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Multiple DNA-associated processes such as DNA repair, replication, and recombination are crucial for the maintenance of genome integrity. Here, we show a novel interaction between the transcription elongation factor Bur1-Bur2 and replication protein A (RPA), the eukaryotic single-stranded DNA-binding protein with functions in DNA repair, recombination, and replication. Bur1 interacted via its C-terminal domain with RPA, and bur1-ΔC mutants showed a deregulated DNA damage response accompanied by increased sensitivity to DNA damage and replication stress as well as increased levels of persisting Rad52 foci. Interestingly, the DNA damage sensitivity of an rfa1 mutant was suppressed by bur1 mutation, further underscoring a functional link between these two protein complexes. The transcription elongation factor Bur1-Bur2 interacts with RPA and maintains genome integrity during DNA replication stress.

The transcriptional coactivator Sub1 has been implicated in several aspects of mRNA metabolism in yeast, such as activation of transcription, termination, and 3'-end formation. Here, we present evidence that Sub1 plays a significant role in controlling phosphorylation of the RNA polymerase II large subunit C-terminal domain (CTD). We show that SUB1 genetically interacts with the genes encoding all four known CTD kinases, SRB10, KIN28, BUR1, and CTK1, suggesting that Sub1 acts to influence CTD phosphorylation at more than one step of the transcription cycle. To address this directly, we first used in vitro kinase assays, and we show that, on the one hand, SUB1 deletion increased CTD phosphorylation by Kin28, Bur1, and Ctk1 but, on the other, it decreased CTD phosphorylation by Srb10. Second, chromatin immunoprecipitation assays revealed that SUB1 deletion decreased Srb10 chromatin association on the inducible GAL1 gene but increased Kin28 and Ctk1 chromatin association on actively transcribed genes. Taken together, our data point to multiple roles for Sub1 in the regulation of CTD phosphorylation throughout the transcription cycle.

Cyclin Y is one of the most highly conserved members of the cyclin superfamily of proteins, which are famous for their crucial roles in regulating the cell cycle and transcription. Despite this high degree of conservation, very little was known about Cyclin Y function prior to a handful of studies published in this past year. Cyclins typically function by activating cyclin-dependent kinases (Cdks) and one insight has come from the identification of a Cdk that is activated by Cyclin Y. Yeast two-hybrid data first linked Cyclin Y with Cdk14, known as Eip63E in Drosophila or PFTAIRE1 in vertebrates. In Drosophila, both Cyclin Y and Eip63E are essential at many stages of development, from embryogenesis to metamorphosis and null mutants show a similar spectrum of developmental defects. In cultured cells, Cyclin Y and Eip63E were shown to phosphorylate the Wg/Wnt co-receptor Arrow/LRP6 in a ligand-independent manner. Eip63E is recruited to LRP6 at the plasma membrane by interacting with Cyclin Y, which is tethered to the membrane through an N-terminal myristoylation. Cyclin Y-dependent LRP6 phosphorylation appears to prime the receptor for subsequent ligand-dependent phosphorylation and activation of the canonical Wnt signaling pathway. Interestingly, Wnt receptor phosphorylation and signaling is maximal in G₂/M when Cyclin Y is at its highest levels, suggesting that Cyclin Y may serve to entrain Wnt signaling to the cell cycle. Given the wide range of roles for Wnt signaling during development, these studies may help explain why Cyclin Y is required at several developmental stages and in turn why these proteins are so well conserved in metazoans.

Cyclin Dependent Kinases (CDKs) regulate the cell division cycle, apoptosis, transcription and differentiation in addition to functions in the nervous system. They are regulated by their cyclin partners and by a variety of additional protein effectors (inhibitors, kinases, phosphatases). Each CDK serves its function by means of specific protein recognition properties. These are also responsible for the differential regulation of CDKs/Cyclin couples involved in processes as different as cell cycle and transcription. The structural features determining general and specific properties for CDKs/Cyclin complexes are analyzed. They reside in an overall conserved architecture with divergent spots used by the complexes to present themselves to specific substrates or other protein effectors.

Following the recent discovery and development of 2-anilino-4-(thiazol-5-yl)pyrimidine cyclin dependent kinase (CDK) inhibitors, a program was initiated to evaluate related ring-constrained analogues, specifically, 2-methyl- and 2-amino-N-aryl-4,5-dihydrothiazolo[4,5-h]quinazolin-8-amines for inhibition of CDKs. Here we report the rational design, synthesis, structure-activity relationships (SARs), and cellular mode-of-action profile of these second generation CDK inhibitors. Many of the analogues from this chemical series inhibit CDKs with very low nanomolar K(i) values. The most potent compound reported in this study inhibits CDK2 with an IC(50) of 0.7 nM ([ATP] = 100 microM). Furthermore, an X-ray crystal structure of 2-methyl-N-(3-(nitro)phenyl)-4,5-dihydrothiazolo[4,5-h]quinazolin-8-amine (11g), a representative from the chemical series in complex with cyclin A-CDK2, is reported, confirming the design rationale and expected binding mode within the CDK2 ATP binding pocket.

The cyclin-dependent kinases (CDKs) have been intensely studied because of their involvement in regulating essential cellular activities that include proliferation and transcription. A series of CDK2-containing structures have informed a general model for the molecular details of CDK activation and regulation. Recent structural studies of other members of the CDK family have lead to a re-appraisal of this model. In this review, we describe alternative CDK-cyclin assemblies taking the recently characterised CDK/cyclin complexes, CDK9/cyclinT1 and CDK4/cyclinD as examples. The differential effects of CDK phosphorylation on CDK activation state and substrate specificity are examined in the light of recent data on CDK2/cyclinA, CDK9/cyclinT, CDK4/cyclinD and Pho85/Pho80. We also present an overview of factors that affect CDK substrate specificity, and, in particular, the contributions that are made by the cyclin subunit. Finally, we review recent results that have helped to unravel the molecular mechanisms underlying the conflicting roles of the Cip/Kip CDK inhibitor family in CDK regulation., (Copyright 2009 Elsevier B.V. All rights reserved.)

Flavopiridol is a potent cyclin-dependant kinase (CDK) inhibitor and is in clinical trials for anticancer treatment. A limiting factor in its drug development has been the high dosage required in human clinical trials. The high dosage is suggested to be necessary because of significant flavopiridol binding to human blood serum. Albumin is the major protein component of blood serum and has been suggested as a likely high affinity binding target. We characterized the binding of human serum albumin to flavopiridol using circular dichroism (hereafter CD). Flavopiridol bound to human serum albumin has a diagnostic CD binding peak at 284 nm. The diagnostic CD binding peak was unobservable for flavopiridol with bovine serum albumin, using the same experimental conditions. However, under higher albumin concentrations a small CD signal is observed confirming, flavopiridol binds to bovine serum albumin as well., (© 2010 Wiley-Liss, Inc.)

Cdc28 is the main cyclin-dependent kinase (CDK) directing the cell cycle in the budding yeast Saccharomyces cerevisiae. Besides cyclin binding, Cdc28 requires phosphorylation by the Cak1 kinase to achieve full activity. We have previously isolated carboxy-terminal cdc28(CST) mutants that are temperature sensitive and exhibit high chromosome instability. Both phenotypes are suppressed by high copy Cak1 in a manner that is independent of its catalytic activity and conversely, combination of cdc28(CST) and cak1 mutations results in synthetic lethality. Altogether, these results suggest that for the Cdc28 complexes to remain stable and active, an interaction with Cak1 is needed via the carboxyl terminus of Cdc28. We report two-hybrid assay data that support this model, and results that indicate that actively growing yeast cells require an optimum Cdc28:Cak1 ratio. While Cak1 is constitutively active and expressed, dividing cells tightly regulate Cak1 protein levels to ensure presence of adequate levels of Cdc28 CDK activity.

Indirubins known to target mammalian cyclin-dependent kinases (CDKs) and glycogen synthase kinase (GSK-3) were tested for their antileishmanial activity. 6-Br-indirubin-3'-oxime (6-BIO), 6-Br-indirubin-3'acetoxime and 6-Br-5methylindirubin-3'oxime (5-Me-6-BIO) were the most potent inhibitors of Leishmania donovani promastigote and amastigote growth (half maximal inhibitory concentration (IC(50)) values < or =1.2 microM). Since the 6-Br substitution on the indirubin backbone greatly enhances the selectivity for mammalian GSK-3 over CDKs, we identified the leishmanial GSK-3 homologues, a short (LdGSK-3s) and a long one, focusing on LdGSK-3s which is closer to human GSK-3beta, for further studies. Kinase assays showed that 5-Me-6-BIO inhibited LdGSK-3s more potently than CRK3 (the CDK1 homologue in Leishmania), whilst 6-BIO was more selective for CRK3. Promastigotes treated with 5-Me-6-BIO accumulated in the S and G2/M cell-cycle phases and underwent apoptosis-like death. Interestingly, these phenotypes were completely reversed in parasites over-expressing LdGSK-3s. This finding strongly supports that LdGSK-3s is: (i) the intracellular target of 5-Me-6-BIO, and (ii) involved in cell-cycle control and in pathways leading to apoptosis-like death. 6-BIO treatment induced a G2/M arrest, consistent with inhibition of CRK3 and apoptosis-like death. These effects were partially reversed in parasites over-expressing LdGSK-3s suggesting that in vivo 6-BIO may also target LdGSK-3s. Molecular docking of 5-Me-6-BIO in CRK3 and 6-BIO in human GSK-3beta and LdGSK-3s active sites predict the existence of functional/structural differences that are sufficient to explain the observed difference in their affinity. In conclusion, LdGSK-3s is validated as a potential drug target in Leishmania and could be exploited for the development of selective indirubin-based leishmanicidals.

Phosphorylation plays essential roles in nearly every aspect of cell life. Protein kinases regulate signalling pathways and cellular processes that mediate metabolism, transcription, cell-cycle progression, differentiation, cytoskeleton arrangement and cell movement, apoptosis, intercellular communication, and neuronal and immunological functions. Protein kinases share a conserved catalytic domain, which catalyses the transfer of the gamma-phosphate of ATP to a serine, threonine or tyrosine residue in protein substrates. The kinase can exist in an active or inactive state regulated by a variety of mechanisms in different kinases that include control by phosphorylation, regulation by additional domains that may target other molecules, binding and regulation by additional subunits, and control by protein-protein association. This Novartis Medal Lecture was delivered at a meeting on protein evolution celebrating the 200th anniversary of Charles Darwin's birth. I begin with a summary of current observations from protein sequences of kinase phylogeny. I then review the structural consequences of protein phosphorylation using our work on glycogen phosphorylase to illustrate one of the more dramatic consequences of phosphorylation. Regulation of protein phosphorylation is frequently disrupted in the diseased state, and protein kinases have become high-profile targets for drug development. Finally, I consider recent advances on protein kinases as drug targets and describe some of our recent work with CDK9 (cyclin-dependent kinase 9)-cyclin T, a regulator of transcription.

Protein kinases enable cellular information processing. Although numerous human phosphorylation sites and their dynamics have been characterized, the evolutionary history and physiological importance of many signaling events remain unknown. Using target phosphoproteomes determined with a similar experimental and computational pipeline, we investigated the conservation of human phosphorylation events in distantly related model organisms (fly, worm, and yeast). With a sequence-alignment approach, we identified 479 phosphorylation events in 344 human proteins that appear to be positionally conserved over approximately 600 million years of evolution and hence are likely to be involved in fundamental cellular processes. This sequence-alignment analysis suggested that many phosphorylation sites evolve rapidly and therefore do not display strong evolutionary conservation in terms of sequence position in distantly related organisms. Thus, we devised a network-alignment approach to reconstruct conserved kinase-substrate networks, which identified 778 phosphorylation events in 698 human proteins. Both methods identified proteins tightly regulated by phosphorylation as well as signal integration hubs, and both types of phosphoproteins were enriched in proteins encoded by disease-associated genes. We analyzed the cellular functions and structural relationships for these conserved signaling events, noting the incomplete nature of current phosphoproteomes. Assessing phosphorylation conservation at both site and network levels proved useful for exploring both fast-evolving and ancient signaling events. We reveal that multiple complex diseases seem to converge within the conserved networks, suggesting that disease development might rely on common molecular networks.

The C-terminal domain (CTD) of RNA polymerase II regulates transcription through spatially and temporally coordinated events. Previous work had established that the CTD binds DNA but the significance of this interaction has not been determined. The present work shows that the CTD binds DNA in its unphosphorylated form, the form in which it is present in the pre-initiation complex. The CTD/DNA complex is recognized by and is phosphorylated by Cdk7 but not by Cdk9. Model-building studies indicate the structural mechanism underlying such specificity involves interaction of Cdk7 with DNA in the context of the CTD/DNA complex. The model has been tested by mutagenesis experiments. CTD dissociates from DNA following phosphorylation by Cdk7, allowing transcription initiation. The CTD then becomes accessible for further phosphorylation by Cdk9 that drives the transition to transcription elongation.

The four proteins CDK8, cyclin C, Med12, and Med13 can associate with Mediator and are presumed to form a stable "CDK8 subcomplex" in cells. We describe here the isolation and enzymatic activity of the 600-kDa CDK8 subcomplex purified directly from human cells and also via recombinant expression in insect cells. Biochemical analysis of the recombinant CDK8 subcomplex identifies predicted (TFIIH and RNA polymerase II C-terminal domain [Pol II CTD]) and novel (histone H3, Med13, and CDK8 itself) substrates for the CDK8 kinase. Notably, these novel substrates appear to be metazoan-specific. Such diverse targets imply strict regulation of CDK8 kinase activity. Along these lines, we observe that Mediator itself enables CDK8 kinase activity on chromatin, and we identify Med12--but not Med13--to be essential for activating the CDK8 kinase. Moreover, mass spectrometry analysis of the endogenous CDK8 subcomplex reveals several associated factors, including GCN1L1 and the TRiC chaperonin, that may help control its biological function. In support of this, electron microscopy analysis suggests TRiC sequesters the CDK8 subcomplex and kinase assays reveal the endogenous CDK8 subcomplex--unlike the recombinant submodule--is unable to phosphorylate the Pol II CTD.

The complex CDK3-cyclin is involved in the control of the progression of G0. While the mechanisms governing early and late G1 progression are well understood, very little is known about the G0-G1 transition. Human CDK3 is closely related to cyclin-dependent kinase 2 (CDK2). Since there is no crystallographic structure of human CDK3, this work describes for the first time a molecular model of human CDK3 complexed with several inhibitors. Comparison of the binary complexes with different inhibitors strongly indicates that those inhibitors should inhibit CDK3 as well as CDK2.

Background: Intra-cellular information exchange, propelled by cascades of interacting signalling proteins, is essential for the proper functioning and survival of cells. Now that the interactome of several organisms is being mapped and several structural mechanisms of cooperativity at the molecular level in proteins have been elucidated, the formalization of this fundamental quantity, i.e. information, in these very diverse biological contexts becomes feasible., Results: We show here that Shannon's mutual information quantifies information in biological system and more specifically the cooperativity inherent to the assembly of macromolecular complexes. We show how protein complexes can be considered as particular instances of noisy communication channels. Further we show, using a portion of the p27 regulatory pathway, how classical equilibrium thermodynamic quantities such as binding affinities and chemical potentials can be used to quantify information exchange but also to determine engineering properties such as channel noise and channel capacity. As such, this information measure identifies and quantifies those protein concentrations that render the biochemical system most effective in switching between the active and inactive state of the intracellular process., Conclusion: The proposed framework provides a new and original approach to analyse the effects of cooperativity in the assembly of macromolecular complexes. It shows the conditions, provided by the protein concentrations, for which a particular system acts most effectively, i.e. exchanges the most information. As such this framework opens the possibility of grasping biological qualities such as system sensitivity, robustness or plasticity directly in terms of their effect on information exchange. Although these parameters might also be derived using classical thermodynamic parameters, a recasting of biological signalling in terms of information exchange offers an alternative framework for visualising network cooperativity that might in some cases be more intuitive.

Cell cycle progression requires interaction between cyclin-dependent kinase B (CDKB) and cyclin B (CYCB). The seasonal expression patterns of the CDKB and CYCB homologues from Populus tomentosa Carr. were investigated, and effects of temperature and exogenous indole-3-acetic acid (IAA) on their expression were further studied in water culture experiments. Based on the differential responses of dormant cambium cells to exogenous IAA, four stages of cambium dormancy were confirmed for P. tomentosa: quiescence 1 (Q1), rest, quiescence 2-1 (Q2-1), and quiescence 2-2 (Q2-2). PtoCDKB and PtoCYCB transcripts were strongly expressed in the active phases, weakly in Q1, and almost undetectable from rest until late Q2-2. Climatic data analysis showed a correlation between daily air temperature and PtoCDKB and PtoCYCB expression patterns. Water culture experiments with temperature treatment further showed that a low temperature (4 degrees C) kept PtoCDKB and PtoCYCB transcripts at undetectable levels, while a warm temperature (25 degrees C) induced their expression in the cambium region. Meanwhile, water culture experiments with exogenous IAA treatment showed that induction of PtoCDKB and PtoCYCB transcription was independent of exogenous IAA. The results suggest that, in deciduous hardwood P. tomentosa growing in a temperate zone, the temperature in early spring is a vital environmental factor for cambium reactivation. The increasing temperature in early spring may induce CDKB and CYCB homologue transcription in the cambium region, which is necessary for cambium cell division.

Progression through the cell division cycle is controlled by a family of cyclin-dependent kinases (CDKs), the activity of which depends on their binding to regulatory partners (cyclins A-H). Deregulation of the activity of CDKs has been associated with the development of infectious, neurodegenerative, and proliferative diseases such as Alzheimer's, Parkinson's, or cancer. Most cancer cells contain mutations in the pathways that control the activity of CDKs. This observation led this kinase family to become a central target for the development of new drugs for cancer therapy. A range of structurally diverse molecules has been shown to inhibit the activity of CDKs through their activity as ATP antagonists. Nevertheless, the ATP binding sites on CDKs are highly conserved, limiting the kinase specificity of these inhibitors. Various genetic and crystallographic approaches have provided essential information about the mechanism of formation and activation of CDK-cyclin complexes, providing new ways to implement novel research strategies toward the discovery of new, more effective and selective drugs. Herein we review the progress made in the development of ATP-noncompetitive CDK-cyclin inhibitors.

Pho85 cyclins (Pcls), activators of the yeast cyclin-dependent kinase (CDK) Pho85, belong together with the p35 activator of mammalian CDK5 to a distinct structural cyclin class. Different Pcls target Pho85 to distinct substrates. Pcl5 targets Pho85 specifically to Gcn4, a yeast transcription factor involved in the response to amino acid starvation, eventually causing the degradation of Gcn4. Pcl5 is itself highly unstable, an instability that was postulated to be important for regulation of Gcn4 degradation. We used hybrids between different Pcls to circumscribe the substrate recognition function to the core cyclin box domain of Pcl5. Furthermore, the cyclin hybrids revealed that Pcl5 degradation is uniquely dependent on two distinct degradation signals: one N-terminal and one C-terminal to the cyclin box domain. Whereas the C-terminal degradation signal is independent of Pho85, the N-terminal degradation signal requires phosphorylation of a specific threonine residue by the Pho85 molecule bound to the cyclin. This latter mode of degradation depends on the SCF ubiquitin ligase. Degradation of Pcl5 after self-catalyzed phosphorylation ensures that activity of the Pho85/Pcl5 complex is self-limiting in vivo. We demonstrate the importance of this mechanism for the regulation of Gcn4 degradation and for cell growth under conditions of amino acid starvation.

CDC25 phosphatases are considered as attractive targets for anti-cancer therapy. To date, quinone derivatives are among the most potent inhibitors of CDC25 phosphatase activity. We present in this paper the synthesis and the biological evaluation of new quinolinedione and naphthoquinone derivatives, containing carboxylic or malonic acids groups introduced to mimic the role of the phosphate moieties of Cyclin-Dependent Kinase complexes. The most efficient compounds show inhibitory activity against CDC25B with IC(50) values in the 10 microM range, and are cytotoxic against HeLa cells.

Myocardial infarction (MI) is often followed by heart failure (HF), but the mechanisms precipitating the transition to HF remain largely unknown. A genomic profile was performed in a monkey model of MI, from the myocardium adjacent to chronic (2-month) MI followed by 3 weeks of pacing to develop HF. The transcript of the gene encoding the cell cycle-related kinase (CCRK) was down-regulated by 50% in HF heart compared with control (p<0.05), which was confirmed by quantitative PCR. The CCRK sequence cloned from a heart library showed a conservation of the N-terminal kinase domain when compared with the "generic" isoform cloned previously but a different C-terminal half due to alternative splicing with frameshift. The homology of the cardiac sequence was 100% between mice and humans. Expression of the corresponding protein, measured upon generation of a monoclonal antibody, was limited to heart, liver, and kidney. Upon overexpression in cardiac myocytes, both isoforms promote cell growth and reduce apoptosis by chelerythrine (p<0.05 versus control). Using a yeast two-hybrid screening, we found an interaction of the generic but not the cardiac CCRK with cyclin H and casein kinase 2. In addition, only the generic CCRK phosphorylates the cyclin-dependent kinase 2, which was accompanied by a doubling of myocytes in the S and G(2) phases of the cell cycle (p < 0.05 versus control). Therefore, the heart expresses a splice variant of CCRK, which promotes cardiac cell growth and survival; differs from the generic isoform in terms of protein-protein interactions, substrate specificity and regulation of the cell cycle; and is down-regulated significantly in HF.

Mediator is an essential transcriptional cofactor of RNA polymerase II (Pol II) in eukaryotes. This cofactor is a large complex containing up to 30 subunits and consisting of four modules: head, middle, tail, and CDK/Cyclin. Generally, Mediator connects transcriptional regulators, cofactors, chromatin regulators, and chromatin remodellers, with the pre-initiation complex to provide a platform for the assembly of these factors. Many previous studies have revealed that CDK8, a subunit of the CDK/Cyclin module, is one of the key subunits mediating the pivotal roles of Mediator in transcriptional regulation. In addition to CDK8, CDK11 is conserved among vertebrates as a Mediator subunit and closely resembles CDK8. While the role of CDK8 has been studied extensively, little is known of the role of CDK11 in Mediator. We purified human CDK11 (hCDK11)-containing protein complexes from an epitope-tagged hCDK11-expressing HeLa cell line and found that hCDK11 could independently form Mediator complexes devoid of human CDK8 (hCDK8). To investigate the in vivo transcriptional activity of the complex, we employed a luciferase assay. Although hCDK11 has nearly 80% amino acid sequence identity to hCDK8, siRNA-knockdown study revealed that hCDK8 and hCDK11 possess opposing functions in viral activator VP16-dependent transcriptional regulation.

An estimated one-third of all proteins in higher eukaryotes are regulated by phosphorylation by protein kinases (PKs). Although plant genomes encode more than 1000 PKs, the substrates of only a small fraction of these kinases are known. By mass spectrometry of peptides from cytoplasmic- and nuclear-enriched fractions, we determined 303 in vivo phosphorylation sites in Arabidopsis proteins. Among 21 different PKs, 12 were phosphorylated in their activation loops, suggesting that they were in their active state. Immunoblotting and mutational analysis confirmed a tyrosine phosphorylation site in the activation loop of a GSK3/shaggy-like kinase. Analysis of phosphorylation motifs in the substrates suggested links between several of these PKs and many target sites. To perform quantitative phosphorylation analysis, peptide arrays were generated with peptides corresponding to in vivo phosphorylation sites. These peptide chips were used for kinome profiling of subcellular fractions as well as H 2O 2-treated Arabidopsis cells. Different peptide phosphorylation profiles indicated the presence of overlapping but distinct PK activities in cytosolic and nuclear compartments. Among different H 2O 2-induced PK targets, a peptide of the serine/arginine-rich (SR) splicing factor SCL30 was most strongly affected. SRPK4 (SR protein-specific kinase 4) and MAPKs (mitogen-activated PKs) were found to phosphorylate this peptide, as well as full-length SCL30. However, whereas SRPK4 was constitutively active, MAPKs were activated by H 2O 2. These results suggest that SCL30 is targeted by different PKs. Together, our data demonstrate that a combination of mass spectrometry with peptide chip phosphorylation profiling has a great potential to unravel phosphoproteome dynamics and to identify PK substrates.

CDK2 inhibitors containing the related bicyclic heterocycles pyrazolopyrimidines and imidazopyrazines were discovered through high-throughput screening. Crystal structures of inhibitors with these bicyclic cores and two more related ones show that all but one have a common binding mode featuring two hydrogen bonds (H-bonds) to the backbone of the kinase hinge region. Even though ab initio computations indicated that the imidazopyrazine core would bind more tightly to the hinge, pyrazolopyrimidines gain an advantage in potency through participation of N4 in an H-bond network involving two catalytic residues and bridging water molecules. Further insight into inhibitor/CDK2 interactions was gained from analysis of additional crystal structures. Significant gains in potency were obtained by optimizing the fit of hydrophobic substituents to the gatekeeper region of the ATP binding site. The most potent inhibitors have good selectivity.

Cyclin-dependent kinases (CDK) play a key role in coordinating cell division in all eukaryotes. We investigated the capability of cyclin-dependent kinases CDKA and CDKB from the green alga Chlamydomonas reinhardtii to complement a Saccharomyces cerevisiae cdc28 temperature-sensitive mutant. The full-length coding regions of algal CDKA and CDKB cDNA were amplified by RT-PCR and cloned into the yeast expression vector pYES-DEST52, yielding pYD52-CDKA and pYD52-CDKB. The S. cerevisiae cdc28-1N strain transformed with these constructs exhibited growth at 36 degrees C in inducing (galactose) medium, but not in repressing (glucose) medium. Microscopic observation showed that the complemented cells had the irregular cylindrical shape typical for G2 phase-arrested cells when grown on glucose at 36 degrees C, but appeared as normal budded cells when grown on galactose at 36 degrees C. Sequence analysis and complementation tests proved that both CDKA and CDKB are functional CDC28/cdc2 homologs in C. reinhardtii. The complementation of the mitotic phenotype of the S. cerevisiae cdc28-1N mutant suggests a mitotic role for both of the kinases.

The ability to sense and respond appropriately to environmental changes is a primary requirement of all living organisms. In response to phosphate limitation, Saccharomyces cerevisiae induces transcription of a set of genes involved in the regulation of phosphate acquisition from the ambient environment. A signal transduction pathway (the PHO pathway) mediates this response, with Pho85-Pho80 playing a vital role. Here we report the X-ray structure of Pho85-Pho80, a prototypic structure of a CDK-cyclin complex functioning in transcriptional regulation in response to environmental changes. The structure revealed a specific salt link between a Pho85 arginine and a Pho80 aspartate that makes phosphorylation of the Pho85 activation loop dispensable and that maintains a Pho80 loop conformation for possible substrate recognition. It further showed two sites on the Pho80 cyclin for high-affinity binding of the transcription factor substrate (Pho4) and the CDK inhibitor (Pho81) that are markedly distant to each other and the active site.

Evolutionary change in gene regulation is a key mechanism underlying the genetic component of organismal diversity. Here, we study evolution of regulation at the posttranslational level by examining the evolution of cyclin-dependent kinase (CDK) consensus phosphorylation sites in the protein subunits of the pre-replicative complex (RC). The pre-RC, an assembly of proteins formed during an early stage of DNA replication, is believed to be regulated by CDKs throughout the animals and fungi. Interestingly, although orthologous pre-RC components often contain clusters of CDK consensus sites, the positions and numbers of sites do not seem conserved. By analyzing protein sequences from both distantly and closely related species, we confirm that consensus sites can turn over rapidly even when the local cluster of sites is preserved, consistent with the notion that precise positioning of phosphorylation events is not required for regulation. We also identify evolutionary changes in the clusters of sites and further examine one replication protein, Mcm3, where a cluster of consensus sites near a nucleocytoplasmic transport signal is confined to a specific lineage. We show that the presence or absence of the cluster of sites in different species is associated with differential regulation of the transport signal. These findings suggest that the CDK regulation of MCM nuclear localization was acquired in the lineage leading to Saccharomyces cerevisiae after the divergence with Candida albicans. Our results begin to explore the dynamics of regulatory evolution at the posttranslational level and show interesting similarities to recent observations of regulatory evolution at the level of transcription.

Cyclin dependent kinases (CDK) associate with cyclins to regulate cell cycle progression and gene transcription by phosphorylating key proteins. The different cyclin-CDK complexes display differences in substrate specificities with substrates binding across a shallow, hydrophobic, substrate-binding pocket known as the cyclin groove. However the mechanism underlying this differential substrate recognition remains largely unknown and cannot be explained merely on the basis of sequence variability. A subset of cyclins, cyclins A2, E1 and B1 despite being structurally and functionally similar, show marked differences in their interactions with recruitment peptides derived from their substrate or inhibitor proteins p27, p21, p57, E2F1, p53, pRb and p107. While these peptides (characterized by a cyclin binding motif of four residues ZRXL where Z and X are cationic residues) inhibit the activity of cyclins A2 and E1, no such inhibition is observed for cyclin B1. Electrostatic potentials of cyclins A2, E1 and B1 show that anionic regions of cyclins A2 and E1 enable them to bind peptides while cationic regions at homologous locations in cyclin B1 abrogate binding. These arise from charged residues that are conserved. Mutations that switch these characters are suggested. Computed energetics of binding confirms this. Deregulation of the enzymatic activity of this class of enzymes is a ubiquitous feature of human neoplasia, but attempts to exploit this therapeutically have been confounded by a lack of understanding of the precise specificity of the different cyclin complexes. Here we begin to clarify this issue by explaining the mechanism by which cyclin B1 escapes regulation by the p21 family of CDKIs.

Although activation of A-type cyclin-dependent kinase (CDKA) is required for plant cell division, little is known about how CDKA is activated before commitment to cell division. Here, we show that auxin is required for the formation of active CDKA-associated complexes, promoting assembly of the complex in tobacco suspension culture Bright Yellow-2 (BY-2) cells. Protein gel blot analysis revealed that CDKA levels increased greatly after stationary-phase BY-2 cells were subcultured into fresh medium to re-enter the cell cycle. However, these increasing levels subsided when cells were subcultured into auxin-deprived medium, and a subtle increase was observed after subculturing into sucrose-deprived medium. Additionally, p13(suc1)-associated kinase activity did not increase significantly after subculturing into either auxin- or sucrose-deprived medium, but increased strongly after subculturing into medium containing both auxin and sucrose. Using gel filtration, we found that p13(suc1)-associated kinase activity against tobacco retinoblastoma-related protein was maximal in fractions corresponding to the molecular mass of the cyclin/CDKA complex. Interestingly, this peak distribution of high molecular-mass fractions of CDKA disappeared after cells were subcultured into auxin-deprived medium. These findings suggest an important role for auxin in the assembly of active CDKA-associated complexes.

Protein kinases represent promising anticancer drug targets. We describe here the meriolins, a new family of inhibitors of cyclin-dependent kinases (CDK). Meriolins represent a chemical structural hybrid between meridianins and variolins, two families of kinase inhibitors extracted from various marine invertebrates. Variolin B is currently in preclinical evaluation as an antitumor agent. A selectivity study done on 32 kinases showed that, compared with variolin B, meriolins display enhanced specificity toward CDKs, with marked potency on CDK2 and CDK9. The structures of pCDK2/cyclin A/variolin B and pCDK2/cyclin A/meriolin 3 complexes reveal that the two inhibitors bind within the ATP binding site of the kinase, but in different orientations. Meriolins display better antiproliferative and proapoptotic properties in human tumor cell cultures than their parent molecules, meridianins and variolins. Phosphorylation at CDK1, CDK4, and CDK9 sites on, respectively, protein phosphatase 1alpha, retinoblastoma protein, and RNA polymerase II is inhibited in neuroblastoma SH-SY5Y cells exposed to meriolins. Apoptosis triggered by meriolins is accompanied by rapid Mcl-1 down-regulation, cytochrome c release, and activation of caspases. Meriolin 3 potently inhibits tumor growth in two mouse xenograft cancer models, namely, Ewing's sarcoma and LS174T colorectal carcinoma. Meriolins thus constitute a new CDK inhibitory scaffold, with promising antitumor activity, derived from molecules initially isolated from marine organisms.

Background Information: Mitosis during the dinoflagellate cell cycle is unusual in that the nuclear envelope remains intact and segregation of the permanently condensed chromosomes uses a cytoplasmic mitotic spindle. To examine regulation of the dinoflagellate cell cycle in the context of these unusual nuclear features, it is necessary to isolate and characterize cell cycle regulators such as CDK (cyclin-dependent kinase)., Results: We report the characterization of a CDK from the dinoflagellate Lingulodinium polyedrum. This CDK reacts with an anti-PSTAIRE antibody and was identified by protein microsequencing after partial purification. The protein microsequence shows homology toward the Pho85/CDK5 clade of CDKs. Neither the amount nor the phosphorylation state changed over the course of the cell cycle, in agreement with results reported for CDK5 family members in other systems., Conclusions: We conclude we have probably isolated a dinoflagellate CDK5-like protein. The data reported here support the identification of this protein as a CDK5 homologue, and suggest that dinoflagellates may contain several CDK families.

Several derivatives of the native Srb10 proteins from Saccharomyces cerevisiae and Kluyveromyces lactis, with removed selected motifs, have been constructed in order to test their role in Srb10p function. It has been demonstrated that the ATP binding site is necessary for repression of FLO11, CYC7 and SPI1. Yeast Srb10p specific motifs CM-I and CM-II, outside the kinase domain, are also necessary to complement two mutant phenotypes in S. cerevisiae Deltasrb10 strains, the failure to growth in galactose at 37 degrees C and flocculation. They are also required to keep transcriptional repression of FLO11 in non-flocculants, and for aerobic repression of CYC7 and SPI1. Two-hybrid analyses revealed that, in Srb10p derivatives, the absence of these motifs decreases the interaction of Srb10p with its cyclin partner Srb11p and with the component Tup1p of the general co-repressor complex Tup1p-Ssn6p.

Cyclins, cyclin-dependent kinases, and a number of other proteins control the progression of plant cell cycle. Although extensive studies have revealed the roles of some cell cycle regulators and the underlying mechanisms in Arabidopsis, relatively a small number of cell cycle regulators were functionally analyzed in rice. In this study, we describe 41 regulators in the rice genome. Our results indicate that the rice genome contains a less number of the core cell cycle regulators than the Arabidopsis one does, although the rice genome is much larger than the Arabidopsis one. Eight groups of CDKs similar to those in Arabidopsis were identified in the rice genome through phylogenetic analysis, and the corresponding members in the different groups include E2F, CKI, Rb, CKS and Wee. The structures of the core cell regulators were relatively conserved between the rice and Arabidopsis genomes. Furthermore, the expression of the majority of the core cell cycle genes was spatially regulated, and the most closely related ones showed very similar patterns of expression, suggesting functional redundancy and conservation between the highly similar core cell cycle genes in rice and Arabidopsis. Following auxin or cytokinin treatment, the expression of the core cell cycle genes was either upregulated or downregulated, suggesting that auxin and/or cytokinin may directly regulate the expression of the core cell cycle genes. Our results provide basic information to understand the mechanism of cell cycle regulation and the functions of the rice cell cycle genes.

Cdk7, a member of the cyclin dependent protein kinase family, regulates the activities of other Cdks through phosphorylation on their activation segment, and hence contributes to control of the eukaryotic cell cycle. Cdk7 is itself phosphorylated on the activation segment. Cdk7 phosphorylates Cdk1, Cdk2, Cdk4, and Cdk6, but only Cdk1 and Cdk2 can phosphorylate Cdk7 and none of them is able to auto-phosphorylate. The activation segments of the Cdks are very similar in sequence. Their specificity does not appear to be dictated by the sequences surrounding the phosphorylation sites but by structural determinants at remote sites. Through mutagenesis studies, we have identified regions in Cdk2 responsible for its interaction with Cdk7. A model has been built that explains the molecular basis for the specificity observed in Cdk recognition. The two kinases are arranged in a quasi-symmetric head-to-tail arrangement in which the N-terminal lobe from one kinase docks against the C-terminal lobe from the other kinase, and the activation segments are within reach of the opposite catalytic sites. Further experiments demonstrate that cyclin A hydrophobic pocket is not a recruitment site for Cdk7., (2007 Wiley-Liss, Inc.)

We previously described the identification of the 3'end of an unknown gene CDK7 using differential display which appeared to be up-regulated in diabetic kidneys [R.A. Page, C.A. Morris, J.D. Williams, C.J. von Ruhland, A.N. Malik, Isolation of diabetes-associated kidney genes using differential display, Biochem. Biophys. Res. Commun. 232 (1997) 49-53]. Here we show that CDK7 is a putative thiol related gene which is regulated by glucose in human and rat renal cells. CDK7 mRNA increased by >threefold in cultured human mesangial cells grown in high glucose for 4 days. In the kidneys of the GK rat, a model of type II diabetes, CDK7 showed a steady age-related increase in mRNA, increasing to >sixfold in 40 week GK rats compared to normoglycemic age-matched Wistar rat kidneys, this increase correlates with progressive hyperglycemia. CDK7 mRNA is widely expressed, showing particularly high levels of expression in rat and human liver, and encodes a putative 338 amino acids highly conserved peptide with several conserved domains, including a cys-pro-arg-cys domain conserved in 15 diverse species which is similar to the catalytic centre of thioredoxin, suggesting a role in oxidative stress.

p27, an important cell cycle regulator, blocks the G(1)/S transition in cells by binding and inhibiting Cdk2/cyclin A and Cdk2/cyclin E complexes (Cdk2/E). Ubiquitination and subsequent degradation play a critical role in regulating the levels of p27 during cell cycle progression. Here we provide evidence suggesting that both Cdk2/E and phosphorylation of Thr(187) on p27 are essential for the recognition of p27 by the SCF(Skp2/Cks1) complex, the ubiquitin-protein isopeptide ligase (E3). Cdk2/E provides a high affinity binding site, whereas the phosphorylated Thr(187) provides a low affinity binding site for the Skp2/Cks1 complex. Furthermore, binding of phosphorylated p27/Cdk2/E to the E3 complex showed positive cooperativity. Consistently, p27 is also ubiquitinated in a similarly cooperative manner. In the absence of p27, Cdk2/E and Cks1 increase Skp2 phosphorylation. This phosphorylation enhances Skp2 auto-ubiquitination, whereas p27 inhibits both phosphorylation and auto-ubiquitination of Skp2.

Little is known about how cells regulate the size of their organelles. In this study, we find that proper flagellar length control in Chlamydomonas reinhardtii requires the activity of a new member of the cyclin-dependent kinase (CDK) family, which is encoded by the LF2 (long flagella 2) gene. This novel CDK contains all of the important residues that are essential for kinase activity but lacks the cyclin-binding motif PSTAIRE. Analysis of genetic lesions in a series of lf2 mutant alleles and site-directed mutagenesis of LF2p reveals that improper flagellar length and defective flagellar assembly correlate with the extent of disruption of conserved kinase structures or residues by mutations. LF2p appears to interact with both LF1p and LF3p in the cytoplasm, as indicated by immunofluorescence localization, sucrose density gradients, cell fractionation, and yeast two-hybrid experiments. We propose that LF2p is the catalytic subunit of a regulatory kinase complex that controls flagellar length and flagellar assembly.

As in other eukaryotes, progression through the cell cycle in plants is governed by cyclin-dependent kinases. Phosphorylation of a canonical Thr residue in the T-loop of the kinases is required for high enzyme activity in animals and yeast. We show that the Arabidopsis thaliana Cdc2(+)/Cdc28 homolog CDKA;1 is also phosphorylated in the T-loop and that phosphorylation at the conserved Thr-161 residue is essential for its function. A phospho-mimicry T161D substitution restored the primary defect of cdka;1 mutants, and although the T161D substitution displayed a dramatically reduced kinase activity with a compromised ability to bind substrates, homozygous mutant plants were recovered. The rescue by the T161D substitution, however, was not complete, and the resulting plants displayed various developmental abnormalities. For instance, even though flowers were formed, these plants were completely sterile as a result of a failure of the meiotic program, indicating that different requirements for CDKA;1 function are needed during plant development.

Transient protein-protein interactions have key regulatory functions in many of the cellular processes that are implicated in cancerous growth, particularly the cell cycle. Targeting these transient interactions as therapeutic targets for anticancer drug development seems like a good idea, but it is not a trivial task. This Review discusses the issues and difficulties that are encountered when considering these transient interactions as drug targets, using the example of the cell division cycle 25 (Cdc25) phosphatases and their cyclin-dependent kinase (CDK)-cyclin protein substrates.

Leafy spurge (Euphorbia esula L.) is a deep-rooted perennial weed that propagates both by seeds and underground adventitious buds located on the crown and roots. To enhance our understanding of growth and development during seed germination and vegetative propagation, a leafy spurge gene (Accession No. AF230740) encoding a CDK-activating kinase (Ee;CDKF;1) involved in cell-cycle progression was identified, and its function was confirmed based on its ability to rescue a yeast temperature-sensitive CAK mutant (GF2351) and through in vitro kinase assays. Site-directed mutagenesis of Ee;CDKF;1 indicated that two threonine residues (Thr291 and Thr296) were mutually responsible for intra-molecular autophosphorylation and for phosphorylating its substrate protein, cyclin-dependent kinase (CDK). Polyclonal antibodies generated against the Ee;CDKF;1 protein or against a phosphorylated Ee;CDKF;1 peptide [NERYGSL(pT)SC] were used to examine abundance and phosphorylation of CDKF;1 during seed germination and bud growth. The levels of CDKF;1 were lower in dry or imbibed seeds than in germinating seeds or seedlings. Differences in CDKF;1 were also observed during adventitious bud development; small buds appeared to have greater levels of CDKF;1 than large buds. Similar patterns of CDKF;1 expression were detected with either the polyclonal antibody developed using the CDKF;1 protein or the phosphorylated peptide. These results indicated that Thr291 is constitutively phosphorylated in vivo and associated with Ee;CDKF;1 activity. Our results further suggest that a certain level of CDKF;1 activity is maintained in most tissues and may be an important phenomenon for enzymes that regulate early steps in cell-cycle signaling pathways.

Molecular dynamics simulations were performed to elucidate the interactions of CDK2 and CDK5 complexes with three inhibitors: R-roscovitine, S-roscovitine, and indirubin-3'-oxime. The preference of the two complexes for R-roscovitine over the S enantiomer, as reported by the experiment, was also found by the simulations. More importantly, the simulations showed that the cause of the stronger affinity for the R enantiomer is the presence of an important hydrogen bond between R-roscovitine and the kinases not found with S-roscovitine. The simulations also showed two amino acid mutations in the active site of CDK5/R-roscovitine that favor binding-enhanced electrostatic contributions, making the inhibitor more effective for CDK5 than for CDK2. This suggests that the effectiveness of roscovitine-like inhibitors can be improved by enhancing their electrostatic interaction with the kinases. Finally, molecular mechanics-Possion-Boltzmann/surface area calculations of the CDK5/indirubin-3'-oxime system in both water-excluded and water-included environments gave significantly different electrostatic contributions to the binding. The simulations detected the displacement of a water molecule in the active site of the water-included CDK/indirubin-3'-oxime system. This resulted in a more conserved binding pattern than the water-excluded structure. Hence, in the design of new indirubin-like inhibitors, it is important to include the water molecule in the analysis.

D-type cyclins are key regulators of the cell division cycle. In association with Cyclin Dependent Kinases (CDK) 2/4/6, they control the G1/S-phase transition in part by phosphorylation and inactivation of tumor suppressor of retinoblastoma family. Defective regulation of the G1/S transition is a well-known cause of cancer, making the cyclin D1-CDK4/6 complex a promising therapeutic target. Our objective is to develop inhibitors that would block the formation or the activation of the cyclin D1-CDK4/6 complex, using in silico docking experiments on a structural homology model of the cyclin D1-CDK4/6 complex. To this end we focused on the cyclin subunit in three different ways: (1) targeting the part of the cyclin D1 facing the N-terminal domain of CDK4/6, in order to prevent the dimer formation; (2) targeting the part of the cyclin D1 facing the C-terminal domain of CDK4/6, in order to prevent the activation of CDK4/6 by blocking the T-loop in an inactive conformation, and also to destabilize the dimer; (3) targeting the groove of cyclin D1 where p21 binds, in order to mimic its inhibition mode by preventing binding of cyclin D1-CDK4/6 complex to its targets. Our strategy, and the tools we developed, will provide a computational basis to design lead compounds for novel cancer therapeutics, targeting a broad range of proteins involved in the regulation of the cell cycle.

Skp2 is the substrate recognition subunit of the multi-subunit ubiquitin ligase SCF(Skp2). It consists of an N-terminal F-box domain that binds to the Skp1 subunit and thereby tethers it to the SCF catalytic core, and an elongated C-terminal domain comprising ten Leucine-rich repeats (LRR) that binds the substrate. A small accessory protein, Cks1, is required for SCF(Skp2) to target certain substrates, including the Cyclin-dependent kinase inhibitor p27. Here we have used hydrogen/deuterium exchange monitored by mass spectrometry to investigate the mode of action of Cks1 on SCF(Skp2). We show that complex formation between Cks1 and Skp2 causes conformational changes in both proteins in regions distant from the respective binding sites. We find that Skp2 interacts with a localised region of Cks1 but the interaction causes a global change in the hydrogen exchange behaviour of Cks1. Also, whilst Cks1 binds to the most C-terminal LRRs of the elongated Skp2 molecule, the interaction induces conformational changes at the distant N-terminal LRRs, close to the F-box motif. Further, binding of Cks1 to Skp2 significantly stabilises the interaction between Skp2 and Skp1. The results reveal that the C-terminal substrate recognition region of Skp2 is coupled to the N-terminal Skp1-binding region and thereby to the SCF catalytic core; this result adds to the model proposed previously that, whilst the principal function of the F-box protein is to recruit the substrate, an additional function may be to help position the substrate in an optimal way within the SCF complex to enable efficient ubiquitin transfer.

CDK8 (cyclin-dependent kinase 8), along with CycC, Med12, and Med13, form a repressive module (the Cdk8 module) that prevents RNA polymerase II (pol II) interactions with Mediator. Here, we report that the ability of the Cdk8 module to prevent pol II interactions is independent of the Cdk8-dependent kinase activity. We use electron microscopy and single-particle reconstruction to demonstrate that the Cdk8 module forms a distinct structural entity that binds to the head and middle region of Mediator, thereby sterically blocking interactions with pol II.

In budding yeast, Gin4, a Nim1-related kinase, plays an important role in proper organization of the septin ring at the mother-bud neck, a filamentous structure that is critical for diverse cellular processes including mitotic entry and cytokinesis. How Gin4 kinase activity is regulated is not known. Here we showed that a neck-associated Ser/Thr kinase Elm1, which is important for septin assembly, is critical for proper modification of Gin4 and its physiological substrate Shs1. In vitro studies with purified recombinant proteins demonstrated that Elm1 directly phosphorylates and activates Gin4, which in turn phosphorylates Shs1. Consistent with these observations, acute inhibition of Elm1 activity abolished mitotic Gin4 phosphorylation and Gin4-dependent Shs1 modification in vivo. In addition, a gin4 mutant lacking the Elm1-dependent phosphorylation sites exhibited an impaired localization to the bud-neck and, as a result, induced a significant growth defect with an elongated bud morphology. Thus, Elm1 regulates the septin assembly-dependent cellular events by directly phosphorylating and activating the Gin4-dependent pathway(s).

Protein kinases in general are known to be very flexible macromolecules. In this article, the conformational plasticity of the ATP binding site in cyclin dependent kinases is analyzed. Movement of the two lysine residues lining the ATP binding site are shown to play a major role in the conformational variability of the site. Linear models are developed to identify and quantify ligand properties that maximally influence the lysine side chain conformations. A few simple properties of the ligands are shown to account for more than 70% of the variation in the lysine conformations. The results are validated using test data and molecular simulation studies. Illustrative applications of the results of this analysis to finding the appropriate crystal structure for molecular docking and binding mode predictions of novel ligands are provided. This work provides a new approach to quantify ligand-induced conformational changes in the active sites of flexible proteins and to find the appropriate crystal structure for docking novel ligands.

The mechanism by which the epidermal growth factor receptor (EGFR) is activated upon dimerization has eluded definition. We find that the EGFR kinase domain can be activated by increasing its local concentration or by mutating a leucine (L834R) in the activation loop, the phosphorylation of which is not required for activation. This suggests that the kinase domain is intrinsically autoinhibited, and an intermolecular interaction promotes its activation. Using further mutational analysis and crystallography we demonstrate that the autoinhibited conformation of the EGFR kinase domain resembles that of Src and cyclin-dependent kinases (CDKs). EGFR activation results from the formation of an asymmetric dimer in which the C-terminal lobe of one kinase domain plays a role analogous to that of cyclin in activated CDK/cyclin complexes. The CDK/cyclin-like complex formed by two kinase domains thus explains the activation of EGFR-family receptors by homo- or heterodimerization.

A study by Zhang et al. (2006) in this issue of Cell provides compelling evidence that the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) is activated by the formation of an asymmetric dimer, with one kinase domain in the EGF-mediated dimer activating the other through an allosteric mechanism.

Tobacco (Nicotiana tabacum L.) cv Bright Yellow-2 (BY-2) cells are the most highly synchronizable plant cell culture, and previously we used them to analyze cell cycle regulation of cyclin-dependent kinases (CDKs) containing the cyclin binding motifs PSTAIRE (CDKA) and PPTA/TLRE (CDKB). Here we describe the analysis of tobacco CycD3 cyclins whose transcripts predominantly accumulate during G2 to M phase, which represents a unique feature of this type of cyclin D in plants. Although protein levels of CycD3s fluctuate with different patterns during the cell cycle, kinase assays revealed that the CycD3-associated kinases phosphorylate histone H1 and the tobacco retinoblastoma related protein (NtRBR1) with two peaks at the G1/S and G2/M boundaries. In vitro pull-down assays revealed that cell cycle-regulated CycD3s bind to CDKA, but more weakly than does CycD3;3, and that they also bind to CDKB and the CDK inhibitor NtKIS1a. Mutations in the cyclin box of the CycD3s showed that two amino acids are required for binding with CDKA and NtKIS1a, but no diminished interaction was observed with CDKB. A reconstituted kinase assay was adapted for use with bacterially produced GST-CycD3s, and kinase activity could be activated by incubation of extracts from exponentially growing BY-2 cells. Such activated complexes contained CDKA and CDKB, and the reconstituted GST-CycD3 mutants, retaining binding ability to CDKB, showed kinase activity, suggesting that these cell cycle-regulated CycD3s form active complexes with both A- and B-type CDKs in vitro.

Pctaire1, a member of the cyclin-dependent kinase (Cdk)-related family, has recently been shown to be phosphorylated and regulated by Cdk5/p35. Although Pctaire1 is expressed in both neuronal and non-neuronal cells, its precise functions remain elusive. We performed a yeast two-hybrid screen to identify proteins that interact with Pctaire1. N-Ethylmaleimide-sensitive fusion protein (NSF), a crucial factor in vesicular transport and membrane fusion, was identified as one of the Pctaire1 interacting proteins. We demonstrate that the D2 domain of NSF, which is required for the oligomerization of NSF subunits, binds directly to and is phosphorylated by Pctaire1 on serine 569. Mutation of this phosphorylation site on NSF (S569A) augments its ability to oligomerize. Moreover, inhibition of Pctaire1 activity by transfecting its kinase-dead (KD) mutant into COS-7 cells enhances the self-association of NSF. Interestingly, Pctaire1 associates with NSF and synaptic vesicle-associated proteins in adult rat brain. To investigate whether Pctaire1 phosphorylation of NSF is involved in regulation of Ca(2+)-dependent exocytosis, we examined the effect of expressing Pctaire1 or NSF phosphorylation mutants on the regulated secretion of growth hormone from PC12 cells. Interestingly, expression of either Pctaire1-KD or NSF-S569A in PC12 cells significantly increases high K(+)-stimulated growth hormone release. Taken together, our findings provide the first demonstration that Pctaire1 phosphorylation of NSF regulates the ability of NSF to oligomerize, implicating an unexpected role of this kinase in modulating exocytosis. These findings open a new avenue of research in studying the functional roles of Pctaire1 in the nervous system.