Cyclin-dependent kinase control of centrosome duplication (original) (raw)
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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
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 E in centrosome duplication and reduplication in sea urchin zygotes
Journal of Cellular Physiology, 2008
When protein synthesis is completely blocked from before fertilization, the sea urchin zygote arrests in first S phase and the paternal centrosome reduplicates multiple times. However, when protein synthesis is blocked starting in prophase of first mitosis, the zygote divides and the blastomeres arrest in a G1-like state. The centrosome inherited from this mitosis duplicates only once in each blastomere for reasons that are not understood. The late G1 rise in cyclin E/cdk2 kinase activity initiates centrosome duplication in mammalian cells and its activity is needed for centrosome duplication in Xenopus egg extracts. Since the half-time for cyclin E turnover is normally $1 h in sea urchin zygotes, the different behaviors of centrosomes during G1 and S phase arrests could be due to differential losses of cyclin E and its associated kinase activities at these two arrest points. To better understand the mechanisms that limit centrosome duplication, we characterize the levels of cyclin E and its associated kinase activity at the S phase and G1 arrest points. We first demonstrate that cyclin E/cdk2 kinase activity is required for centrosome duplication and reduplication in sea urchin zygotes. Next we find that cyclin E levels and cyclin E/cdk2 kinase activities are both constitutively and equivalently elevated during both the S phase and G1 arrests. This indicates that centrosome duplication during the G1 arrest is limited by a block to reduplication under conditions permissive for duplication. The cytoplasmic conditions of S phase, however, abrogate this block to reduplication.
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.
Cyclin B1–Cdk1 Activation Continues after Centrosome Separation to Control Mitotic Progression
PLOS Biology, 2007
Activation of cyclin B1-cyclin-dependent kinase 1 (Cdk1), triggered by a positive feedback loop at the end of G2, is the key event that initiates mitotic entry. In metaphase, anaphase-promoting complex/cyclosome-dependent destruction of cyclin B1 inactivates Cdk1 again, allowing mitotic exit and cell division. Several models describe Cdk1 activation kinetics in mitosis, but experimental data on how the activation proceeds in mitotic cells have largely been lacking. We use a novel approach to determine the temporal development of cyclin B1-Cdk1 activity in single cells. By quantifying both dephosphorylation of Cdk1 and phosphorylation of the Cdk1 target anaphase-promoting complex/cyclosome 3, we disclose how cyclin B1-Cdk1 continues to be activated after centrosome separation. Importantly, we discovered that cytoplasmic cyclin B1-Cdk1 activity can be maintained even when cyclin B1 translocates to the nucleus in prophase. These experimental data are fitted into a model describing cyclin B1-Cdk1 activation in human cells, revealing a striking resemblance to a bistable circuit. In line with the observed kinetics, cyclin B1-Cdk1 levels required to enter mitosis are lower than the amount of cyclin B1-Cdk1 needed for mitotic progression. We propose that gradually increasing cyclin B1-Cdk1 activity after centrosome separation is critical to coordinate mitotic progression.
Proceedings of the National Academy of Sciences, 1991
Heterologous centrosomes from diversed species including humans promote egg cleavage when injected into metaphase-arrested Xenopus eggs. We have recently isolated centrosomes from calf thymocytes and shown that they were unable to induce egg cleavage, an inability that was apparently correlated with the peculiar structure of these centrosomes rather than with a lack of microtubule-nucleating activity: the two centrioles were associated in a colinear orientation by their proximal ends. To promote cleavage, a heterologous centrosome probably is required to duplicate, although this has not yet been demonstrated. Therefore, we designed an in vitro assay that would enable us to directly observe the duplication process. We show that competent centrosomes from KE37 cells synchronized in G1 phase initiate procentriole budding in interphasic extracts from Xenopus eggs in the absence of protein synthesis, whereas calf thymocyte centrosomes do not. Since calf thymocyte centrosomes do not suppo...