Cyclin-dependent kinases (Cdks) are serine/threonine kinases and their catalytic activities are modulated by interactions with cyclins and Cdk inhibitors (CKIs). Close cooperation between this trio is necessary for ensuring orderly progression through the cell cycle. In addition to their well-established function in cell cycle control, it is becoming increasingly apparent that mammalian Cdks, cyclins and CKIs play indispensable roles in processes such as transcription, epigenetic regulation, metabolism, stem cell self-renewal, neuronal functions and spermatogenesis. Even more remarkably, they can accomplish some of these tasks individually, without the need for Cdk/cyclin complex formation or kinase activity. In this Review, we discuss the latest revelations about Cdks, cyclins and CKIs with the goal of showcasing their functional diversity beyond cell cycle regulation and their impact on development and disease in mammals.

Cdks, cyclins and CKIs: roles beyond cell cycle regulation

Maintenance of pluripotency is crucial to the mammalian embryo's ability to generate the extra-embryonic and embryonic tissues that are needed for intrauterine survival and foetal development. The recent establishment of embryonic stem cells from human blastocysts (hESCs) provides an opportunity to identify the factors supporting pluripotency at early stages of human development. Using this in vitro model, we have recently shown that Nodal can block neuronal differentiation, suggesting that TGFβ family members are involved in cell fate decisions of hESCs, including preservation of their pluripotency. Here, we report that Activin/Nodal signalling through Smad2/3 activation is necessary to maintain the pluripotent status of hESCs. Inhibition of Activin/Nodal signalling by follistatin and by overexpression of Lefty or Cerberus-Short, or by the Activin receptor inhibitor SB431542, precipitates hESC differentiation. Nevertheless, neither Nodal nor Activin is sufficient to sustain long-term hESC growth in a chemically defined medium without serum. Recent studies have shown that FGF2 can also maintain long-term expression of pluripotency markers, and we find that inhibition of the FGF signalling pathway by the tyrosine kinase inhibitor SU5402 causes hESC differentiation. However, this effect of FGF on hESC pluripotency depends on Activin/Nodal signalling, because it is blocked by SB431542. Finally, long-term maintenance of in-vitro pluripotency can be achieved with a combination of Activin or Nodal plus FGF2 in the absence of feeder-cell layers, conditioned medium or Serum Replacer. These findings suggest that the Activin/Nodal pathway maintains pluripotency through mechanism(s) in which FGF acts as a competence factor and therefore provide further evidence of distinct mechanisms for preservation of pluripotency in mouse and human ESCs.

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Activin/Nodal and FGF pathways cooperate to maintain pluripotency of human embryonic stem cells.

Entry and progression through mitosis has traditionally been linked directly to the activity of cyclin-dependent kinase 1 (Cdk1). In this study we utilized low doses of the Cdk1-specific inhibitor, RO3306 from early G2 phase onwards. Addition of low doses of RO3306 in G2 phase induced minor chromosome congression and segregation defects. In contrast, mild doses of RO3306 during G2 phase resulted in cells entering an aberrant mitosis, with cells fragmenting centrosomes and failing to fully disassemble the nuclear envelope. Cells often underwent cytokinesis and metaphase simultaneously, despite the presence of an active spindle assembly checkpoint, which prevented degradation of cyclin B1 and securin, resulting in the random partitioning of whole chromosomes. This highly aberrant mitosis produced a significant increase in the proportion of viable polyploid cells present up to 3 days post-treatment. Furthermore, cells treated with medium doses of RO3306 were only able to reach the threshold of Cdk1 substrate phosphorylation required to initiate nuclear envelope breakdown, but failed to reach the levels of phosphorylation required to correctly complete pro-metaphase. Treatment with low doses of Okadaic acid, which primarily inhibits PP2A, rescued the mitotic defects and increased the number of cells that completed a normal mitosis. This supports the current model that PP2A is the primary phosphatase that counterbalances the activity of Cdk1 during mitosis. Taken together these results show that continuous and subtle disruption of Cdk1 activity from G2 phase onwards has deleterious consequences on mitotic progression by disrupting the balance between Cdk1 and PP2A.

Partial inhibition of Cdk1 in G2 phase overrides the SAC and decouples mitotic events

Inflammasomes are intracellular protein complexes that drive the activation of inflammatory caspases. So far, four inflammasomes involving NLRP1, NLRP3, NLRC4 and AIM2 have been described that recruit the common adaptor protein ASC to activate caspase-1, leading to the secretion of mature IL-1β and IL-18 proteins. The NLRP3 inflammasome has been implicated in the pathogenesis of several acquired inflammatory diseases as well as cryopyrin-associated periodic fever syndromes (CAPS) caused by inherited NLRP3 mutations. Potassium efflux is a common step that is essential for NLRP3 inflammasome activation induced by many stimuli. Despite extensive investigation, the molecular mechanism leading to NLRP3 activation in response to potassium efflux remains unknown. Here we report the identification of NEK7, a member of the family of mammalian NIMA-related kinases (NEK proteins), as an NLRP3-binding protein that acts downstream of potassium efflux to regulate NLRP3 oligomerization and activation. In the absence of NEK7, caspase-1 activation and IL-1β release were abrogated in response to signals that activate NLRP3, but not NLRC4 or AIM2 inflammasomes. NLRP3-activating stimuli promoted the NLRP3-NEK7 interaction in a process that was dependent on potassium efflux. NLRP3 associated with the catalytic domain of NEK7, but the catalytic activity of NEK7 was shown to be dispensable for activation of the NLRP3 inflammasome. Activated macrophages formed a high-molecular-mass NLRP3-NEK7 complex, which, along with ASC oligomerization and ASC speck formation, was abrogated in the absence of NEK7. NEK7 was required for macrophages containing the CAPS-associated NLRP3(R258W) activating mutation to activate caspase-1. Mouse chimaeras reconstituted with wild-type, Nek7(-/-) or Nlrp3(-/-) haematopoietic cells showed that NEK7 was required for NLRP3 inflammasome activation in vivo. These studies demonstrate that NEK7 is an essential protein that acts downstream of potassium efflux to mediate NLRP3 inflammasome assembly and activation.

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NEK7 is an essential mediator of NLRP3 activation downstream of potassium efflux

The circadian rhythms are daily oscillations in various biological processes that are regulated by an endogenous clock. Disruption of these rhythms has been associated with cancer in humans. One of the cellular processes that is regulated by circadian rhythm is cell proliferation, which often shows asynchrony between normal and malignant tissues. This asynchrony highlights the importance of the circadian clock in tumour suppression in vivo and is one of the theoretical foundations for cancer chronotherapy. Investigation of the mechanisms by which the circadian clock controls cell proliferation and other cellular functions might lead to new therapeutic targets.

The circadian clock: pacemaker and tumour suppressor

The microtubule-organizing activity of the centrosome oscillates during the cell cycle, reaching its highest level at mitosis. At the onset of mitosis, the centrosome undergoes maturation, which is characterized by a drastic expansion of the pericentriolar matrix (PCM) and a robust increase in microtubule-organizing activity. It is known that PLK1 is critical for the initiation of centrosome maturation. In this paper, we report that pericentrin (PCNT), a PCM protein, was specifically phosphorylated by PLK1 during mitosis. Phosphoresistant point mutants of PCNT did not recruit centrosomal proteins, such as CEP192, GCP-WD (γ-complex protein with WD repeats), γ-tubulin, Aurora A, and PLK1, into the centrosome during mitosis. However, centrosomal recruitment of CEP215 depended on PCNT irrespective of its phosphorylation status. Furthermore, ectopic expression of PLK1-PCNT fusion proteins induced the centrosomal accumulation of CEP192, GCP-WD, and γ-tubulin even in interphase cells, mimicking centrosome maturation. Based on these results, we propose that PLK1-mediated phosphorylation of PCNT initiates centrosome maturation by organizing the spindle pole–specific PCM lattice.

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PLK1 phosphorylation of pericentrin initiates centrosome maturation at the onset of mitosis

Centrosomes play an important role in various cellular processes, including spindle formation and chromosome segregation. They are composed of two orthogonally arranged centrioles, whose duplication occurs only once per cell cycle. Accurate control of centriole numbers is essential for the maintenance of genomic integrity. Although it is well appreciated that polo-like kinase 4 (Plk4) plays a central role in centriole biogenesis, how it is recruited to centrosomes and whether this step is necessary for centriole biogenesis remain largely elusive. Here we showed that Plk4 localizes to distinct subcentrosomal regions in a temporally and spatially regulated manner, and that Cep192 and Cep152 serve as two distinct scaffolds that recruit Plk4 to centrosomes in a hierarchical order. Interestingly, Cep192 and Cep152 competitively interacted with the cryptic polo box of Plk4 through their homologous N-terminal sequences containing acidic-α-helix and N/Q-rich motifs. Consistent with these observations, the expression of either one of these N-terminal fragments was sufficient to delocalize Plk4 from centrosomes. Furthermore, loss of the Cep192- or Cep152-dependent interaction with Plk4 resulted in impaired centriole duplication that led to delayed cell proliferation. Thus, the spatiotemporal regulation of Plk4 localization by two hierarchical scaffolds, Cep192 and Cep152, is critical for centriole biogenesis.

https://www.pnas.org/content/pnas/110/50/E4849.full.pdf

Hierarchical recruitment of Plk4 and regulation of centriole biogenesis by two centrosomal scaffolds, Cep192 and Cep152.

The vast majority of mammalian testes are located outside the body cavity for proper thermoregulation. Heat has an adverse effect on mammalian spermatogenesis and eventually leads to sub- or infertility. Recent studies have provided insights into the molecular response of male germ cells to high temperatures. Here, we review the effects of heat on male germ cells and discuss the mechanisms underlying germ cell loss and impairment. We also discuss the role of translational control in male germ cells as a potential protective mechanism against heat-induced germ cell apoptosis.

Heat stress response of male germ cells

The Aspergillus nimA gene encodes a Ser/Thr protein kinase which is required for mitosis, in addition to Cdc2, and which has been suggested to have a role in chromosomal condensation. In this study, we isolated a potential murine homologue of nimA, Nek2, which was shown to be expressed most abundantly in the testis of the adult tissues examined. Its expression in the testis was restricted to the germ cells, with highest levels detected in spermatocytes at pachytene and diplotene stages. Immunohistochemical analysis revealed that Nek2 localized to nuclei, exhibiting a non-uniform distribution within the nucleus. Nek2 appeared to be associated with meiotic chromosomes, an association that was better defined by immunolocalization to hypotonically dispersed meiotic chromosomes. This localization was more apparent in regions of dense chromatin, including the sex vesicle, and was also obvious at some of the chromosome ends. The presence of Nek2 protein was not unique to male germ cells, as it was found in meiotic pachytene stage oocytes as well. Furthermore, in an in vitro experimental setting in which meiotic chromosome condensation was induced with okadaic acid, a concomitant induction of Nek2 kinase activity was observed. The expression of Nek2 in meiotic prophase is consistent with the hypothesis that in vivo, Nek2 is involved in the G2/M phase transition of the cell cycle. Our results further provide evidence that in vivo, mouse Nek2 is involved in events of meiosis, including but not limited to chromosomal condensation.

The NIMA-related kinase 2, Nek2, is expressed in specific stages of the meiotic cell cycle and associates with meiotic chromosomes

Centriole disengagement is considered an essential step for licensing a new round of centriole duplication in the next cell cycle. Separase is critical for centriole disengagement. Here, we showed that pericentrin B (PCNTB) is specifically cleaved by separase at the exit of mitosis. The cleavage-resistant PCNTB mutant blocks the centriole disengagement and duplication. We also observed that an artificial cleavage of PCNTB during M phase induced premature disengagement of centrioles. Based on these results, we concluded that the separase-dependent cleavage of PCNTB is necessary and sufficient for centriole disengagement during mitosis.

https://www.tandfonline.com/doi/pdf/10.4161/cc.20878

Kunsoo Rhee

Papers

PLK1 phosphorylation of pericentrin initiates centrosome maturation at the onset of mitosis

Hierarchical recruitment of Plk4 and regulation of centriole biogenesis by two centrosomal scaffolds, Cep192 and Cep152.

Heat stress response of male germ cells

The NIMA-related kinase 2, Nek2, is expressed in specific stages of the meiotic cell cycle and associates with meiotic chromosomes

Separase-dependent cleavage of pericentrin B is necessary and sufficient for centriole disengagement during mitosis.