Identification of Cdk targets that control cytokinesis

The final event of the eukaryotic cell cycle is cytokinesis, when two new daughter cells are born. How the timing and execution of cytokinesis is controlled is poorly understood. Here, we show that downregulation of cyclin-dependent kinase (Cdk) activity, together with upregulation of its counteracting phosphatase Cdc14, controls each of the sequential steps of cytokinesis, including furrow ingression, membrane resolution and cell separation in budding yeast. We use phosphoproteome analysis of mitotic exit to identify Cdk targets that are dephosphorylated at the time of cytokinesis. We then apply a new and widely applicable tool to generate conditionally phosphorylated proteins to identify those whose dephosphorylation is required for cytokinesis. This approach identifies Aip1, Ede1 and Inn1 as cytokinetic regulators. Our results suggest that cytokinesis is coordinately controlled by the master cell cycle regulator Cdk together with its counteracting phosphatase and that it is executed by concerted dephosphorylation of Cdk targets involved in several cell biological processes.     

Prediction of cyclin-dependent kinase phosphorylation substrates

Protein phosphorylation, mediated by a family of enzymes called cyclin-dependent kinases (Cdks), plays a central role in the cell-division cycle of eukaryotes. Phosphorylation by Cdks directs the cell cycle by modifying the function of regulators of key processes such as DNA replication and mitotic progression. Here, we present a novel computational procedure to predict substrates of the cyclin-dependent kinase Cdc28 (Cdk1) in the Saccharomyces cerevisiae. Currently, most computational phosphorylation site prediction procedures focus solely on local sequence characteristics. In the present procedure, we model Cdk substrates based on both local and global characteristics of the substrates. Thus, we define the local sequence motifs that represent the Cdc28 phosphorylation sites and subsequently model clustering of these motifs within the protein sequences. This restraint reflects the observation that many known Cdk substrates contain multiple clustered phosphorylation sites. The present strategy defines a subset of the proteome that is highly enriched for Cdk substrates, as validated by comparing it to a set of bona fide, published, experimentally characterized Cdk substrates which was to our knowledge, comprehensive at the time of writing. To corroborate our model, we compared its predictions with three experimentally independent Cdk proteomic datasets and found significant overlap. Finally, we directly detected in vivo phosphorylation at Cdk motifs for selected putative substrates using mass spectrometry.     

Molecular basis for the specificity of p27 toward cyclin-dependent kinases that regulate cell division

The cyclin-dependent kinase inhibitors (CKIs) bind to and directly regulate the catalytic activity of cyclin-dependent kinase (Cdk)/cyclin complexes involved in cell cycle control and do not regulate other, closely related Cdks. We showed previously that the CKI, p27, binds to Cdk2/cyclin A though a sequential mechanism that involves folding-on-binding. The first step in the kinetic mechanism is interaction of a small, highly dynamic domain of p27 (domain 1) with the cyclin subunit of the Cdk2/cyclin A complex, followed by much slower binding of a more lengthy and less flexible domain (domain 2) to Cdk2. The second step requires folding of domain 2 into the kinase inhibitory conformation. Rapid binding of p27 domain 1 to cyclin A tethers the inhibitor to the binary Cdk2/cyclin A complex, which reduces the entropic barrier associated with slow binding of domain 2 to the catalytic subunit. We show here that p27/cyclin interactions are an important determinant of p27 specificity towards cell cycle Cdks. We used surface plasmon resonance, limited proteolysis, mass spectrometry, and NMR spectroscopy to study the interaction of p27 with Cdk2/cyclin A, and with another Cdk complex, Cdk5/p25, that is involved in neurodegeneration. Importantly, Cdk5/p35 (the parent complex of Cdk5/p25) is not regulated by p27 in neurons. Our results show that p27 binds to Cdk5 and Cdk2 with similar, slow kinetics. However, p27 fails to interact with p25 within the Cdk5/p25 complex, which we believe prevents formation of a kinetically trapped, inhibited p27/Cdk5/p25 complex in vivo. The helical topology of p25 is very similar to that of cyclin A. However, p25 lacks the MRAIL sequence in one helix that, in the cell cycle cyclins, mediates specific interactions with domain 1 of p21 and p27. Our results strongly suggest that p21 and p27, related Cdk inhibitors, select their cell cycle regulatory Cdk targets by binding specifically to the cyclin subunit of these Cdk/cyclin complexes as a first step in a sequential, folding-on-binding mechanism.     

Cdk5 phosphorylation of FAK regulates centrosome-associated miocrotubules and neuronal migration

Cdk5 is a member of the cyclin-dependent kinase (Cdk) family. Unlike other Cdks that promote cell cycle, Cdk5 is activated in postmitotic neurons and critically regulates neuronal migration by phosphorylating its substrates during brain development. Recently, we found that Cdk5 phosphorylates focal adhesion kinase (FAK) at Serine 732 in vitro and is responsible for this phosphorylation in the developing brain. Our experiments using a phospho-specific antibody and an S732-unphosphorylatable mutant FAK suggest that S732 phosphorylation may regulate a centrosome-associated microtubule structure to promote nuclear translocation, a critical step in neuronal migration. S732 phosphorylation does not directly impact on the kinase activity of FAK, but appears to prevent the accumulation of FAK at the centrosome. Our study reveals a similarity between Cdk5 and Cdk1 in the regulation of neuronal migration and cell division, respectively. In addition, our study implicates FAK in a signaling pathway that directly regulates microtubules.     

Identification of Mcm2 phosphorylation sites by S-phase-regulating kinases

Minichromosome maintenance 2-7 proteins play a pivotal role in replication of the genome in eukaryotic organisms. Upon entry into S-phase several subunits of the MCM hexameric complex are phosphorylated. It is thought that phosphorylation activates the intrinsic MCM DNA helicase activity, thus allowing formation of active replication forks. Cdc7, Cdk2, and ataxia telangiectasia and Rad3-related kinases regulate S-phase entry and S-phase progression and are known to phosphorylate the Mcm2 subunit. In this work, by in vitro kinase reactions and mass spectrometry analysis of the products, we have mapped phosphorylation sites in the N terminus of Mcm2 by Cdc7, Cdk2, Cdk1, and CK2. We found that Cdc7 phosphorylates Mcm2 in at least three different sites, one of which corresponds to a site also reported to be phosphorylated by ataxia telangiectasia and Rad3-related. Three serine/proline sites were identified for Cdk2 and Cdk1, and a unique site was phosphorylated by CK2. We raised specific anti-phosphopeptide antibodies and found that all the sites identified in vitro are also phosphorylated in cells. Importantly, although all the Cdc7-dependent Mcm2 phosphosites fluctuate during the cell cycle with kinetics similar to Cdc7 kinase activity and Cdc7 protein levels, phosphorylation of Mcm2 in the putative cyclin-dependent kinase (Cdk) consensus sites is constant during the cell cycle. Furthermore, our analysis indicates that the majority of the Mcm2 isoforms phosphorylated by Cdc7 are not stably associated with chromatin. This study forms the basis for understanding how MCM functions are regulated by multiple kinases within the cell cycle and in response to external perturbations.     

Cyclin dependent kinase 5 and its interacting proteins in cell death induced in vivo by cyclophosphamide in developing mouse embryos

Activation or inactivation of members of the cyclin-dependent kinase family is important during cell cycle progression. However, Cdk5, a member of this family that was originally identified because of its high structural homology to Cdc2, is activated during cell differentiation and cell death but not during cell cycle progression. We previously demonstrated a correlation between the up-regulation of Cdk5 protein and kinase activity and cell death during development and pathogenesis. We report here that cyclophosphamide (CP) induces massive apoptotic cell death in mouse embryos and that Cdk5 is expressed in apoptotic cells displaying fragmented DNA. During CP-induced cell death, Cdk5 protein expression is substantially increased as detected by immunohistochemistry but not by Western blot, while its mRNA level remains the same as control, and its kinase activity is markedly elevated. The up-regulation of Cdk5 during CP-induced cell death is not due to de novo protein synthesis. We also examined p35, a regulatory protein of Cdk5 in neuronal differentiation. Using a yeast two-hybrid system, we isolated p35, a neuronal differentiation specific protein, as a protein that interacts with Cdk5 in CP-treated embryos. p35 mRNA level does not change, but the protein expression of p25, a truncated form of p35, is elevated during cell death in vivo, as established here, as well as during cell death in vitro. Our results suggest a role for Cdk5 and its regulatory proteins during CP induced cell death. These results further support the view that Cdk5 and its regulation may be key players in the execution of cell death regardless of how the cell dies, whether through biological mechanisms, disease states such as Alzheimer's disease, or induction by CP.     

Minimal Models for Cell-Cycle Control Based on Competitive Inhibition and?Multisite Phosphorylations of Cdk Substrates

The eukaryotic cell cycle is characterized by alternating oscillations in the activities of cyclin-dependent kinase (Cdk) and the anaphase-promoting complex (APC). Successful completion of the cell cycle is dependent on the precise, temporally ordered appearance of these activities. A modest level of Cdk activity is sufficient to initiate DNA replication, but mitosis and APC activation require an elevated Cdk activity. In present-day eukaryotes, this temporal order is provided by a complex network of regulatory proteins that control both Cdk and APC activities via sharp thresholds, bistability, and time delays. Using simple computational models, we show here that these dynamical features of cell-cycle organization could emerge in a control system driven by a single Cdk/cyclin complex and APC wired in a negative-feedback loop. We show that ordered phosphorylation of cellular proteins could be explained by multisite phosphorylation/dephosphorylation and competition of substrates for interconverting kinase (Cdk) and phosphatase. In addition, the competition of APC substrates for ubiquitylation can create and maintain sustained oscillations in cyclin levels. We propose a sequence of models that gets closer and closer to a realistic model of cell-cycle control in yeast. Since these models lack the elaborate control mechanisms characteristic of modern eukaryotes, they suggest that bistability and time delay may have characterized eukaryotic cell divisions before the current cell-cycle control network evolved in all its complexity.     

Cell Cycle Control by a Minimal Cdk Network

In present-day eukaryotes, the cell division cycle is controlled by a complex network of interacting proteins, including members of the cyclin and cyclin-dependent protein kinase (Cdk) families, and the Anaphase Promoting Complex (APC). Successful progression through the cell cycle depends on precise, temporally ordered regulation of the functions of these proteins. In light of this complexity, it is surprising that in fission yeast, a minimal Cdk network consisting of a single cyclin-Cdk fusion protein can control DNA synthesis and mitosis in a manner that is indistinguishable from wild type. To improve our understanding of the cell cycle regulatory network, we built and analysed a mathematical model of the molecular interactions controlling the G1/S and G2/M transitions in these minimal cells. The model accounts for all observed properties of yeast strains operating with the fusion protein. Importantly, coupling the model’s predictions with experimental analysis of alternative minimal cells, we uncover an explanation for the unexpected fact that elimination of inhibitory phosphorylation of Cdk is benign in these strains while it strongly affects normal cells. Furthermore, in the strain without inhibitory phosphorylation of the fusion protein, the distribution of cell size at division is unusually broad, an observation that is accounted for by stochastic simulations of the model. Our approach provides novel insights into the organization and quantitative regulation of wild type cell cycle progression. In particular, it leads us to propose a new mechanistic model for the phenomenon of mitotic catastrophe, relying on a combination of unregulated, multi-cyclin-dependent Cdk activities.     

Probing the Role of Nascent Helicity in p27 Function as a Cell Cycle Regulator

p27 regulates the activity of Cdk complexes which are the principal governors of phase transitions during cell division. Members of the p27 family of proteins, which also includes p21 and p57, are called the Cip/Kip cyclin-dependent kinase regulators (CKRs). Interestingly, the Cip/Kip CKRs play critical roles in cell cycle regulation by being intrinsically unstructured, a characteristic contrary to the classical structure-function paradigm. They exhibit nascent helicity which has been localized to a segment referred to as sub-domain LH. The nascent helicity of this sub-domain is conserved and we hypothesize that it is an important determinant of their functional properties. To test this hypothesis, we successfully designed and prepared p27 variants in which domain LH was either more or less helical with respect to the wild-type protein. Thermal denaturation experiments showed that the ternary complexes of the p27 variants bound to Cdk2/Cyclin A were less stable compared to the wild-type complex. Isothermal titration calorimetry experiments showed a decrease in the enthalpy of binding for all the mutants with respect to p27. The free energies of binding varied within a much narrower range. In vitro Cdk2 inhibition assays showed that the p27 variants exhibited disparate inhibitory potencies. Furthermore, when over-expressed in NIH 3T3 mouse fibroblast cells, the less helical p27 variants were less effective in causing cell cycle arrest relative to the wild-type p27. Our results indicate that the nascent helicity of sub-domain LH plays a key role mediating the biological function of p27.     

Evidence of key role of Cdk2 overexpression in pemphigus vulgaris

The pathogenesis of pemphigus vulgaris (PV) is still poorly understood. Autoantibodies present in PV patients can promote detrimental effects by triggering altered transduction of signals, which results in a final acantholysis. To investigate mechanisms involved in PV, cultured keratinocytes were treated with PV serum. PV sera were able to promote the cell cycle progression, inducing the accumulation of cyclin-dependent kinase 2 (Cdk2). Microarray analysis on keratinocytes detected that PV serum induced important changes in genes coding for one and the same proteins with known biological functions involved in PV disease (560 differentially expressed genes were identified). Then, we used two different approaches to investigate the role of Cdk2. First, small interfering RNA depletion of Cdk2 prevented cell-cell detachment induced by PV sera. Second, pharmacological inhibition of Cdk2 activity through roscovitine prevented blister formation and acantholysis in the mouse model of the disease. In vivo PV serum was found to alter multiple different pathways by microarray analysis (1463 differentially expressed genes were identified). Major changes in gene expression induced by roscovitine were studied through comparison of effects of PV serum alone and in association with roscovitine. The most significantly enriched pathways were cell communication, gap junction, focal adhesion, adherens junction, and tight junction. Our data indicate that major Cdk2-dependent multiple gene regulatory events are present in PV. This alteration may influence the evolution of PV and its therapy.     

Incomplete folding upon binding mediates Cdk4/cyclin D complex activation by tyrosine phosphorylation of inhibitor p27 protein

p27(Kip1) (p27), an intrinsically disordered protein, regulates the various Cdk/cyclin complexes that control cell cycle progression. The kinase inhibitory domain of p27 contains a cyclin-binding subdomain (D1), a Cdk-binding subdomain (D2), and a linker helix subdomain that connects D1 and D2. Here, we report that, despite extensive sequence conservation between Cdk4/cyclin D1 (hereafter Cdk4/cyclin D) and Cdk2/cyclin A, the thermodynamic details describing how the individual p27 subdomains contribute to equally high affinity binding to these two Cdk/cyclin complexes are strikingly different. Differences in enthalpy/entropy compensation revealed that the D2 subdomain of p27 folds incompletely when binding Cdk4/cyclin D versus Cdk2/cyclin A. Incomplete binding-induced folding exposes tyrosine 88 of p27 for phosphorylation by the nonreceptor tyrosine kinase Abl. Importantly, tyrosine phosphorylation (of p27) relieves Cdk inhibition by p27, enabling cell cycle entry. Furthermore, the interaction between a conserved hydrophobic patch on cyclin D and subdomain D1 is much weaker than that with cyclin A; consequently, a construct containing subdomains D1 and LH (p27-D1LH) does not inhibit substrate binding to Cdk4/cyclin D as it does to Cdk2/cyclin A. Our results provide a mechanism by which Cdk4 (within the p27/Cdk4/cyclin D complex) is poised to be activated by extrinsic mitogenic signals that impinge upon p27 at the earliest stage of cell division. More broadly, our results further illustrate the regulatory versatility of intrinsically disordered proteins.     

A Novel Member of the Cyclin-Dependent Kinase Family in Paramecium tetraurelia

Passage through the cell cycle in eukaryotes requires the successive activation of different cyclin-dependent protein kinases. Here, we describe the identification and characterization of a novel class of cyclin-dependent protein kinase, termed Cdk2, in the ciliate Paramecium tetraurelia. It is 301 amino acids long, 7 amino acids shorter than Cdk1, the CDK that is associated with macronuclear DNA synthesis. All the catalytic domains typical of protein kinases can be located within the sequence and putative regulatory phosphorylation sites equivalent to Thr14, Tyr15, and Thr161 in human CDK1 are also conserved. The 'PSTAIRE' region characteristic of most CDKs is perfectly conserved. Cdk2 shares only 48% homology to Cdk1 at the amino acid level, suggesting that the evolutionary separation of Cdk1 and Cdk2 is ancient, and implying that they have different roles in cell cycle regulation. Like Cdk1, Cdk2 does not bind to yeast p13suc1, even though it has better conservation of p13suc1 binding sites than Cdk1 does. The Cdk2 protein level is relatively constant throughout the vegetative cell cycle. Cdk2 exhibits kinase activity towards bovine histone H1 in vitro with the maximal level late in the cell cycle, suggesting it may be involved in the regulation of cytokinesis. Our results further support the view that an analogue of the cyclin-dependent kinase cell cycle regulatory system like that of yeast and higher eukaryotic cells operates in Paramecium and that a family of cyclin-dependent kinases may control different aspects of the Paramecium cell cycle.     

Dual G1 and G2 phase inhibition by a novel,selective Cdc25 inhibitor 6-chloro-7-[corrected](2-morpholin-4-ylethylamino)-quinoline-5,8-dione

The Cdc25 dual specificity phosphatases coordinate cell cycle progression, but potent and selective inhibitors have generally been unavailable. In the present study, we have examined one potential inhibitor, 6-chloro-7-(2-morpholin-4-ylethylamino)-quinoline-5,8-dione (NSC 663284), that was identified in the compound library of the National Cancer Institute [corrected]. We found that NSC 663284 arrested synchronized cells at both G(1) and G(2)/M phase, and blocked dephosphorylation and activation of Cdk2 and Cdk1 in vivo, as predicted for a Cdc25 inhibitor. Using the natural Cdc25A substrate, Tyr(15)-phosphorylated Cdk2/cyclin A, we demonstrated that NSC 663284 blocked reactivation of Cdk2/cyclin A kinase by Cdc25A catalytic domain in vitro. In-gel trypsin digestion followed by capillary liquid chromatography-electrospray ionization mass spectrometry and tandem mass spectrometry revealed the direct binding of NSC 663284 to one of the two serine residues in the active site loop HCEFSSER of the Cdc25A catalytic domain. Cdc25 binding and inhibition could contribute to the anti-proliferative activity of NSC 663284 and its ability to arrest cell cycle progression. Moreover, NSC 663284 should be a valuable reagent to probe the actions of Cdc25 phosphatases within cells and may also be useful structure for the design of more potent and selective antiproliferative agents.     

CDK activation by non-cyclin proteins

Progression through the cell cycle is regulated by cyclin-dependent kinases (CDKs), which associate with activating partners, named cyclins, to phosphorylate substrates efficiently. Cyclins are periodically synthesized and degraded during the cell cycle, playing a key role in the precise activation and inactivation of CDKs. However, CDKs can also be activated by other proteins, which lack sequence similarity to cyclins. These include the RINGO/Speedy proteins, which were originally identified as regulators of the meiotic cell cycle in Xenopus oocytes. Recently, five different mammalian RINGO/Speedy family members have been reported, all of which can bind to and directly activate Cdk1 and Cdk2.