Search

CrkRS is a Cdc2-related protein kinase that contains an arginine- and serine-rich (SR) domain, a characteristic of the SR protein family of splicing factors, and is proposed to be involved in RNA processing. However, whether it acts together with a cyclin and at which steps it may function to regulate RNA processing are not clear. Here, we report that CrkRS interacts with cyclin L1 and cyclin L2, and thus rename it as the long form of cyclin-dependent kinase 12 (CDK12(L)). A shorter isoform of CDK12, CDK12(S), that differs from CDK12(L) only at the carboxyl end, was also identified. Both isoforms associate with cyclin L1 through interactions mediated by the kinase domain and the cyclin domain, suggesting a bona fide CDK/cyclin partnership. Furthermore, CDK12 isoforms alter the splicing pattern of an E1a minigene, and the effect is potentiated by the cyclin domain of cyclin L1. When expression of CDK12 isoforms is perturbed by small interfering RNAs, a reversal of the splicing choices is observed. The activity of CDK12 on splicing is counteracted by SF2/ASF and SC35, but not by SRp40, SRp55, and SRp75. Together, our findings indicate that CDK12 and cyclin L1/L2 are cyclin-dependent kinase and cyclin partners and regulate alternative splicing.

Progress has been made recently in the structure-based optimization of novel cell cycle antitumor therapeutics based on cyclin-dependent kinase (CDK) inhibition. A novel inhibitor series based on the 2-amino-4-heteroaryl-pyrimidine scaffold was discovered using the LIDAEUS high-throughput docking methodology, and was subsequently optimized for CDK2 potency through information provided by crystallographic complex structures. A computational study of CDK4 inhibitors led to the incorporation of selectivity determinants into a pyrimidine pharmacophore to generate isoform-specific inhibitors. In addition, molecules from the inhibitor series have been crystallized in complex structures with both monomeric inactive CDK2 and an active complex of CDK2 bound to cyclin A or E. This crystallization revealed that significant differences exist in the affinity of the inhibitors for active and inactive states of CDK2. Information on differences in affinity facilitates the prediction of experimental binding of inhibitors and allows for the further development of structure-guided design.

The synthesis, characterization and biological activity of the first zinc(II) complexes with potent inhibitors of cyclin-dependent kinases (CDKs) derived from 6-benzylaminopurine are described. Based on the results following from elemental analyses, infrared, NMR and ES+MS (electrospray mass spectra in the positive ion mode) spectroscopies, conductivity data, thermal analysis and X-ray structures, the tetrahedral Zn(II) complexes of the compositions [Zn(Olo)Cl(2)](n) (1), [Zn(iprOlo)Cl(2)](n) (2), [Zn(BohH(+))Cl(3)] x H(2)O (3) and [Zn(iprOloH(+))Cl(3)] x H(2)O (4) have been prepared, where Olo=2-(2-hydroxyethylamino)-6-benzylamino-9-methylpurine (Olomoucine), iprOlo=2-(2-hydroxyethylamino)-6-benzylamino-9-isopropylpurine (i-propyl-Olomoucine), Boh=2-(3-hydroxypropylamino)-6-benzylamino-9-isopropylpurine (Bohemine). The 1D-polymeric chain structure for [Zn(Olo)Cl(2)](n) (1) as well as the monomeric one for [Zn(BohH(+))Cl(3)] x H(2)O (3) and [Zn(iprOloH(+))Cl(3)] x H(2)O (4) have been revealed unambiguously by single crystal X-ray analyses. The 1D-polymeric chain of 1 consists of Zn(Olo)Cl(2) monomeric units in which the Zn(II) ion is coordinated by two chlorine atoms and one oxygen atom of the 2-hydroxyethylamino group of Olomoucine. The next monomeric unit is bonded to Zn(II) through the N7 atom of a purine ring. Thus, each of Zn(II) ions is tetrahedrally coordinated and a ZnCl(2)NO chromophore occurs in the complex 1. The complexes 3 and 4 are mononuclear species with a distorted tetrahedral arrangement of donor atoms around the Zn(II) ion with a ZnCl(3)N chromophore. The corresponding CDK inhibitor, i.e., both Boh and iprOlo, is coordinated to Zn(II) via the N7 atom of the purine ring in 3 and 4. The cytotoxicity of the zinc(II) complexes against human melanoma, sarcoma, leukaemia and carcinoma cell lines has been determined as well as the inhibition of the CDK2/cyclin E kinase. A relationship between the structure and biological activity of the complexes is also discussed.

Cdk7 performs two essential but distinct functions as a CDK-activating kinase (CAK) required for cell-cycle progression and as the RNA polymerase II (Pol II) CTD kinase of general transcription factor IIH. To investigate the substrate specificity underlying this dual function, we created an analog-sensitive (AS) Cdk7 able to use bulky ATP derivatives. Cdk7-AS-cyclin H-Mat1 phosphorylates approximately 10-15 endogenous polypeptides in nuclear extracts. We identify seven of these as known and previously unknown Cdk7 substrates that define two classes: proteins such as Pol II and transcription elongation factor Spt5, recognized efficiently only by the fully activated Cdk7 complex, through sequences surrounding the site of phosphorylation; and CDKs, targeted equivalently by all active forms of Cdk7, dependent on substrate motifs remote from the phosphoacceptor residue. Thus, Cdk7 accomplishes dual functions in cell-cycle control and transcription not through promiscuity but through distinct, stringent modes of substrate recognition.

Cyclin-dependent protein kinases (CDKs) are attractive targets for drug discovery and efforts have led to the identification of novel CDK selective inhibitors in the development of treatments for cancers, neurological disorders, and infectious diseases. More recently, they have become the focus of rational drug design programs for the development of new antimalarial agents. CDKs are valid targets as they function as essential regulators of cell growth and differentiation. To date, several CDKs have been characterized from the genome of the malaria-causing protozoan Plasmodium falciparum. Our approach employs experimental and virtual screening methodologies to identify and refine chemical inhibitors of the parasite CDK Pfmrk, a sequence homologue of human CDK7. Chemotypes of Pfmrk inhibitors include the purines, quinolinones, oxindoles, and chalcones, which have sub-micromolar IC50 values against the parasite enzyme, but not the human CDKs. Additionally, we have developed and validated a pharmacophore, based on Pfmrk inhibitors, which contains two hydrogen bond acceptor functions and two hydrophobic sites, including one aromatic ring hydrophobic site. This pharmacophore has been exploited to identify additional compounds that demonstrate significant inhibitory activity against Pfmrk. A molecular model of Pfmrk designed using the crystal structure of human CDK7 highlights key amino acid substitutions in the ATP binding pocket. Molecular modeling and docking of the active site pocket with selective inhibitors has identified possible receptor-ligand interactions that may be responsible for inhibitor specificity. Overall, the unique biochemical characteristics associated with this protein, to include distinctive active site amino acid residues and variable inhibitor profiles, distinguishes the Pfmrk drug screen as a paradigm for CDK inhibitor analysis in the parasite.

Eukaryotic cell proliferation is controlled by growth factors and essential nutrients. In their absence, cells may enter into a quiescent state (G0). In Saccharomyces cerevisiae, the conserved protein kinase A (PKA) and rapamycin-sensitive TOR (TORC1) pathways antagonize G0 entry in response to carbon and/or nitrogen availability primarily by inhibiting the PAS kinase Rim15 function. Here, we show that the phosphate-sensing Pho80-Pho85 cyclin-cyclin-dependent kinase (CDK) complex also participates in Rim15 inhibition through direct phosphorylation, thereby effectively sequestering Rim15 in the cytoplasm via its association with 14-3-3 proteins. Inactivation of either Pho80-Pho85 or TORC1 causes dephosphorylation of the 14-3-3-binding site in Rim15, thus enabling nuclear import of Rim15 and induction of the Rim15-controlled G0 program. Importantly, we also show that Pho80-Pho85 and TORC1 converge on a single amino acid in Rim15. Thus, Rim15 plays a key role in G0 entry through its ability to integrate signaling from the PKA, TORC1, and Pho80-Pho85 pathways.

Cyclin-dependent kinases (CDKs) have been identified as potential targets for development of drugs, mainly against cancer. These studies generated a vast library of chemical inhibitors of CDKs, and some of these molecules can also inhibit kinases identified in the Plasmodium falciparum genome. Here we describe structural models for Protein Kinase 6 from P. falciparum (PfPK6) complexed with Roscovitine and Olomoucine. These models show clear structural evidence for differences observed in the inhibition, and may help designing inhibitors for PfPK6 generating new potential drugs against malaria.

The ubiquitin-mediated proteolysis of the Cdk2 inhibitor p27(Kip1) plays a central role in cell cycle progression, and enhanced degradation of p27(Kip1) is associated with many common cancers. Proteolysis of p27(Kip1) is triggered by Thr187 phosphorylation, which leads to the binding of the SCF(Skp2) (Skp1-Cul1-Rbx1-Skp2) ubiquitin ligase complex. Unlike other known SCF substrates, p27(Kip1) ubiquitination also requires the accessory protein Cks1. The crystal structure of the Skp1-Skp2-Cks1 complex bound to a p27(Kip1) phosphopeptide shows that Cks1 binds to the leucine-rich repeat (LRR) domain and C-terminal tail of Skp2, whereas p27(Kip1) binds to both Cks1 and Skp2. The phosphorylated Thr187 side chain of p27(Kip1) is recognized by a Cks1 phosphate binding site, whereas the side chain of an invariant Glu185 inserts into the interface between Skp2 and Cks1, interacting with both. The structure and biochemical data support the proposed model that Cdk2-cyclin A contributes to the recruitment of p27(Kip1) to the SCF(Skp2)-Cks1 complex.

Proteins participate in complex sets of interactions that represent the mechanistic foundation for much of the physiology and function of the cell. These protein-protein interactions are organized into exquisitely complex networks. The architecture of protein-protein interaction networks was recently proposed to be scale-free, with most of the proteins having only one or two connections but with relatively fewer 'hubs' possessing tens, hundreds or more links. The high level of hub connectivity must somehow be reflected in protein structure. What structural quality of hub proteins enables them to interact with large numbers of diverse targets? One possibility would be to employ binding regions that have the ability to bind multiple, structurally diverse partners. This trait can be imparted by the incorporation of intrinsic disorder in one or both partners. To illustrate the value of such contributions, this review examines the roles of intrinsic disorder in protein network architecture. We show that there are three general ways that intrinsic disorder can contribute: First, intrinsic disorder can serve as the structural basis for hub protein promiscuity; secondly, intrinsically disordered proteins can bind to structured hub proteins; and thirdly, intrinsic disorder can provide flexible linkers between functional domains with the linkers enabling mechanisms that facilitate binding diversity. An important research direction will be to determine what fraction of protein-protein interaction in regulatory networks relies on intrinsic disorder.

The production of mRNAs in all living organisms is an extremely complex process that includes multiple catalytic activities such as transcription, capping, splicing, polyadenylation, cleavage and export. All of these processes are controlled by a large group of proteins which form very dynamic complexes interacting with DNA and pre-mRNAs to coordinate these activities. Phosphorylations play a central role in regulating formation, activation and inactivation of these complexes. A growing number of protein kinases have been identified that are capable of phosphorylating proteins involved in mRNA production. Among them, Cyclin-dependent Kinases (CDKs) represent a family of serine/threonine protein kinases that become active upon binding to a cyclin regulatory partner. CDK/cyclin complexes were first identified as crucial regulators of cell cycle progression. More recently, CDK/cyclin complexes have also been implicated in transcription and mRNA processing leading to the concept of an intricate network of CDK/cyclin complexes regulating cell cycle, transcription and mRNA processing via cross-talk between multiple CDKs. In this review, we discuss the role of CDK/cyclin-dependent phosphorylation in the regulation of transcription and RNA splicing and highlight recent findings that indicate the involvement of CDK/cyclin complexes in connecting transcription and RNA splicing.

Malaria, with 300-500 million clinical cases resulting in 1-3 million fatalities a year, is one of the most deadly tropical diseases. As current antimalarial therapeutics become increasingly ineffective due to parasitic resistance, there exists an urgent need to develop and pursue new therapeutic strategies. Recent genome sequencing and molecular cloning projects have identified several enzymes from Plasmodium (P.) falciparum that may represent novel drug targets, including a family of proteins that are homologous to the mammalian cyclin-dependent kinases (CDKs). CDKs are essential for the control of the mammalian cell cycle and, based on the conservation of the CDKs across species, the plasmodial CDKs are expected to play a crucial role in parasitic growth. Here we present a 3D structural model of Pfmrk, a putative human CDK activating kinase (CAK) homolog in P. falciparum. Notable features of the present structural model include: (1) parameterization of the Mg2+ hexacoordination system using ab initio quantum chemical calculations to accurately represent the ATP-kinase interaction; and (2) comparison between the docking scores and measured binding affinities for a series of oxindole-based Pfmrk inhibitors of known activity. Detailed analysis of inhibitor-Pfmrk binding interactions enabled us to identify specific residues (viz. Met66, Met75, Met91, Met94 and Phe143) within the Pfmrk binding pocket that may play an important role in inhibitor binding affinity and selectivity. The availability of this Pfmrk structural model, together with insights gained from analysis of ligand-receptor interactions, should promote the rational design of potent and selective Pfmrk inhibitors as antimalarial therapeutics.

Although best known for its ability to inhibit Cyclin/Cdk complexes and the replication protein PCNA, p21(WAF1/CIP1) is a multifunctional protein that interacts with many cellular binding partners, including a number of transcriptional regulators. Previously, we characterized p21 derepression of the p300 sumoylation-dependent transcriptional repression domain, CRD1. Such repression domains are at least partially dependent upon recruitment of histone deacetylase (HDAC) complexes but the mechanism through which p21 selectively disrupts CRD1 activity remains unknown. Here, we demonstrate that distinct motifs in the C-terminus of p21 are required for regulation of p300 CRD1 function and that this effect does not correlate with Cyclin or PCNA binding. Through the creation of N-terminal glutathione-s-transferase fusion proteins, which also overcome the problems of instability that result from many p21 mutations, we investigated p21 binding to HDACs. Although p21 binds both Class I and Class II HDACs in vitro, only weak association with HDAC1 and 2 is seen in cells. Mutation of the p21 PCNA binding domain significantly increases this interaction suggesting that binding is mutually exclusive and only naturally occurs under certain conditions. Binding of HDACs also failed to correlate with CRD1 inducibility, suggesting that p21 targets other transcriptional repression complexes to mediate this effect.

Cyclin C binds the cyclin-dependent kinases CDK8 and CDK3, which regulate mRNA transcription and the cell cycle, respectively. The crystal structure of cyclin C reveals two canonical five-helix repeats and a specific N-terminal helix. In contrast to other cyclins, the N-terminal helix is short, mobile, and in an exposed position that allows for interactions with proteins other than the CDKs. A model of the CDK8/cyclin C pair reveals two regions in the interface with apparently distinct roles. A conserved region explains promiscuous binding of cyclin C to CDK8 and CDK3, and a non-conserved region may be responsible for discrimination of CDK8 against other CDKs involved in transcription. A conserved and cyclin C-specific surface groove may recruit substrates near the CDK8 active site. Activation of CDKs generally involves phosphorylation of a loop at a threonine residue. In CDK8, this loop is longer and the threonine is absent, suggesting an alternative mechanism of activation that we discuss based on a CDK8-cyclin C model.

Cyclin-dependent kinase 5 (Cdk5) is widely viewed as a possible target for a wide variety of neurological disorders. One pathological role attributed to Cdk5 is the abnormal phosphorylation of tau that may lead to the neuronal inclusions known as neurofibrillary tangles. A high through-put screen for inhibitors of Cdk5-mediated phosphorylation of tau resulted in three compounds with distinct mechanisms of action. One compound is competitive with ATP and has a high affinity for the Cdk5 ATP binding pocket. The second compound also competes with ATP, is noncompetitive with tau, and (uniquely among this class of inhibitors) displaces adjacent amino acid residues to make room for the nitrophenyl group. A third compound did not compete with ATP, but did compete with tau at low concentrations of tau. The SAR and charge optimization derived from cocrystals of the two ATP competitors along with cocrystals of three other ATP competitors map out the importance of filling and properly charging different regions of the ATP binding pocket. Taken together, this analysis shows how the structure of Cdk5 constrains the space of potential inhibitors and reveals a pocket unfilled in all of the structures. These leads could be a starting point for structure-based drug design of more potent and selective inhibitors.

Dynamin function is mediated in part through association of its proline-rich domain (PRD) with the Src homology 3 (SH3) domains of several putative binding proteins. To assess the specificity and kinetics of this process, we undertook surface plasmon resonance studies of the interaction between isolated PRDs of dynamin-1 and -2 and several purified SH3 domains. Glutathione S-transferase-linked SH3 domains bound with high affinity (K(D) approximately 10 nm to 1 microm) to both dynamin-1 and -2. The simplest interaction appeared to take place with the amphiphysin-SH3 domain; this bound to a single high affinity site (K(D) approximately 10 nm) in the C terminus of dynamin-1 PRD, as predicted by previous studies. Binding to the dynamin-2 PRD was also monophasic but with a slightly lower affinity (K(D) approximately 25 nm). Endophilin-SH3 binding to both dynamin-1 and -2 PRDs was biphasic, with one high affinity site (K(D) approximately 14 nm) in the N terminus of the PRD and another lower affinity site (K(D) approximately 60 nm) in the C terminus of dynamin-1. The N-terminal site in dynamin-2 PRD had a 10-fold lower affinity for endophilin-SH3. Preloading of dynamin-1 PRD with the amphiphysin-SH3 domain partially occluded binding of the endophilin-SH3 domain, indicating overlap between the binding sites in the C terminus, but endophilin was still able to interact with the high affinity N-terminal site. This shows that more than one SH3 domain can simultaneously bind to the PRD and suggests that competition probably occurs in vivo between different SH3-containing proteins for the limited number of PXXP motifs. Endophilin-SH3 binding to the high affinity site was disrupted when dynamin-1 PRD was phosphorylated with Cdk5, indicating that this site overlaps the phosphorylation sites, but amphiphysin-SH3 binding was unaffected. Other SH3 domains showed similarly complex binding characteristics, and substantial differences were noted between the PRDs from dynamin-1 and -2. For example, SH3 domains from c-Src, Grb2, and intersectin bound only to the C-terminal half of dynamin-2 PRD but to both the N- and C-terminal portions of dynamin-1 PRD. Thus, differential binding of SH3 domain-containing proteins to dynamin-1 and -2 may contribute to the distinct functions performed by these isoforms.

In order to understand the mechanisms of ligand binding and the interaction between the ligand and the cyclin-dependent kinase 10 (CDK10), a three-dimensional (3D) model of the CDK10 is generated based on the crystal structure of the cyclin-dependent kinase 2 (CDK2) (PDB code 1AQ1) by using InsightII/Homology module. With the aid of the molecular mechanics and molecular dynamics methods, the last refined model is obtained and is further assessed by PROFILE-3D and PROSTAT, which show that the refined model is reliable. With this model, a flexible docking study is performed and the results indicate that the Lys39 and Asp94 form hydrogen bonds and have strong nonbonding interaction with adenosine 5'-triphosphate (ATP). From the docking studies, we also suggest that the Leu141, Tyr21, and Val24 in CDK10 are three important determinant residues in binding as they have strong nonbonding interaction with ATP. The hydrogen bonding interactions also play an important role for the stability of the complex. Our results may be helpful for further experimental investigations.

Transporter ProP of Escherichia coli, a member of the major facilitator superfamily (MFS), acts as an osmosensor and an osmoregulator in cells and after purification and reconstitution in proteoliposomes. H(+)-osmoprotectant symport via ProP is activated when medium osmolality is elevated with membrane impermeant osmolytes. The three-dimensional structure of ProP was modeled with the crystal structure of MFS member GlpT as a template. This GlpT structure represents the inward (or cytoplasm)-facing conformation predicted by the alternating access model for transport. LacZ-PhoA fusion analysis and site-directed fluorescence labeling substantiated the membrane topology and orientation predicted by this model and most hydropathy analyses. The model predicts the presence of a proton pathway within the N-terminal six-helix bundle of ProP (as opposed to the corresponding pathway found within the C-terminal helix bundle of its paralogue, LacY). Replacement of residues within the N-terminal helix bundle impaired the osmotic activation of ProP, providing the first indication that residues outside the C-terminal domain are involved in osmosensing. Some residues that were accessible from the periplasmic side, as predicted by the structural model, were more susceptible to covalent labeling in permeabilized membrane fractions than in intact bacteria. These residues may be accessible from the cytoplasmic side in structures not represented by our current model, or their limited exposure in vivo may reflect constraints on transporter structure that are related to its osmosensory mechanism.

Inhibition of the cell cycle kinase, cyclin-dependent kinase-4 (Cdk4), is expected to provide an effective method for the treatment of proliferative diseases such as cancer. The pyrido[2,3-d]pyrimidin-7-one template has been identified previously as a privileged structure for the inhibition of ATP-dependent kinases, and good potency against Cdks has been reported for representative examples. Obtaining selectivity for individual Cdk enzymes, particularly Cdk4, has been challenging. Here, we report that the introduction of a methyl substituent at the C-5 position of the pyrido[2,3-d]pyrimidin-7-one template is sufficient to confer excellent selectivity for Cdk4 vs other Cdks and representative tyrosine kinases. Further optimization led to the identification of highly potent and selective inhibitors of Cdk4 that exhibit potent antiproliferative activity against human tumor cells in vitro. The most selective Cdk4 inhibitors were evaluated for antitumor activity against MDA-MB-435 human breast carcinoma xenografts in mice.

Yeast accumulate glycogen in response to nutrient limitation. The key enzymes of glycogen synthesis and degradation, glycogen synthase, and phosphorylase, are regulated by reversible phosphorylation. Phosphorylation inactivates glycogen synthase but activates phosphorylase. The kinases and phosphatases that control glycogen synthase are well characterized whilst the enzymes modifying phosphorylase are poorly defined. Here, we show that the cyclin-dependent protein kinase, Pho85p, which we have previously found to regulate glycogen synthase also controls the phosphorylation state of phosphorylase.

We describe a method to create ligands specific for a given protein family. The method is applied to generate ligand candidates for the cyclin-dependent kinase (CDK) family. The CDK family of proteins is involved in regulating the cell cycle by alternately activating and deactivating the cell's progression through the cycle. CDKs are activated by association with cyclin and are inhibited by complexation with small molecules. X-ray crystal structures are available for three of the thirteen known CDK family members: CDK2, CDK5 and CDK 6. In this work, we use novel computational approaches to design ligand candidates that are potentially inhibitory across the three CDK family members as well as more specific molecules which can potentially inhibit one or any combination of two of the three CDK family members. We define a new scoring term, SpecScore, to quantify the potential inhibitory power of the generated structures. According to a search of the World Drug Alerts, the highest scoring SpecScore molecule that is specific for the three CDK family members shows very similar chemical characteristics and functional groups to numerous molecules known to deactivate several members of the CDK family.

The yeast cyclin-dependent kinase Srb10 phosphorylates various transcriptional activators as they activate transcription, and acidic transcriptional activating domains found on several activators directly bind Srb10. Here we show that the interaction between Srb10 (with its associated cyclin Srb11) and each of several different activating regions, in vitro, leads to the phosphorylation of peptide sequences attached to but outside of the activating regions themselves. In some cases, residues within the activating regions are also phosphorylated. The results define a mechanism by which a kinase is recruited to alternate substrates with diverse physiological consequences.

Cyclin-dependent kinase 11 (CDK11; also named PITSLRE) is part of the large family of p34(cdc2)-related kinases whose functions appear to be linked with cell cycle progression, tumorigenesis, and apoptotic signaling. The mechanism that CDK11(p58) induces apoptosis is not clear. Some evidences suggested beta1,4-galactosyltransferase 1 (beta1,4-GT 1) might participate in apoptosis induced by CDK11(p58). In this study, we demonstrated that ectopically expressed beta1,4-GT 1 increased CDK11(p58)-mediated apoptosis induced by cycloheximide (CHX). In contrast, RNAi-mediated knockdown of beta1,4-GT 1 effectively inhibited apoptosis induced by CHX in CDK11(p58)-overexpressing cells. For example, the cell morphological and nuclear changes were reduced; the loss of cell viability was prevented and the number of cells in sub-G1 phase was decreased. Knock down of beta1,4-GT 1 also inhibited the release of cytochrome c from mitochondria and caspase-3 processing. Therefore, the cleavage of CDK11(p58) by caspase-3 was reduced. We proposed that beta1,4-GT 1 might contribute to the pro-apoptotic effect of CDK11(p58). This may represent a new mechanism of beta1,4-GT 1 in CHX-induced apoptosis of CDK11(p58)-overexpressing cells.

The cyclin-dependent kinases (CDK) CDK1, CDK2, CDK4, and CDK6 are serine/threonine protein kinases targeted in cancer therapy due to their role in cell cycle progression. The postmitotic CDK5 is involved in biological pathways important for neuronal migration and differentiation. CDK5 represents an attractive pharmacological target as its deregulation is implicated in various neurodegenerative diseases such as Alzheimer's, Parkinson's, and Niemann-Pick type C diseases, ischemia, and amyotrophic lateral sclerosis. We have generated an improved crystal form of CDK5 in complex with p25, a segment of the p35 neuronal activator. The crystals were used to solve the structure of CDK5/p25 with (R)-roscovitine and aloisine at a resolution of 2.2 and 2.3 A, respectively. The structure of CDK5/p25/roscovitine provides a rationale for the preference of CDK5 for the R over the S stereoisomer. Furthermore, roscovitine stabilized an unusual collapsed conformation of the glycine-rich loop, an important site of CDK regulation, and we report an investigation of the effects of glycine-rich loop phosphorylation on roscovitine binding. The CDK5/p25 crystals represent a valuable new tool for the identification and optimization of selective CDK inhibitors.

Cyclin-dependent kinases (CDKs) play a central role in cell cycle control, apoptosis, transcription, and neuronal functions. They are important targets for the design of drugs with antimitotic or antineurodegenerative effects. CDK4 and CDK6 form a subfamily among the CDKs in mammalian cells, as defined by sequence similarities. Compared to CDK2 and CDK5, structural information on CDK4 and CDK6 is sparse. We describe here the crystal structure of human CDK6 in complex with a viral cyclin and a flavonol inhibitor, fisetin. Fisetin binds to the active form of CDK6, forming hydrogen bonds with the side chains of residues in the binding pocket that undergo large conformational changes during CDK activation by cyclin binding. The 4-keto group and the 3-hydroxyl group of fisetin are hydrogen bonded with the backbone in the hinge region between the N-terminal and C-terminal kinase domain, as has been observed for many CDK inhibitors. However, CDK2 and HCK kinase in complex with other flavone inhibitors such as quercetin and flavopiridol showed a different binding mode with the inhibitor rotated by about 180 degrees. The structural information of the CDK6-fisetin complex is correlated with the binding affinities of different flavone inhibitors for CDK6. This complex structure is the first description of an inhibitor complex with a kinase from the CDK4/6 subfamily and can provide a basis for selecting and designing inhibitor compounds with higher affinities and specificities.

The interactions between the tumor suppressor protein p21WAF1 and the cyclin-dependent kinase (CDK) complexes and with proliferating cell nuclear antigen (PCNA) regulate and coordinate the processes of cell-cycle progression and DNA replication. We present the x-ray crystal structure of PCNA complexed with a 16-mer peptide related to p21 that binds with a Kd of 100 nM. Two additional crystal structures of native PCNA provide previously undescribed structures of uncomplexed human PCNA and show that significant changes on ligand binding include rigidification of a number of flexible regions on the surface of PCNA. In the competitive binding experiments described here, we show that a 20-mer sequence from p21 can be associated simultaneously with PCNA and CDK/cyclin complexes. A structural model for this quaternary complex is presented in which the C-terminal sequence of p21 acts like double-sided tape and docks to both the PCNA and cyclin molecules. The quaternary complex shows little direct interaction between PCNA and cyclin, giving p21 the role of an adaptor molecule. Taken together, the biochemical and structural results delineate a druggable inhibitor site on the surface of PCNA that may be exploited in the design of peptidomimetics, which will act independently of cyclin-groove inhibitors.

Cyclin-dependent kinases (CDKs) play a key role in regulating the cell cycle. The cyclins, their activating agents, and endogenous CDK inhibitors are frequently mutated in human cancers, making CDKs interesting targets for cancer chemotherapy. Our aim is the discovery of selective CDK4/cyclin D1 inhibitors. An ATP-competitive pyrazolopyrimidinone CDK inhibitor was identified by HTS and docked into a CDK4 homology model. The resulting binding model was consistent with available SAR and was validated by a subsequent CDK2/inhibitor crystal structure. An iterative cycle of chemistry and modeling led to a 70-fold improvement in potency. Small substituent changes resulted in large CDK4/CDK2 selectivity changes. The modeling revealed that selectivity is largely due to hydrogen-bonded interactions with only two kinase residues. This demonstrates that small differences between enzymes can efficiently be exploited in the design of selective inhibitors.

Most cyclin-dependent kinases are negatively regulated by phosphorylation of two residues, a threonine at residue 14 and a tyrosine at residue 15. These residues are dephosphorylated by the cdc25 family of dual-specificity phosphatases leading to cell cycle progression. These phosphatases are inactivated by cellular checkpoint pathways in response to DNA damage leading to cell cycle arrest. Checkpoint pathways regulate the function of these phosphatases by regulating their stability, localization, association with substrate, and their activity. Hence, determining these properties for the cdc25 family of phosphatases becomes crucial for understanding how checkpoint pathways regulate the function of the cdc25 family members and, hence, cell cycle progression. This chapter describes methods to determine the activity, levels, phosphorylation status, and localization of both endogenous and overexpressed cdc25 proteins.

All cyclin-dependent kinases (CDKs) involved in eukaryotic cell cycle control require phosphorylation at a conserved threonine (or serine) residue within the activation- or T-loop to attain full enzymatic activity. The enzyme responsible for this activating phosphorylation, the CDK-activating kinase (CAK), is therefore essential for proliferation of all eukaryotic cells. We describe methods to assess the T-loop phosphorylation state of the major mammalian CDKs in vivo; to measure the levels of CAK activity in cell-free extracts; and to analyze the abundance, subunit composition, and phosphorylation state of CAK complexes in metazoan cells. When derangement of normal CDK regulation is suspected as a cause of disturbed cell cycle progression, the combination of these methodologies can ascertain whether a primary CAK defect is the explanation.

Since a prior review of cell cycle inhibitors developed at the National Cancer Institute, continued progress in the application of these molecules has been pursued. Evidence of preliminary activity on the part of flavopiridol in certain chronic leukemias has pointed to that disease area as of potential interest, but likely by affecting transcriptional regulation through non-cell cycle-related CDKs. Brief duration infusion early phase trials with UCN-01, and combination studies with cytotoxics are commencing. Emerging structural data has refined the basis for screening strategies directed at cell cycle regulatory kinases, including cdks, chk kinases and most recently the mitotic phase aurora kinases. This interval progress report will review and update progress in these related but distinct drug discovery and development interest areas.

Targeting viral proteins has lead to many successful antivirals. Yet, such antivirals rapidly select for resistance, tend to be active against only a few related viruses, and require previous characterization of the target proteins. Alternatively, antivirals may be targeted to cellular proteins. Replication of many viruses requires cellular CDKs and pharmacological CDK inhibitors (PCIs), such as the purine-based roscovitine (Rosco), are proving safe in clinical trials against cancer. Rosco inhibits replication of wild-type or (multi-)drug resistant HIV, HCMV, EBV, VZV, and HSV-1 and 2. However, the antiviral mechanisms of purine PCIs remain unknown. Our objective is to characterize these mechanisms using HSV as a model We have shown that Rosco prevents initiation of transcription from viral, but not cellular, genomes. This inhibition is promoter independent, but genome dependent, and requires no viral proteins. This is a novel antiviral mechanism and a previously unknown activity for purine PCIs.

CDK11p110 (cyclin-dependent kinase 11p110, formerly known as PITSLRE) is a member of the CDK superfamily. It associates with cyclin L and is involved in the regulation of transcription and in premRNA splicing. During staurosporine-, Fas- and tumour necrosis factor a-induced apoptosis, CDK11p110, is cleaved by caspases to generate smaller 46-50 kDa proteins containing the catalytic kinase domain. Ectopic expression of the caspase-processed form CDK11p46 induces apoptosis. The mechanisms that regulate activation and stability of CDK11 isoforms are still unclear. In the present study, we demonstrate that in human melanoma cells CDK11p110 and CDK11p46 interact with Hsp90 (heat-shock protein 90) and its co-chaperone cdc37. Furthermore, we show that the treatment of cells with the Hsp90-specific inhibitor geldanamycin leads to ubiquitination and enhanced degradation of both CDK11p110 and CDK11p46 through a proteasome-dependent pathway. We also determined that geldanamycin-triggered degradation of CDK11p46 slows down the progression of apoptosis. These results indicate that Hsp90 and cdc37 stabilize CDK11 kinase, and suggest that this stabilization is crucial for its pro-apoptotic function.

The design and discovery of selective cyclin-dependent kinase 4 (CDK4) inhibitors have been actively pursued in order to develop therapeutic cancer treatments. By means of a consecutive computational protocol involving homology modeling, docking experiments, and molecular dynamics simulations, we examine the characteristic structural and dynamic properties that distinguish CDK4 from CDK2 in its complexation with selective inhibitors. The results for all three CDK4-selective inhibitors under investigation show that the large-amplitude motion of a disordered loop of CDK4 is damped out in the presence of the inhibitors whereas their binding in the CDK2 active site has little effect on the loop flexibility. It is also found that the binding preference of CDK4- selective inhibitors for CDK4 over CDK2 stems from the reduced solvent accessibility in the active site of the former due to the formation of a stable hydrogen-bond triad by the Asp99, Arg101, and Thr102 side chains at the top of the active-site gorge. Besides the differences in loop flexibility and solvent accessibility, the dynamic stabilities of the hydrogen bonds between the inhibitors and the side chain of the lysine residue at the bottom of the active site also correlate well with the relative binding affinities of the inhibitors for the two CDKs. These results highlight the usefulness of this computational approach in evaluating the selectivity of a CDK inhibitor, and demonstrate the necessity of considering protein flexibility and solvent effects in designing new selective CDK4-selective inhibitors.

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 also assists in the regulation of transcription as part of the transcription factor TFIIH complex. For maximum activity and stability, CDK7 requires phosphorylation, association with cyclin H, and association with a third protein, MAT1. We have determined the crystal structure of human CDK7 in complex with ATP at 3 A resolution. The kinase is in the inactive conformation, similar to that observed for inactive CDK2. The activation segment is phosphorylated at Thr170 and is in a defined conformation that differs from that in phospho-CDK2 and phospho-CDK2/cyclin A. The functional properties of the enzyme against CDK2 and CTD as substrates are characterized through kinase assays. Experiments confirm that CDK7 is not a substrate for kinase-associated phosphatase.

Over the past decade and a half, the paradigm has emerged of cardiac hypertrophy and ensuing heart failure as fundamentally a problem in signal transduction, impinging on the altered expression or function of gene-specific transcription factors and their partners, which then execute the hypertrophic phenotype. Strikingly, RNA polymerase II (RNAPII) is itself a substrate for two protein kinases-the cyclin-dependent kinases Cdk7 and Cdk9--that are activated by hypertrophic cues. Phosphorylation of RNAPII in the carboxyl terminal domain (CTD) of its largest subunit controls a number of critical steps subsequent to transcription initiation, among them enabling RNAPII to overcome its stalling in the promoter-proximal region and to engage in efficient transcription elongation. Here, we summarize our current understanding of the RNAPII-directed protein kinases in cardiac hypertrophy. Cdk9 activation is essential in tissue culture for myocyte enlargement and sufficient in transgenic mice for hypertrophy to occur and yet is unrelated to the "fetal" gene program that is typical of pathophysiological heart growth. Although this trophic effect of Cdk9 appears benign superficially, pathophysiological levels of Cdk9 activity render myocardium remarkably susceptible to apoptotic stress. Cdk9 interacts adversely with Gq-dependent pathways for hypertrophy, impairing the expression of numerous genes for mitochondrial proteins, and, in particular, suppressing master regulators of mitochondrial biogenesis and function, perioxisome proliferator-activated receptor-gamma coactivator-1 (PGC-1), and nuclear respiratory factor-1 (NRF-1). Given the dual transcriptional roles of Cdk9 in hypertrophic growth and mitochondrial dysfunction, we suggest the potential usefulness of Cdk9 as a target in heart failure drug discovery.

The cell division cycle is regulated by a family of cyclin-dependent protein kinases (CDKs) that are functionally conserved among many eukaryotic species. The characterization of plasmodial CDKs has identified them as a leading antimalarial drug target in our laboratory. We have developed a three-dimensional QSAR pharmacophore model for inhibition of a Plasmodium falciparum CDK, known as Pfmrk, from a set of fifteen structurally diverse kinase inhibitors with a wide range of activity. The model was found to contain two hydrogen bond acceptor functions and two hydrophobic sites including one aromatic-ring hydrophobic site. Although the model was not developed from X-ray structural analysis of the known CDK2 structure, it is consistent with the structure-functional requirements for binding of the CDK inhibitors in the ATP binding pocket. Using the model as a template, a search of the in-house three-dimensional multiconformer database resulted in the discovery of sixteen potent Pfmrk inhibitors. The predicted inhibitory activities of some of these Pfmrk inhibitors from the molecular model agree exceptionally well with the experimental inhibitory values from the in vitro CDK assay.

As new drugs rapidly advance into clinical trials, comprehensive identification of their intracellular targets becomes fundamental for the full understanding of the molecular basis of their efficacy and toxicity. This is particularly important when the targets belong to a large family and the inhibitors recognize a conserved site among different members of the class. A typical example is the kinase family, where efforts are aimed at the development of inhibitors of distinct kinases for therapeutic applications in oncology, inflammation and other disease areas. In this case, inhibitors targeting the ATP pocket may cross react with different kinases, as well as with other proteins that bind ATP. This review critically discusses the available approaches for kinase selectivity profiling. It also reviews some examples of inhibitor affinity chromatography applied to inhibitors of kinases and other protein families as a tool to identify and characterize their intracellular targets.

The protozoan parasite Trypanosoma brucei undergoes a complex developmental cycle coordinated with cell cycle control. These processes in eukaryotes are frequently regulated through mitogen-activated protein kinases (MAPKs) and cyclin-dependent protein kinases (CDKs), respectively. We have discovered a novel protein kinase which shares features of both ERK-type MAPKs and CDKs (T. brucei ERK-like, CDK-like protein kinase). This molecule, named TbECK1, is similar to the unusual mammalian KKIAMRE protein kinase family. Moreover, TbECK1 possesses a long C-terminal extension reminiscent of those found in mammalian ERK5, ERK7 and ERK8. Expression analyses demonstrate that TbECK1 is constitutively expressed during the trypanosome life cycle at both RNA and protein level. In transgenic parasites we demonstrate that expression of a mutant of TbECK1 that lacks the C-terminal extension produces a slow growth phenotype, associated with the appearance of cells with aberrant karyotypes. Using this as an assay we further demonstrate that the phenotype is dependent upon the potential for catalytic activity of TbECK1 and on the integrity of at least one of the phosphorylable amino acids in its phosphorylation lip. C-terminal extensions are a common feature of kinetoplastid protein kinases. Our results demonstrate for the first time that this domain has a regulatory function., (Copyright 2004 Blackwell Publishing Ltd)

The c-type cytochromes are defined by the occurrence of heme covalently linked to the polypeptide via thioether bonds between heme and the cysteine sulfhydryls in the CXXCH motif of apocytochrome. Maintenance of apocytochrome sulfhydryls in a reduced state is a prerequisite for covalent ligation of heme to the CXXCH motif. In bacteria, a thiol disulfide transporter and a thioredoxin are two components in a thio-reduction pathway involved in c-type cytochrome assembly. We have identified in photosynthetic eukaryotes nucleus-encoded homologs of a prokaryotic thiol disulfide transporter, CcdA, which all display an N-terminal extension with respect to their bacterial counterparts. The extension of Arabidopsis CCDA functions as a targeting sequence, suggesting a plastid site of action for CCDA in eukaryotes. Using PhoA and LacZ as topological reporters, we established that Arabidopsis CCDA is a polytopic protein with within-membrane strictly conserved cysteine residues. Insertional mutants in the Arabidopsis CCDA gene were identified, and loss-of-function alleles were shown to impair photosynthesis because of a defect in cytochrome b(6)f accumulation, which we attribute to a block in the maturation of holocytochrome f, whose heme binding domain resides in the thylakoid lumen. We postulate that plastid cytochrome c maturation requires CCDA, thioredoxin HCF164, and other molecules in a membrane-associated trans-thylakoid thiol-reducing pathway.

The immunosuppressant rapamycin has been shown to inhibit G(1)/S transition of the cell cycle. This inhibition is thought to be mediated by maintenance of the threshold levels of cyclin-dependent kinase (CDK) inhibitor p27(Kip1) (p27) and inhibition of p70 s6 kinase (p70(s6k)). However, recent evidence suggests that cells still remain sensitive to rapamycin in the absence of functional p27 or p70(s6k). Here, we show that rapamycin represses cyclin D3 levels in activated human T lymphocytes with no inhibitory effects on cyclin D2. Furthermore, rapamycin elicits similar cyclin D3 modulatory effects in B lymphocytes. The overall effect of rapamycin on cyclin D3 leads to impaired formation of active complexes with Cdk4 or Cdk6 and subsequent inhibition of cyclin D3/CDK kinase activity. Decrease in cyclin D3 protein levels is due to translational repression and not due to attenuated transcription of the cyclin D3 gene. Importantly, stable overexpression of cyclin D3 (2-2.5 fold) in Jurkat T cell transfectants renders them resistant to lower doses (1-10 ng/ml) of rapamycin. These results point to a critical role of cyclin D3 in rapamycin-mediated immunosuppressive effects in T cells and cell cycle regulation in lymphocytes in general.

Cyclin-dependent kinases (CDKs) control cell cycle progression through timely coordinated phosphorylation events. Two kinesin-like proteins that interact with CDKA;1 were identified and designated KCA1 and KCA2. They are 81% identical and have a similar three-partite domain organization. The N-terminal domain contains an ATP and microtubule-binding site typical for kinesin motors. A green fluorescent protein (GFP) fusion of the N-terminal domain of KCA1 decorated microtubules in Bright Yellow-2 cells, demonstrating microtubule-binding activity. During cytokinesis the full-length GFP-fusion protein accumulated at the midline of young and mature expanding phragmoplasts. Two-hybrid analysis and coimmunoprecipitation experiments showed that coiled-coil structures of the central stalk were responsible for homo- and heterodimerization of KCA1 and KCA2. By western-blot analysis, high molecular mass KCA molecules were detected in extracts from Bright Yellow-2 cells overproducing the full-length GFP fusion. Treatment of these cultures with the phosphatase inhibitor vanadate caused an accumulation of these KCA molecules. In addition to dimerization, interactions within the C-terminally located tail domain were revealed, indicating that the tail could fold onto itself. The tail domains of KCA1 and KCA2 contained two adjacent putative CDKA;1 phosphorylation sites, one of which is conserved in KCA homologs from other plant species. Site-directed mutagenesis of the conserved phosphorylation sites in KCA1 resulted in a reduced binding with CDKA;1 and abolished intramolecular tail interactions. The data show that phosphorylation of the CDKA;1 site provokes a conformational change in the structure of KCA with implications in folding and dimerization.

Cyclins are regulatory proteins that interact with cyclin-dependent kinases (CDKs) to control progression through the cell cycle. In Arabidopsis thaliana, 34 cyclin genes have been described, grouped into five different types (A, B, D, H, and T). A novel class of seven cyclins was isolated and characterized in Arabidopsis, designated P-type cyclins (CYCPs). They all share a conserved central region of 100 amino acids ("cyclin box") displaying homology to the corresponding region of the PHO80 cyclin from Saccharomyces cerevisiae and the related G1 cyclins from Trypanosoma cruzi and T. brucei. The CYCP4;2 gene was able to partially re-establish the phosphate-dependent expression of the PHO5 gene in a pho80 mutant strain of yeast. The CYCPs interact preferentially with CDKA;1 in vivo and in vitro as shown by yeast two-hybrid analysis and co-immunoprecipitation experiments. P-type cyclins were mostly expressed in proliferating cells, albeit also in differentiating and mature tissues. The possible role of CYCPs in linking cell division, cell differentiation, and the nutritional status of the cell is discussed.

A procedure for determining the extent of phosphorylation at individual sites of multiply phosphorylated proteins was developed and applied to two polyphosphorylated proteins. The protocol, using simple chemical (Fischer methyl-esterification) and enzymatic (phosphatase) modification steps and an accessible isotopic labeling reagent (methyl alcohol-d(4)), is described in detail. Site-specific phosphorylation stoichiometries are derived from the comparison of chemically identical but isotopically distinct peptide species analyzed by microspray liquid chromatography-mass spectrometry (microLC-MS) using a Micromass Q-TOF2 mass spectrometer. Ten phosphorylation sites were unambiguously identified in tryptic digests of both proteins, and phosphorylation stoichiometries were determined for eight of the ten sites using the isotope-coded strategy. The extent of phosphorylation was also estimated from the mass spectral peak areas for the phosphorylated and unmodified peptides, and these estimates, when compared with stoichiometries determined using the isotope-coded technique, differed only marginally (within approximately 20%).

Previous studies have shown that p34(SEI-1), also known as TRIP-Br1, is involved in cell cycle regulations by interacting with a number of important proteins including CDK4. However, the detailed mechanism and structural basis of the interaction remains to be determined. We report the use of in vitro studies to address these problems. First, it was shown that p34(SEI-1) binds to CDK4 directly, and the binding does not compete directly with p16. In the presence of p16, a quaternary complex is formed between p34(SEI-1), CDK4, cyclin D2, and p16. Second, it was found that p34(SEI-1) activates the kinase activity of CDK4 at lower concentrations (reaching the maximum at 500 nM) but inhibits the same activity at higher concentrations, implying that p34(SEI-1)-mediated CDK4 activation is dose-dependent. Again, the effects of p34(SEI-1) and p16 are independent of each other. Third, it was shown that p34(SEI-1) possesses a LexA-mediated transactivation activity. Finally, a set of truncation mutants were used to dissect the structural elements responsible for the different functions of p34(SEI-1). The results indicate that the fragment 30-160 can bind, activate, and inhibit CDK4; the fragment 30-132 can bind and activate but does not inhibit CDK4, while the fragment 30-88 cannot bind, activate, or inhibit but retains the LexA-mediated transactivation activity.

Membrane protein topology predictions can be markedly improved by the inclusion of even very limited experimental information. We have recently introduced an approach for the production of reliable topology models based on a combination of experimental determination of the location (cytoplasmic or periplasmic) of a protein's C terminus and topology prediction. Here, we show that determination of the location of a protein's C terminus, rather than some internal loop, is the best strategy for large-scale topology mapping studies. We further report experimentally based topology models for 31 Escherichia coli inner membrane proteins, using methodology suitable for genome-scale studies.

A number of selective inhibitors of the CDK4/cyclin D1 complex have been reported recently. Due to the absence of an experimental CDK4 structure, the ligand and protein determinants contributing to CDK4 selectivity are poorly understood at present. Here, we report the use of computational methods to elucidate the characteristics of selectivity and to derive the structural basis for specific, high-affinity binding of inhibitors to the CDK4 active site. From these data, the hypothesis emerged that appropriate incorporation of an ionizable function into a CDK2 inhibitor results in more favorable binding to CDK4. This knowledge was applied to the design of compounds in the otherwise CDK2-selective 2-anilino-4-(thiazol-5-yl)pyrimidine pharmacophore that are potent and highly selective ATP antagonists of CDK4/cyclin D1. The findings of this study also have significant implications in the design of CDK4 mimic structures based on CDK2.

In this study, we describe the effect of red and blue light on the timing of cell division, DNA synthesis, and activity and presence of cyclin-dependent kinases (CDKs), in synchronous cultures of the unicellular green alga Chlamydomonas reinhardtii. Cell division and DNA synthesis were found to occur later in cells grown in blue or white light, than in red light. CDK-like activity, measured using a histone H1 kinase assay, correspondingly occurred later in cultures that were grown in blue light compared to cultures grown in red light. The amount of CDK-like proteins, as detected using an antibody against the PSTAIRE motif, showed a maximum during the division phase. We conclude that the mechanism that causes the delay in the timing of cell division in blue light has its action before DNA replication takes place and also precedes the increase in CDK-like activity.

Cdc37 is a molecular chaperone that is important for the stability and activity of several protein kinases, including Cdk4 and Raf1. We first determined, using in vitro assays, that Cdc37 binds to the amino-terminal lobe of Cdk4. Subsequent mutagenesis revealed that Gly-15 (G15A) and Gly-18 (G18A) were critical for Cdc37-Cdk4 complex formation. Gly-15 and Gly-18 of Cdk4 are within the conserved Gly-X-Gly-X-X-Gly motif that is required for ATP binding to the kinase. Mutation of either glycine at the equivalent positions of Raf1 (G358A and G361A) also inhibited Cdc37 binding to Raf1. Replacing another conserved residue critical for ATP binding and kinase activity, Lys-35 (K35A), reduced Cdc37-Cdk4 complex formation but to a lesser extent. The interaction of Cdk4 with Cdc37 in vitro was not sensitive to changes in ATP levels. Cell-based assays indicated that Cdk4(G15A) and Cdk4(G18A) were present at the same level as wild type Cdk4. Equivalent amounts of p16 bound to Cdk4(G15A) and Cdk4(G18A) relative to wild type Cdk4, suggesting that Cdk4(G15A) and Cdk4(G18A) adopt significant tertiary structure. However, in contrast to wild type Cdk4, Cdk4(G15A), and Cdk4(G18A) had greatly reduced binding of cyclin D1, Cdc37, and Hsp90. Importantly, overexpression of Cdc37 not only stimulated cyclin D1 binding to wild type Cdk4 but also restored its binding to Cdk4(G15A). Under the same conditions, p16 binding to wild type Cdk4 was suppressed. Our findings show that the interaction of Cdc37 with its client protein kinases requires amino acid residues within a motif that is present in many protein kinases.

The C-terminal domain (CTD) of the largest subunit of RNA polymerase II plays critical roles in the initiation, elongation and processing of primary transcripts. These activities are at least partially regulated by the phosphorylation of the CTD by three cyclin-dependent protein kinases (CDKs), namely CDK7, CDK8 and CDK9. In this study, we systematically compared the phosphorylation of different recombinant CTD substrates by recombinant CDK7/CycH/MAT1, CDK8/CycC and CDK9/CycT1 kinases. We showed that CDK7, CDK8 and CDK9 produce different patterns of phosphorylation of the CTD. CDK7/CycH/MAT1 generates mostly hyperphosphorylated full-length and truncated CTD peptides, while CDK8/CycC and CDK9/CycT1 generate predominantly hypophosphorylated peptides. Total activity towards different parts of the CTD also differs between the three kinases; however, these differences did not correlate with their ability to hyperphosphorylate the substrates. The last 10 repeats of the CTD can act as a suppressor of the activity of the kinases in the context of longer peptides. Our results indicate that the three kinases possess different biochemical properties that could reflect their actions in vivo.

In budding yeast, Cla4 and Ste20, two p21-activated kinases, contribute to numerous morphogenetic processes. Loss of Ste20 or Cla4 individually confers distinct phenotypes, implying that they regulate different processes. However, loss of both proteins is lethal, suggesting some functional overlap. To explore the role(s) of Cla4, we and others have sought mutations that are lethal in a cla4 Delta strain. These mutations define >60 genes. Recently, both Ste20 and Cla4 have been implicated in mitotic exit. Here, we identify a genetic interaction between PHO85, which encodes a cyclin-dependent kinase, and CLA4. We further show that the Pho85-coupled G(1) cyclins Pcl1 and Pcl2 contribute to this Pho85 role. We performed a two-hybrid screen with Pcl1. Three Pcl1-interacting proteins were identified: Ncp1, Hms1, and a novel ATPase dubbed Epa1. Each of these proteins interacts with Pcl1 in GST pull-down experiments and is specifically phosphorylated by Pcl1.Pho85 complexes. NCP1, HMS1, and EPA1 also genetically interact with CLA4. Like Cla4, the proteins Hms1, Ncp1, and Pho85 appear to affect mitotic exit, a conclusion that follows from the mislocalization of Cdc14, a key mitotic regulator, in strains lacking these proteins. We propose a model in which the G(1) Pcl1.Pho85 complex regulates mitotic exit machinery.

This study investigates molecular mechanisms underlying cell cycle arrest when cells are exposed to high levels of oxygen (hyperoxia). Hyperoxia has previously been shown to increase expression of the cell cycle regulators p53 and p21. In the current study, we found that p53-deficient human lung adenocarcinoma H1299 cells failed to induce p21 or growth arrest in G(1) when exposed to 95% oxygen. Instead, cells arrested in S and G(2). Stable expression of p53 restored induction of p21 and G(1) arrest without affecting mRNA expression of the other Cip or INK4 G(1) kinase inhibitors. To confirm the role of p21 in G(1) arrest, we created H1299 cells with tetracycline-inducible expression of enhanced green fluorescent protein (EGFP), EGFP fused to p21 (EGFp21), or EGFP fused to p27 (EGFp27), a related cell cycle inhibitor. The amino terminus of p21 and p27 bind cyclin-dependent kinases (Cdk), whereas the carboxy terminus of p21 binds the sliding clamp proliferating cell nuclear antigen (PCNA). EGFp21 or EGFp27, but not EGFP by itself, restored G(1) arrest during hyperoxia. When separately overexpressed, the amino-terminal Cdk and carboxy-terminal PCNA binding domains of p21 each prevented cells from exiting G(1) during exposure. These findings demonstrate that exposure in vitro to hyperoxia exerts G(1) arrest through p53-dependent induction of p21 that suppresses Cdk and PCNA activity. Because PCNA also participates in DNA repair, these results raise the possibility that p21 also affects repair of oxidized DNA.