome cycle is unknown. Using a gene knockout approach, we report that Cdk2 and Cdk4 are critical to the centrosome cycle, since centrosome separation and duplication are premature in Cdk2 ؊/؊ mouse embryonic fibroblasts (MEFs) and are compromised in Cdk4 ؊/؊ MEFs. Additionally, ablation of Cdk4 or Cdk2 abrogates centrosome amplification and chromosome instability in p53-null MEFs. Absence of Cdk2 or Cdk4 prevents centrosome amplification by abrogating excessive centriole duplication. Furthermore, hyperactive Cdk2 and Cdk4 deregulate the licensing of the centrosome duplication cycle in p53-null cells by hyperphosphorylating nucleophosmin (NPM) at Thr199, as evidenced by observations that ablation of Cdk2, Cdk4, or both Cdk2 and Cdk4 abrogates that excessive phosphorylation. Since a mutant form of NPM lacking the G 1 Cdk phosphorylation site (NPM T199A ) prevents centrosome amplification to the same extent as ablation of Cdk2 or Cdk4, we conclude that the Cdk2/Cdk4/NPM pathway is a major guardian of centrosome dysfunction and genomic integrity.
Functional Relationship among PLK2, PLK4 and ROCK2 to Induce Centrosome Amplification
Cell Cycle, 2015
The presence of more than 2 centrosomes (centrosome amplification) leads to defective mitosis and chromosome segregation errors, is frequently found in a variety of cancer types, and believed to be the major cause of chromosome instability. One mechanism for generation of amplified centrosomes is over-duplication of centrosomes in a single cell cycle, which is expected to occur when cells are temporarily arrested. There are a growing number of kinases that are critical for induction and promotion of centrosome amplification in the cell cycle-arrested cells, including Rhoassociated kinase (ROCK2), Polo-like kinase 2 (PLK2) and PLK4. Here, we tested whether these kinases induce centrosome amplification in a linear pathway or parallel pathways. We first confirmed that ROCK2, PLK2 and PLK4 are all essential for centrosomes to re-duplicate in the cells arrested by exposure to DNA synthesis inhibitor. Using the centrosome amplification rescue assay, we found that PLK2 indirectly activates ROCK2 via phosphorylating nucleophosmin (NPM), and PLK4 functions downstream of ROCK2 to drive centrosome amplification in the arrested cells.
The Centrosome and Its Duplication Cycle
Cold Spring Harbor Perspectives in Biology, 2015
The centrosome was discovered in the late 19th century when mitosis was first described. Long recognized as a key organelle of the spindle pole, its core component, the centriole, was realized more than 50 or so years later also to comprise the basal body of the cilium. Here, we chart the more recent acquisition of a molecular understanding of centrosome structure and function. The strategies for gaining such knowledge were quickly developed in the yeasts to decipher the structure and function of their distinctive spindle pole bodies. Only within the past decade have studies with model eukaryotes and cultured cells brought a similar degree of sophistication to our understanding of the centrosome duplication cycle and the multiple roles of this organelle and its component parts in cell division and signaling. Now as we begin to understand these functions in the context of development, the way is being opened up for studies of the roles of centrosomes in human disease.
Nucleophosmin/B23 Is a Target of CDK2/Cyclin E in Centrosome Duplication
Cell, 2000
Carroll et al., 1999). and Anatomy A family of serine/threonine kinases known as cyclin-† Infectious Disease Division dependent kinases (CDKs) controls the onset of the ma-University of Cincinnati College of Medicine jor cell cycle events such as DNA synthesis and mitosis Cincinnati, Ohio 45267 (Heichman and Roberts, 1994; Nurse, 1994; Sherr, 1994). ‡ Department of Pharmacology The activity of CDKs is regulated by their association Baylor College of Medicine with different cyclins, which are temporally expressed Houston, Texas 77030 at specific cell cycle stages. For example, expression § Children's Hospital Research Foundation of cyclin E, which associates with CDK2, is maximal in Cincinnati, Ohio 45229 late G1 (Koff et al., 1992; Dulic et al., 1992), and the formation of active CDK2/cyclin E complex is essential for S phase entry (Ohtsubo et al., 1993; van den Heuvel Summary and Harlow, 1993). Centrosome duplication consists of three distinct steps: (1) loss of orthogonal configuration and separa-In animal cells, duplication of centrosomes and DNA tion of the paired centrioles, (2) synthesis of a procentriis coordinated. Since CDK2/cyclin E triggers initiation ole next to each preexisting centriole, and (3) elongation of both events, activation of CDK2/cyclin E is thought of the procentrioles (reviewed in Lange and Gull, 1996). to link these two events. We identified nucleophos-It has been found that the activation of CDK2/cyclin E is min (NPM/B23) as a substrate of CDK2/cyclin E in necessary for initiation of centrosome duplication (both centrosome duplication. NPM/B23 associates specifiseparation of centrioles and procentriole formation) cally with unduplicated centrosomes, and NPM/B23 (Hinchcliffe et al., 1999; Lacey et al., 1999). Moreover, dissociates from centrosomes by CDK2/cyclin E-mediconstitutive activation of CDK2/cyclin E in cells results ated phosphorylation. An anti-NPM/B23 antibody, in uncoupling of initiation of centrosome and DNA dupliwhich blocks this phosphorylation, suppresses the inication (Mussman et al., 2000). In these cells, centrotiation of centrosome duplication in vivo. Moreover, somes duplicate immediately after entry into G1, much expression of a nonphosphorylatable mutant NPM/ before the onset of DNA synthesis, indicating that ini-B23 in cells effectively blocks centrosome duplication. tiation of centrosome duplication primarily depends on Thus, NPM/B23 is a target of CDK2/cyclin E in the activation of CDK2/cyclin E, while initiation of DNA initiation of centrosome duplication. replication requires additional events before being triggered by CDK2/cyclin E. In normal cells, the activation Introduction of CDK2/cyclin E at late G1 triggers initiation of both centrosome and DNA duplication, and thus coordinates The centrosome in animal cells consists of a pair of these two events. Moreover, inhibition of CDK2 by CDK centrioles (the core structure of the centrosome) and an inhibitors abolishes the ability of CDK2/cyclin E to inamorphous pericentriolar region composed of a number duce initiation of centrosome duplication, demonstraof different proteins, and acts as a microtubule organizting that the kinase activity of CDK2 is required (Hinching center (for a recent review, see Lange and Gull, cliffe et al., 1999; Lacey et al., 1999; Matsumoto et al., 1996). During mitosis, the centrosomes function as spin-1999). Thus, it is reasonable to predict that certain dle poles, directing the formation of bipolar mitotic spincentrosomal protein(s) are phosphorylated by CDK2/ dles and determining the cleavage furrow plane, both cyclin E, and that this phosphorylation event may trigger of which are essential for accurate chromosome transthe initiation of centrosome duplication. mission to daughter cells. Since each daughter cell re-Using isolated centrosomes as substrates, we found ceives one centrosome upon cytokinesis, it must duplithat nucleophosmin (NPM/B23) is phosphorylated by cate prior to the next mitosis, and do so only once. Thus, CDK2/cyclin E. Moreover, NPM/B23 is associated with centrosome duplication must occur in coordination with unduplicated, but not with duplicated centrosomes, and other cell cycle events, including DNA synthesis. Indeed, dissociates from centrosomes upon phosphorylation by duplication of centrioles begins near the G1/S boundary CDK2/cyclin E. Microinjection of anti-NPM/B23 antiand centrosome duplication is completed in G2 (Vandre body, which blocks this phosphorylation, suppresses and Borisy, 1989; Tournier and Bornens, 1994). Abrogathe initiation of centrosome duplication. Moreover, extion of the regulation that coordinates centrosome and pression of the NPM/B23 deletion mutant, which is un-DNA duplication will likely increase the frequency of able to be phosphorylated by CDK2/cyclin E, blocks the initiation of centrosome duplication. These findings show that dissociation of NPM/B23 from centrosomes