Live cell imaging reveals distinct roles in cell cycle regulation for Nek2A and Nek2B

Fletcher, Lynda; Cerniglia, George J.; Yen, Tim J.; Muschel, Ruth J.

doi:10.1016/j.bbamcr.2005.01.007

Cited by 38 publications

(35 citation statements)

References 9 publications

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“…Its downregulation in HeLa cells results in prolongation of mitosis, which could suggest a potential role of this protein as an accelerator of MPF inactivation during mitosis in these cells. 69 On the other hand, in HeLa cells overexpressing GFP-Nek2A anaphase is delayed, 70 which shows that Nek2A is a more complex player of mitotic regulation. However, in calcium-activated CSF extract of Xenopus, where endogenous Nek2A is absent, non-degradable mutant of this protein prevents cyclins degradation.…”

Section: Discussionmentioning

confidence: 99%

Cyclin B Dissociation from CDK1 Precedes its Degradation Upon MPF Inactivation in Mitotic Extracts of Xenopus laevis Embryos

Chesnel

Bazile²,

Pascal³

et al. 2006

Cell Cycle

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Cyclin B is a regulatory subunit of CDK1 within MPF complex. Degradation of cyclin B via ubiquitin-proteasome pathway seemed to be absolutely required for the M-phase exit. However, inhibition of the proteasome proteolytic activity upon the exit from the meiotic metaphase II-arrest in Xenopus cell-free extract revealed that the proteasome-dependent dissociation of cyclin B from CDK1 is sufficient to inactivate MPF without cyclin B degradation. In this study we analyze whether the same mechanism operates during the exit from mitotic M-phase. We show in Xenopus cell-free extract undergoing the first or the second embryonic mitosis that CDK1 oscillations are not affected by proteasome inhibition with MG132 or ALLN despite effective inhibition of cyclins B degradation. The majority of cyclins B1 and B2 surviving CDK1 inactivation is CDK-free and cyclin B2 becomes resistant to phosphatase λ dephosphorylation. The pool of cyclins B remaining after CDK1 inactivation in the presence of MG132 is mitotically inert, while exogenous or newly synthesized cyclin B activates CDK1. This suggests that cyclins B remain sequestered within the proteasome upon MPF inactivation in the presence of MG132.Comparison of the dynamics of the decline of total and CDK-bound pools of cyclins B1, B2 and B4 upon mitotic exit in absence of protein synthesis reveals that CDK-bound cyclins B diminish clearly faster. Our results thus show that cyclin B dissociation from CDK1 precedes cyclins B degradation upon CDK1 inactivation in mitotic embryo extracts and that proteasome proteolytic activity is dispensable for both activation and inactivation of CDK1 in such extracts.

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Section: Discussionmentioning

confidence: 99%

Cyclin B Dissociation from CDK1 Precedes its Degradation Upon MPF Inactivation in Mitotic Extracts of Xenopus laevis Embryos

Chesnel

Bazile²,

Pascal³

et al. 2006

Cell Cycle

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“…HeLa, T24, AT22IJE and AT22IJE-T, and immortalized MEFs (derived from p53À/À mice) either wild-type or ligase 4À/À (14) (kindly supplied by G. Iliakis, Institute of Medical Radiation Biology, University of Duisberg-Essen, Essen, Germany) were grown in a humidified 37jC incubator with 5% CO 2 level in DMEM supplemented with 10% fetal bovine serum, nonessential amino acids, and 2 mmol/L of Lglutamine. HeLa cells expressing the fusion proteins GFP-H2B or GFPcentrin as described (15,16). AT22IJE (also named FTYZ5) and AT22IJE-T (also named FYPEB37) were kindly provided by Dr. R. Daniels (Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA; ref.…”

Section: Methodsmentioning

confidence: 99%

Abnormal Cytokinesis after X-Irradiation in Tumor Cells that Override the G2 DNA Damage Checkpoint

et al. 2008

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X-irradiation-induced DNA damage perturbs the G 1 , S, and G 2 phases of the cell cycle. The behavior of cells after they have experienced a DNA damage checkpoint delay is poorly characterized. We therefore examined the fates of irradiated tumor cells that have overcome a prolonged G 2 checkpoint delay. Most irradiated cells progressed through mitosis without significant delay, but failed to complete cytokinesis as they remained tethered to each other at the midbody. We observed that the movement of centrioles at the time of cytokinesis was impaired in the irradiated, bridged cells. We attribute the perturbation of centriole dynamics to the presence of chromatin bridges that spanned the daughter cells. The bridged cells exhibited different fates that included death, fusion that formed multinucleated cells, or another round of mitosis with no noticeable cell cycle delays. The presence of ;H2AX foci in the bridge as well as in the separated nuclei indicated that cells were proliferating despite the presence of DNA damage. It seems that DNA damage checkpoints were not reactivated in cells that overrode a prolonged G 2 delay. Cells deficient in ATM, H2AX, XRCC3, or ligase 4 exhibited a higher frequency of radiation-induced bridges than controls, suggesting that the DNA bridges resulted from inadequate DNA repair. These data show a previously unappreciated cytologic hallmark of DNA damage in dividing cells. Chromatin bridges that interfere with cytokinesis are likely to contribute to the replication failure and clonogenic death of cells exposed to irradiation. [Cancer Res 2008;68(10):3724-32]

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“…Nek2 is the most closely related of the human proteins to NIMA based on amino acid conservation within the N-terminal catalytic kinase domain (13). However, depletion studies in human cells do not support an essential role for Nek2 in mitotic entry (14). Rather there is strong evidence that in mitotic cells Nek2 contributes to centrosome separation and perhaps reorganization of the microtubule network at the G 2 /M transition (11,15,16).…”

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confidence: 95%

Alternative Splicing Controls Nuclear Translocation of the Cell Cycle-regulated Nek2 Kinase

Baxter

Wattam

et al. 2007

Journal of Biological Chemistry

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Nek2 is a cell cycle-regulated serine/threonine protein kinase that is up-regulated in human cancers. Functionally, it is implicated in control of centrosome separation and bipolar spindle formation in mitotic cells and chromatin condensation in meiotic cells. Two major splice variants have been described in vertebrates, Nek2A and Nek2B, that differ in their non-catalytic C termini. Recently, a third splice variant, Nek2C, was identified that lacks an eight-amino acid internal sequence within the C-terminal domain of Nek2A. This excision occurs at the same position as the Nek2A/Nek2B splice point. As predicted from their high degree of similarity, we show here that Nek2C shares many properties with Nek2A including kinase activity, dimerization, protein phosphatase 1 interaction, mitotic degradation, microtubule binding, and centrosome localization. Unexpectedly, though, the non-centrosomal pool of protein exhibits a marked difference in distribution for the three splice variants. Nek2C is mainly nuclear, Nek2B is mainly cytoplasmic, and Nek2A is evenly distributed within nuclei and cytoplasm. Mutagenesis experiments revealed a functional bipartite nuclear localization sequence (NLS) that spans the splice site leading to Nek2C having a strong NLS, Nek2A having a weak NLS, and Nek2B having no NLS. Finally, we identified a 28-kDa protein in nuclear extracts as a potential novel substrate of Nek2. Thus, alternative splicing provides an unusual mechanism for modulating Nek2 localization, enabling it to have both nuclear and cytoplasmic functions.

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Live cell imaging reveals distinct roles in cell cycle regulation for Nek2A and Nek2B

Cited by 38 publications

References 9 publications

Cyclin B Dissociation from CDK1 Precedes its Degradation Upon MPF Inactivation in Mitotic Extracts of Xenopus laevis Embryos

Cyclin B Dissociation from CDK1 Precedes its Degradation Upon MPF Inactivation in Mitotic Extracts of Xenopus laevis Embryos

Abnormal Cytokinesis after X-Irradiation in Tumor Cells that Override the G2 DNA Damage Checkpoint

Alternative Splicing Controls Nuclear Translocation of the Cell Cycle-regulated Nek2 Kinase

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