Cdk1: A kinase with changing substrate specificity

Comment on: Kõivomägi M, et al. Mol Cell 2011; 42:610-23.     

Cdk1 coordinates cell-surface growth with the cell cycle

The mechanisms that control cell growth during the cell cycle are poorly understood. In budding yeast, cyclin dependent kinase 1 (Cdk1) triggers polarization of the actin cytoskeleton and bud emergence in late G1 through activation of the Cdc42 GTPase. However, Cdk1 is not thought to be required for subsequent growth of the bud. Here, we show that Cdk1 has an unexpected role in controlling bud growth after bud emergence. Moreover, we show that G1 cyclin-Cdk1 complexes specifically phosphorylate multiple proteins associated with Cdc24, the guanine nucleotide-exchange factor (GEF) that activates the Cdc42 GTPase. A mutant form of a Cdc24-associated protein that fails to undergo Cdk1-dependent phosphorylation causes defects in bud growth. These results provide a direct link between Cdk1 activity and the control of polarized cell growth.     

Dynamics of chromosome positioning during the cell cycle

The arrangement and dynamics of chromosomes inside the nucleus of mammalian cells have been studied intensively over the last two years. Although chromosomes are relatively immobile and occupy non-random positions in interphase, their dynamic movements in mitosis have traditionally been assumed to randomize this arrangement. New methods of live cell imaging now make it possible to follow chromosome movements directly and quantitatively in single cells. Such studies have generated models of chromosome positioning throughout the cell cycle and provide a new basis to address the underlying mechanisms in future experiments.     

Cdk5 levels oscillate during the neuronal cell cycle: Cdh1 ubiquitination triggers proteosome-dependent degradation during S-phase

When cell cycle re-activation occurs in post-mitotic neurons it places them at increased risk for death. The cell cycle/cell death association has been reported in many neurodegenerative diseases including Alzheimer disease (AD), yet the mechanisms by which a normal neuron suppresses the cycle remain largely unknown. Recently, our laboratory has shown that Cdk5 (cyclin-dependent kinase 5) is a key player in this protective function. When a neuron is under stress, Cdk5 is transported to the cytoplasm; this eliminates its cell cycle suppression activity and the neuron re-enters S-phase. In the current study we show that a similar principle applies during a normal cell cycle. When a neuronal cell enters S phase, Cdk5 is transported to the cytoplasm where it is ubiquitinated by the E3 ligase APC-Cdh1. Ubiquitinated Cdk5 is then rapidly degraded by the proteasome. The ubiquitination site of Cdk5 appears to be in the p35 binding area; in the presence of high levels of p35, the ubiquitination of Cdk5 was blocked, and the degradation in S phase was attenuated. The data suggest an unsuspected role for Cdk5 during the progression of a normal cell cycle and offer new pharmaceutical targets for regulating neuronal cell cycling and cell death.     

Dynamics of relative chromosome position during the cell cycle

The position of chromosomal neighborhoods in living cells was followed using three different methods for marking chromosomal domains occupying arbitrary locations in the nucleus; photobleaching of GFP-labeled histone H2B, local UV-marked DNA, and photobleaching of fluorescently labeled DNA. All methods revealed that global chromosomal organization can be reestablished through one cell division from mother to daughters. By simultaneously monitoring cell cycle stage in the cells in which relative chromosomal domain positions were tracked, we observed that chromosomal neighborhood organization is apparently lost in the early G1 phase of the cell cycle. However, the daughter cells eventually regain the general chromosomal organization pattern of their mothers, suggesting an active mechanism could be at play to reestablish chromosomal neighborhoods.     

Selective chemical inhibition as a tool to study Cdk1 and Cdk2 functions in the cell cycle

Cyclin-dependent kinases are highly conserved among all eukaryotes, and have essential roles in the cell cycle. However, these roles are still only poorly understood at a molecular level, partly due to the functional redundance of different Cdk complexes. Indeed, mice knockouts have even thrown into some doubt the assumed essential roles for Cdk2-cyclin E in triggering S-phase, but this is almost certainly due to compensation by Cdk1 complexes. By combining both knockout approaches and chemical Cdk inhibition in Xenopus egg extracts, we have shown that one reason for functional redundancy of Cdk control of S-phase is that Cdk activity required to trigger S-phase is very low. Cdk1 contributes to this activity even in the presence of Cdk2, and Cdk activity at this stage does not show "switch-like" regulation, as at the onset of mitosis. It is important to try to confirm and extend these findings to other cell-types, and to explain why different cells might have evolved different requirements for Cdk activity. In this paper, we present data that suggest that selective chemical Cdk inhibition will be a useful tool towards achieving this goal.     

Cdk5--p39 is a labile complex with the similar substrate specificity to Cdk5--p35

Cyclin-dependent kinase 5 (Cdk5) is a proline-directed Ser/Thr kinase that plays important roles in various neuronal activities, including neuronal migration, synaptic activity, and neuronal cell death. Cdk5 is activated by association with a neuron-specific activator, p35 or its isoform p39, but little is known about the kinase activity of Cdk5--p39. In fact, kinase-active Cdk5--p39 was not prepared from rat brain extracts nor from HEK293 cells expressing Cdk5 and p39 by immunoprecipitation in the presence of non-ionic detergent, under conditions with which active Cdk5--p35 could be isolated. p39 dissociated from Cdk5 in the presence of detergent, indicating that p39 has a lower binding affinity for Cdk5 than p35. We developed a method for purifying kinase-active Cdk5--p39 from Sf9 cells infected with baculovirus encoding Cdk5 and p39. The purified Cdk5--p39 complex showed similar substrate specificity to that of Cdk5--p35, but with opposite sensitivity to detergent. Cdk5--p39 was inactivated by Triton X-100, whereas Cdk5--p35 was activated. The N-terminal deletion from p35 and p39, the amino acid sequences of which are different, did not change the stability or substrate specificity of either Cdk5 complex. The different stability between Cdk5--p35 and Cdk5--p39 suggests their distinct roles under different regulation mechanisms in neurons.     

Drosophila Myt1 is a Cdk1 inhibitory kinase that regulates multiple aspects of cell cycle behavior during gametogenesis

The metazoan Wee1-like kinases Wee1 and Myt1 regulate the essential mitotic regulator Cdk1 by inhibitory phosphorylation. This regulatory mechanism, which prevents Cdk1 from triggering premature mitotic events, is also induced during the DNA damage response and used to coordinate cell proliferation with crucial developmental events. Despite the previously demonstrated role for Myt1 regulation of Cdk1 during meiosis, relatively little is known of how Myt1 functions at other developmental stages. To address this issue, we have undertaken a functional analysis of Drosophila Myt1 that has revealed novel developmental roles for this conserved cell cycle regulator during gametogenesis. Notably, more proliferating cells were observed in myt1 mutant testes and ovaries than controls. This can partly be attributed to ectopic division of germline-associated somatic cells in myt1 mutants, suggesting that Myt1 serves a role in regulating exit from the cell cycle. Moreover, mitotic index measurements suggested that germline stem cells proliferate more rapidly, in myt1 mutant females. In addition, male myt1 germline cells occasionally undergo an extra mitotic division, resulting in meiotic cysts with twice the normal numbers of cells. Based on these observations, we propose that Myt1 serves unique Cdk1 regulatory functions required for efficient coupling of cell differentiation with cell cycle progression.     

Dynamics of the nuclear envelope during cell cycle in plants

Stereology of Allium cepa root meristem cells was done to evaluate changes in the nuclear envelope during cell cycle. A naturally synchronous population was labelled as binucleate by caffeine inhibition of cytokinesis. Growth of the nuclear envelope preferentially occurs from mid G2 to the next mid G1, most probably in relation to the reforming sister nuclei after mitosis. On the other hand, the number of nuclear pores doubles from mid G1 to mid G2, their growth rate being higher in the first half of interphase (from mid G1 to mid S). Hence, the new nuclear envelope probably lacks nuclear pores, which appear later.     

Spatial regulation of greatwall by Cdk1 and PP2A-Tws in the cell cycle

Entry into mitosis requires the phosphorylation of multiple substrates by cyclin B-Cdk1, while exit from mitosis requires their dephosphorylation, which depends largely on the phosphatase PP2A in complex with its B55 regulatory subunit (Tws in Drosophila). At mitotic entry, cyclin B-Cdk1 activates the Greatwall kinase, which phosphorylates Endosulfine proteins, thereby activating their ability to inhibit PP2A-B55 competitively. The inhibition of PP2A-B55 at mitotic entry facilitates the accumulation of phosphorylated Cdk1 substrates. The coordination of these enzymes involves major changes in their localization. In interphase, Gwl is nuclear while PP2A-B55 is cytoplasmic. We recently showed that Gwl suddenly relocalizes from the nucleus to the cytoplasm in prophase, before nuclear envelope breakdown and that this controlled localization of Gwl is required for its function. We and others have shown that phosphorylation of Gwl by cyclin B-Cdk1 at multiple sites is required for its nuclear exclusion, but the precise mechanisms remained unclear. In addition, how Gwl returns to its nuclear localization was not explored. Here we show that cyclin B-Cdk1 directly inactivates a Nuclear Localization Signal in the central region of Gwl. This phosphorylation facilitates the cytoplasmic retention of Gwl, which is exported to the cytoplasm in a Crm1-dependent manner. In addition, we show that PP2A-Tws promotes the return of Gwl to its nuclear localization during cytokinesis. Our results indicate that the cyclic changes in Gwl localization at mitotic entry and exit are directly regulated by the antagonistic cyclin B-Cdk1 and PP2A-Tws enzymes.     

Cdk5 and the non-catalytic arrest of the neuronal cell cycle

Cyclin-dependent kinase 5 (Cdk5) is a nontraditional Cdk that is primarily active in postmitotic neurons. An important core function of Cdk5 involves regulating the migration and maturation of embryonic post-mitotic neurons. Initially there is little evidence indicating a role for Cdk5 in normal cell cycle regulation. These development roles are on its kinase activity. Recent data from our lab, however, suggest that Cdk5 plays a crucial role as a cell cycle suppressor in normal post-mitotic neurons and neuronal cell lines. It performs this foundation in a kinase independent manner. Cdk5 normally found in both nucleus and cytoplasm, but it exits the nucleus in neurons risk to death in an AD patient’s brain. The shift in sub-cellular location is accompanied by cell cycle re-entry and neuronal death. This “new” function of Cdk5 raises cautions in the design of Cdk5-directed drugs for the therapy of neurodegenerative diseases.     

Cdk7- and Cdc25A-independent dephosphorylation of Cdk2 during phorbol ester-mediated cell cycle arrest in U937 cells

The molecular mechanism underlying protein kinase C (PKC)-mediated cell cycle arrest is poorly understood. We undertook to characterize phorbol ester-activated PKC-mediated cell cycle arrest. Treatment with phorbol ester inhibited cell growth of human histiocytic lymphoma U937 cells with 83% of the cells arrested in G1 phase. Reduced activity of cdk2 correlated with cdk2 dephosphorylation and accumulation of cdk2 inhibitor p21Waf in phorbol ester-treated cells. Dephosphorylation of cdk2 was not associated with cdk7 and cdc25A activity in phorbol ester-treated cells. Protein phosphatase inhibitor assays suggest that the dephosphorylation of cdk2 results in the activation of a specific protein tyrosine phosphatase. Thus, dephosphorylation of cdk2 as well as accumulation of cdk2 inhibitor is likely to contribute to the G1 phase arrest in phorbol ester-treated in U937 cells.     

Dynamics of lipids and metabolites during the cell cycle of Chlamydomonas reinhardtii

Metabolites and lipids are the final products of enzymatic processes, distinguishing the different cellular functions and activities of single cells or whole tissues. Understanding these cellular functions within a well‐established model system requires a systemic collection of molecular and physiological information. In the current report, the green alga Chlamydomonas reinhardtii was selected to establish a comprehensive workflow for the detailed multi‐omics analysis of a synchronously growing cell culture system. After implementation and benchmarking of the synchronous cell culture, a two‐phase extraction method was adopted for the analysis of proteins, lipids, metabolites and starch from a single sample aliquot of as little as 10–15 million Chlamydomonas cells. In a proof of concept study, primary metabolites and lipids were sampled throughout the diurnal cell cycle. The results of these time‐resolved measurements showed that single compounds were not only coordinated with each other in different pathways, but that these complex metabolic signatures have the potential to be used as biomarkers of various cellular processes. Taken together, the developed workflow, including the synchronized growth of the photoautotrophic cell culture, in combination with comprehensive extraction methods and detailed metabolic phenotyping has the potential for use in in‐depth analysis of complex cellular processes, providing essential information for the understanding of complex biological systems.     

Cerebral ischemia, cell cycle elements and Cdk5

Stroke is a devastating disorder that significantly contributes to death, disability, and healthcare costs. New therapeutic strategies have been recently focusing on the development of neuroprotective agents that could halt the underlying mechanisms of neuronal death leading to brain damage. Accumulating evidence implicates proteins that are normally involved in the regulation of the cell cycle to neuronal death following ischemic insult, suggesting that these proteins could be suitable targets for stroke therapy. In this brief review, we present in vitro and in vivo arguments linking cell cycle molecules, i.e., cyclins, mitotic cyclin-dependent kinases (Cdks), as well as non-mitotic Cdk5, to ischemic neuronal death. We also report the evaluation of the potential of Cdk inhibitors as neuroprotective strategy for ischemic injury.