Slower CDK4 and faster CDK2 activation in the cell cycle.

Dysregulation of cyclin-dependent kinases (CDKs) impacts cell proliferation, driving cancer. Here, we ask why the cyclin-D/CDK4 complex governs cell cycle progression through the longer G1 phase, whereas cyclin-E/CDK2 regulates the shorter G1/S phase transition. We consider available experimental cellular and structural data including cyclin-E's high-level burst, sustained duration of elevated cyclin-D expression, and explicit solvent molecular dynamics simulations of the inactive monomeric and complexed states, to establish the conformational tendencies along the landscape of the distinct activation scenarios of cyclin-D/CDK4 and cyclin-E/CDK2 in the G1 phase and G1/S transition of the cell cycle, respectively. These lead us to propose slower activation of cyclin-D/CDK4 and rapid activation of cyclin-E/CDK2. We provide the mechanisms through which this occurs, offering innovative CDK4 drug design considerations. Our insightful mechanistic work addresses a compelling cell cycle regulation question and illuminates the distinct activation speeds between the G1 and the G1/S phases, which are crucial for function.

Discriminative SKP2 Interactions with CDK-Cyclin Complexes Support a Cyclin A-Specific Role in p27KIP1 Degradation.

The SCFSKP2 ubiquitin ligase relieves G1 checkpoint control of CDK-cyclin complexes by promoting p27KIP1 degradation. We describe reconstitution of stable complexes containing SKP1-SKP2 and CDK1-cyclin B or CDK2-cyclin A/E, mediated by the CDK regulatory subunit CKS1. We further show that a direct interaction between a SKP2 N-terminal motif and cyclin A can stabilize SKP1-SKP2-CDK2-cyclin A complexes in the absence of CKS1. We identify the SKP2 binding site on cyclin A and demonstrate the site is not present in cyclin B or cyclin E. This site is distinct from but overlapping with features that mediate binding of p27KIP1 and other G1 cyclin regulators to cyclin A. We propose that the capacity of SKP2 to engage with CDK2-cyclin A by more than one structural mechanism provides a way to fine tune the degradation of p27KIP1 and distinguishes cyclin A from other G1 cyclins to ensure orderly cell cycle progression.

USP27-mediated Cyclin E stabilization drives cell cycle progression and hepatocellular tumorigenesis.

Overexpression of Cyclin E has been seen in many types of cancers. However, the underlying mechanism remains enigmatic. Herein, we identified ubiquitin-specific peptidase 27 (USP27) as a Cyclin E interactor. We found that USP27 promoted Cyclin E stability by negatively regulating its ubiquitination. In addition, suppression of USP27 expression resulted in the inhibition of the growth, migration, and invasion of hepatocellular carcinoma. Furthermore, we detected a positive correlation between USP27 and Cyclin E expression in hepatocellular carcinoma tissues. Finally, we found that USP27 expression is inhibited by 5-fluorouracil (5-FU) treatment and USP27 depletion sensitizes Hep3B cells to 5-FU-induced apoptosis. USP27-mediated Cyclin E stabilization is involved in tumorigenesis, suggesting that targeting USP27 may represent a new therapeutic strategy to treat cancers with aberrant overexpression of Cyclin E protein.

Structural prediction of the interaction of the tumor suppressor p27KIP1 with cyclin A/CDK2 identifies a novel catalytically relevant determinant.

BACKGROUND: The cyclin-dependent kinase 2 (CDK2) together with its cyclin E and A partners is a central regulator of cell growth and division. Deregulation of CDK2 activity is associated with diseases such as cancer. The analysis of substrates identified S/T-P-X-R/K/H as the CDK2 consensus sequence. The crystal structure of cyclin A/CDK2 with a short model peptide supports this sequence and identifies key interactions. However, CDKs use additional determinants to recognize substrates, including the RXL motif that is read by the cyclin subunits. We were interested to determine whether additional amino acids beyond the minimal consensus sequence of the well-studied substrate and tumor suppressor p27KIP1 were relevant for catalysis. RESULTS: To address whether additional amino acids, close to the minimal consensus sequence, play a role in binding, we investigate the interaction of cyclin A/CDK2 with an in vivo cellular partner and CDK inhibitor p27KIP1. This protein is an intrinsically unfolded protein and, in particular, the C-terminal half of the protein has not been accessible to structural analysis. This part harbors the CDK2 phosphorylation site. We used bioinformatics tools, including MODELLER, iTASSER and HADDOCK, along with partial structural information to build a model of the C-terminal region of p27KIP1 with cyclin A/CDK2. This revealed novel interactions beyond the consensus sequence with a proline and a basic amino acid at the P + 1 and the P + 3 sites, respectively. We suggest that the lysine at P + 2 might regulate the reversible association of the second counter ion in the active site of CDK2. The arginine at P + 7 interacts with both cyclin A and CDK2 and is important for the catalytic turnover rate. CONCLUSION: Our modeling identifies additional amino acids in p27KIP1 beyond the consensus sequence that contribute to the efficiency of substrate phosphorylation.

Insights on Structural Characteristics and Ligand Binding Mechanisms of CDK2.

Cyclin-dependent kinase 2 (CDK2) is a crucial regulator of the eukaryotic cell cycle. However it is well established that monomeric CDK2 lacks regulatory activity, which needs to be aroused by its positive regulators, cyclins E and A, or be phosphorylated on the catalytic segment. Interestingly, these activation steps bring some dynamic changes on the 3D-structure of the kinase, especially the activation segment. Until now, in the monomeric CDK2 structure, three binding sites have been reported, including the adenosine triphosphate (ATP) binding site (Site I) and two non-competitive binding sites (Site II and III). In addition, when the kinase is subjected to the cyclin binding process, the resulting structural changes give rise to a variation of the ATP binding site, thus generating an allosteric binding site (Site IV). All the four sites are demonstrated as being targeted by corresponding inhibitors, as is illustrated by the allosteric binding one which is targeted by inhibitor ANS (fluorophore 8-anilino-1-naphthalene sulfonate). In the present work, the binding mechanisms and their fluctuations during the activation process attract our attention. Therefore, we carry out corresponding studies on the structural characterization of CDK2, which are expected to facilitate the understanding of the molecular mechanisms of kinase proteins. Besides, the binding mechanisms of CDK2 with its relevant inhibitors, as well as the changes of binding mechanisms following conformational variations of CDK2, are summarized and compared. The summary of the conformational characteristics and ligand binding mechanisms of CDK2 in the present work will improve our understanding of the molecular mechanisms regulating the bioactivities of CDK2.

MicroRNA-16 modulates HuR regulation of cyclin E1 in breast cancer cells.

RNA binding protein (RBPs) and microRNAs (miRNAs or miRs) are post-transcriptional regulators of gene expression that are implicated in development of cancers. Although their individual roles have been studied, the crosstalk between RBPs and miRNAs is under intense investigation. Here, we show that in breast cancer cells, cyclin E1 upregulation by the RBP HuR is through specific binding to regions in the cyclin E1 mRNA 3' untranslated region (3'UTR) containing U-rich elements. Similarly, miR-16 represses cyclin E1, dependent on its cognate binding sites in the cyclin E1 3'UTR. Evidence in the literature indicates that HuR can regulate miRNA expression and recruit or dissociate RNA-induced silencing complexes (RISC). Despite this, miR-16 and HuR do not affect the other's expression level or binding to the cyclin E1 3'UTR. While HuR overexpression partially blocks miR-16 repression of a reporter mRNA containing the cyclin E1 3'UTR, it does not block miR-16 repression of endogenous cyclin E1 mRNA. In contrast, miR-16 blocks HuR-mediated upregulation of cyclin E1. Overall our results suggest that miR-16 can override HuR upregulation of cyclin E1 without affecting HuR expression or association with the cyclin E1 mRNA.

Break CDK2/Cyclin E1 interface allosterically with small peptides.

Most inhibitors of Cyclin-dependent kinase 2 (CDK2) target its ATP-binding pocket. It is difficult, however, to use this pocket to design very specific inhibitors because this catalytic pocket is highly conserved in the protein family of CDKs. Here we report some short peptides targeting a noncatalytic pocket near the interface of the CDK2/Cyclin complex. Docking and molecular dynamics simulations were used to select the peptides, and detailed dynamical network analysis revealed that these peptides weaken the complex formation via allosteric interactions. Our experiments showed that upon binding to the noncatalytic pocket, these peptides break the CDK2/Cyclin complex partially and diminish its kinase activity in vitro. The binding affinity of these peptides measured by Surface Plasmon Resonance can reach as low as 0.5 µM.

Why are the truncated cyclin Es more effective CDK2 activators than the full-length isoforms?

Cell cycle regulating enzymes, CDKs, become activated upon association with their regulatory proteins, cyclins. The G1 cyclin, cyclin E, is overexpressed and present in low molecular weight (LMW) isoforms in breast cancer cells and tumor tissues. In vivo and in vitro studies have shown that these LMW isoforms of cyclin E hyperactivate CDK2 and accelerate the G1-S phase of cell division. The molecular basis of CDK2 hyperactivation due to LMW cyclin E isoforms in cancer cells is, however, unknown. Here, we employ a computational approach, combining homology modeling, bioinformatics analyses, molecular dynamics (MD) simulations, and principal component analyses to unravel the key structural features of CDK2-bound full-length and LMW isoforms of cyclin E1 and correlate those features to their differential activity. Results suggest that the missing N- and C-terminal regions of the cyclin E LMW isoforms constitute the Nuclear Localization Sequence (NLS) and PEST domains and are intrinsically disordered. These regions, when present in the full-length cyclin E/CDK2 complex, weaken the cyclin-CDK interface packing due to the loss of a large number of key interface interactions. Such weakening is manifested in the decreased contact area and increased solvent accessibility at the interface and also by the absence of concerted motions between the two partner proteins in the full-length complex. More effective packing and interactions between CDK2 and LMW cyclin E isoforms, however, produce more efficient protein-protein complexes that accelerate the cell division processes in cancer cells, where these cyclin E isoforms are overexpressed.

Mass spectrometric determination of disulfide bonds in the biologically active recombinant HBx protein of hepatitis B virus.

Many proteins rely on disulfide bonds formed between pairs of cysteines for the stability of their folded state and to keep regulatory control over their functions. The hepatitis B virus-encoded HBx oncoprotein is known to perform an overwhelming array of functions in the cell and has been implicated in the development of hepatocellular carcinoma. However, its structure has not been elucidated. HBx carries nine conserved cysteine residues that have proven to be crucial for its various functions. However, the status of disulfide bonds between the cysteine residues reported in previous studies remains discrepant because of the use of refolded recombinant HBx that may contain non-native disulfides. Now we have determined the disulfide linkages in soluble and biologically active recombinant maltose binding protein-HBx fusion protein using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. We report four disulfide linkages in HBx protein, viz., between Cys(7) and Cys(69), Cys(61) and Cys(115), Cys(78) and Cys(137), and Cys(17) and Cys(143), based on the differential mobility of corresponding disulfide-linked peptide ions under reducing and nonreducing conditions. Cys(148) was observed to be free. Site-directed mutagenesis of Cys(143) and Cys(148) with serine and functional analyses of these mutants affirmed the importance of these residues in the ability of HBx to potentiate Cdk2/cyclin E kinase activity and transcriptionally activate promoter reporter gene activity. Thus, this study identifies native disulfide linkages in the structure of a biologically active viral oncoprotein.

Structure-based discovery of antagonists of nuclear receptor LRH-1.

Liver receptor homolog 1 (nuclear receptor LRH-1, NR5A2) is an essential regulator of gene transcription, critical for maintenance of cell pluripotency in early development and imperative for the proper functions of the liver, pancreas, and intestines during the adult life. Although physiological hormones of LRH-1 have not yet been identified, crystallographic and biochemical studies demonstrated that LRH-1 could bind regulatory ligands and suggested phosphatidylinositols as potential hormone candidates for this receptor. No synthetic antagonists of LRH-1 are known to date. Here, we identify the first small molecule antagonists of LRH-1 activity. Our search for LRH-1 modulators was empowered by screening of 5.2 million commercially available compounds via molecular docking followed by verification of the top-ranked molecules using in vitro direct binding and transcriptional assays. Experimental evaluation of the predicted ligands identified two compounds that inhibit the transcriptional activity of LRH-1 and diminish the expression of the receptor's target genes. Among the affected transcriptional targets are co-repressor SHP (small heterodimer partner) as well as cyclin E1 (CCNE1) and G0S2 genes that are known to regulate cell growth and proliferation. Treatments of human pancreatic (AsPC-1), colon (HT29), and breast adenocarcinoma cells T47D and MDA-MB-468 with the LRH-1 antagonists resulted in the receptor-mediated inhibition of cancer cell proliferation. Our data suggest that specific antagonists of LRH-1 could be used as specific molecular probes for elucidating the roles of the receptor in different types of malignancies.

Molecular dynamics and QM/MM-based 3D interaction analyses of cyclin-E inhibitors.

Abnormal expression of cyclin-dependent kinase 2 (CDK2)/cyclin-E is detected in colorectal, ovarian, breast and prostate cancers. The study of CDK2 with a bound inhibitor revealed CDK2 as a potential therapeutic target for several proliferative diseases. Several highly selective inhibitors of CDK2 are currently undergoing clinical trials, but possibilities remain for the identification and development of novel and improved inhibitors. For example, in silico targeting of ATP-competitive inhibitors of CDKs is of special interest. A series of 3,5-diaminoindazoles was studied using molecular docking and comparative field analyses. We used post-docking short time molecular dynamics (MD) simulation to account for receptor flexibility. The three types of structures, i.e., the highest energy, lowest energy and the structure most resembling the X-ray structure (three complexes) were identified for all ligands. QM/MM energy calculations were performed using a DFT b3lyp/6-31 g* and MM OPLS-2005 force field. Conceptual DFT properties such as the interaction energy of ligand to protein, global hardness (η), HOMO density, electrostatic potential, and electron density were calculated and related to inhibitory activity. CoMFA and CoMSIA were used to account for steric and electrostatic interactions. The results of this study provide insight into the bioactive conformation, interactions involved, and the effect of different drug fragments over different biological activities.

Quantitative proteomics reveals the basis for the biochemical specificity of the cell-cycle machinery.

Cyclin-dependent kinases comprise the conserved machinery that drives progress through the cell cycle, but how they do this in mammalian cells is still unclear. To identify the mechanisms by which cyclin-cdks control the cell cycle, we performed a time-resolved analysis of the in vivo interactors of cyclins E1, A2, and B1 by quantitative mass spectrometry. This global analysis of context-dependent protein interactions reveals the temporal dynamics of cyclin function in which networks of cyclin-cdk interactions vary according to the type of cyclin and cell-cycle stage. Our results explain the temporal specificity of the cell-cycle machinery, thereby providing a biochemical mechanism for the genetic requirement for multiple cyclins in vivo and reveal how the actions of specific cyclins are coordinated to control the cell cycle. Furthermore, we identify key substrates (Wee1 and c15orf42/Sld3) that reveal how cyclin A is able to promote both DNA replication and mitosis.

Peptide-mediated disruption of calmodulin-cyclin E interactions inhibits proliferation of vascular smooth muscle cells and neointima formation.

RATIONALE: Cell cycle progression in vascular smooth muscle cells (VSMCs) is a therapeutic target for restenosis. OBJECTIVE: Having discovered that calmodulin (CaM)-dependent cyclin E/CDK2 activity underlies Ca(2+)-sensitive G(1)-to-S phase transitions in VSMCs, we sought to explore the physiological importance of the CaM-cyclin E interaction. METHODS AND RESULTS: A peptide based on the CaM binding sequence (CBS) of cyclin E was designed to interfere with CaM-cyclin E binding. Compared with control peptides, CBS blocked activating Thr160 phosphorylation of CDK2, decreased basal cyclin E/CDK2 activity, and eliminated Ca(2+)-sensitive cyclin E/CDK2 activity in nuclear extracts from mouse VSMCs. Nucleofection with CBS, or treatment with CBS conjugated to the HIV-1 TAT protein transduction domain to improve bioavailability, inhibited G(1)-to-S cell cycle progression in a dose-dependent manner. These effects were not observed with control peptides. TAT-CBS inhibited (3)H-thymidine incorporation in primary human aortic SMCs (HA-SMCs) in vitro, manifested greater transduction into HA-SMCs compared with endothelial cells in vitro, and limited decreased SM22α expression, neointima formation, and medial thickening without affecting collagen deposition or reendothelialization in a mouse model of carotid artery injury in vivo. The antiproliferative effects of CBS remained evident in mouse embryonic fibroblasts derived from wild-type mice but not cyclin E1/E2 double knockout mice. CONCLUSIONS: A synthetic peptide designed to disrupt CaM-cyclin E binding inhibits Ca(2+)/CaM-dependent CDK2 activity, cell cycle progression, and proliferation in VSMCs and limits arterial remodeling following injury. Importantly, this effect appears to be cyclin E-dependent and may form the basis of a potentially novel therapeutic approach for restenosis.

A novel interaction between HER2/neu and cyclin E in breast cancer.

HER2/neu (HER2) and cyclin E are important prognostic indicators in breast cancer. As both are involved in cell cycle regulation we analyzed whether there was a direct interaction between the two. HER2 and cyclin E expression levels were determined in 395 breast cancer patients. Patients with HER2-overexpression and high levels of cyclin E had decreased 5-year disease-specific survival compared with low levels of cyclin E (14% versus 89%, P<0.0001). In vitro studies were performed in which HER2-mediated activity in HER2-overexpressing breast cancer cell lines was downregulated by transfection with HER2 small interfering RNA or treatment with trastuzumab. Cyclin E expression levels were determined by western blot analysis, and functional effects analyzed using kinase assays, MTT assays were used to assess cell viability as a marker of proliferation and fluorescence-activated cell sorting analysis was used to determine cell cycle profiles. Decreased HER2-mediated signaling resulted in decreased expression of cyclin E, particularly the low molecular weight (LMW) isoforms. Decreased HER2 and LMW cyclin E expression had functional consequences, including decreased cyclin E-associated kinase activity and decreased proliferation, because of increased apoptosis and an increased accumulation of cells in the G1 phase. In vivo studies performed in a HER2-overexpressing breast cancer xenograft model confirmed the effects of trastuzumab on cyclin E expression. Given the relationship between HER2 and cyclin E, in vitro clonogenic assays were performed to assess combination therapy targeting both proteins. Isobologram analysis showed a synergistic interaction between the two agents (trastuzumab targeting HER2 and roscovitine targeting cyclin E). Taken together, these studies show that HER2-mediated signaling effects LMW cyclin E expression, which in turn deregulates the cell cycle. LMW cyclin E has prognostic and predictive roles in HER2-overexpressing breast cancer, warranting further study of its potential as a therapeutic target.

New roles for cyclin E in megakaryocytic polyploidization.

Megakaryocytes are platelet precursor cells that undergo endomitosis. During this process, repeated rounds of DNA synthesis are characterized by lack of late anaphase and cytokinesis. Physiologically, the majority of the polyploid megakaryocytes in the bone marrow are cell cycle arrested. As previously reported, cyclin E is essential for megakaryocyte polyploidy; however, it has remained unclear whether up-regulated cyclin E is an inducer of polyploidy in vivo. We found that cyclin E is up-regulated upon stimulation of primary megakaryocytes by thrombopoietin. Transgenic mice in which elevated cyclin E expression is targeted to megakaryocytes display an increased ploidy profile. Examination of S phase markers, specifically proliferating cell nuclear antigen, cyclin A, and 5-bromo-2-deoxyuridine reveals that cyclin E promotes progression to S phase and cell cycling. Interestingly, analysis of Cdc6 and Mcm2 indicates that cyclin E mediates its effect by promoting the expression of components of the pre-replication complex. Furthermore, we show that up-regulated cyclin E results in the up-regulation of cyclin B1 levels, suggesting an additional mechanism of cyclin E-mediated ploidy increase. These findings define a key role for cyclin E in promoting megakaryocyte entry into S phase and hence, increase in the number of cell cycling cells and in augmenting polyploidization.

Restriction point control of the mammalian cell cycle via the cyclin E/Cdk2:p27 complex.

Numerous top-down kinetic models have been constructed to describe the cell cycle. These models have typically been constructed, validated and analyzed using model species (molecular intermediates and proteins) and phenotypic observations, and therefore do not focus on the individual model processes (reaction steps). We have developed a method to: (a) quantify the importance of each of the reaction steps in a kinetic model for the positioning of a switch point [i.e. the restriction point (RP)]; (b) relate this control of reaction steps to their effects on molecular species, using sensitivity and co-control analysis; and thereby (c) go beyond a correlation towards a causal relationship between molecular species and effects. The method is generic and can be applied to responses of any type, but is most useful for the analysis of dynamic and emergent responses such as switch points in the cell cycle. The strength of the analysis is illustrated for an existing mammalian cell cycle model focusing on the RP [Novak B, Tyson J (2004) J Theor Biol230, 563-579]. The reactions in the model with the highest RP control were those involved in: (a) the interplay between retinoblastoma protein and E2F transcription factor; (b) those synthesizing the delayed response genes and cyclin D/Cdk4 in response to growth signals; (c) the E2F-dependent cyclin E/Cdk2 synthesis reaction; as well as (d) p27 formation reactions. Nine of the 23 intermediates were shown to have a good correlation between their concentration control and RP control. Sensitivity and co-control analysis indicated that the strongest control of the RP is mediated via the cyclin E/Cdk2:p27 complex concentration. Any perturbation of the RP could be related to a change in the concentration of this complex; apparent effects of other molecular species were indirect and always worked through cyclin E/Cdk2:p27, indicating a causal relationship between this complex and the positioning of the RP.

Post-translational modification and stability of low molecular weight cyclin E.

Our laboratory has previously described the presence of five tumor-specific low molecular weight isoforms of cyclin E in both tumor cell lines and breast cancer patient biopsies. We have also shown that one of these low forms arises from an alternate start site, whereas the other four appear as two sets of doublets following cleavage through an elastase-like enzyme. However, the origin of both sets of doublets was unknown. Here, we demonstrate that the larger isoform of each doublet is the result of phosphorylation at a key degradation site. Through site-directed mutagenesis of different phosphorylation sites within the cyclin E protein, we discovered that phosphorylation of threonine 395 is responsible for generating the larger isoform of each doublet. Because phosphorylation of threonine 395 has been linked to the proteasome-mediated degradation of full length cyclin E, we examined the stability of T395A phospho-mutants in both non-tumorigenic mammary epithelial cells and tumor cells. The results revealed that the low molecular weight isoforms appear to be stable in both a tumor cell line and a non-tumor forming cell line regardless of the presence of this critical phosphorylation site. The stability of low molecular weight cyclin E may have implications for both tumorigenesis and treatment of tumors expressing them.

Cyclin E low molecular weight isoforms occur commonly in early-onset gastric cancer and independently predict survival.

BACKGROUND: Post-translational cleavage of full-length cyclin E from the N-terminus can produce low molecular weight (LMW) isoforms of cyclin E containing the C-terminus only. AIM: To assess their presence in early-onset gastric cancer (EOGC), stump cancers and conventional gastric cancers and ascertain how they influence survival in EOGC. METHODS: The expression of full-length and LMW isoforms of cyclin E in 330 gastric cancers, including early-onset gastric cancer (EOGC), stump cancer and conventional gastric cancer (>45 years old) was compared using antibodies targeted to the N- and C-terminals. RESULTS: LMW isoforms were found in 35% of EOGCs, compared to 8% of conventional gastric cancers and 4% of stump cancers; their presence was visualised in cell lines using western blot analysis. In addition, C-terminal staining was a positive predictor of survival in EOGC. In contrast, no correlation with survival was found with the N-terminal antibody which detects only full-length cyclin E. CONCLUSION: EOGCs have a unique molecular phenotype and LMW isoforms of cyclin E may independently influence survival in EOGC.

The crystal structure of human cyclin B.

Cyclin B is the key regulatory protein controlling mitosis in all eukaryotes, where it binds cyclin-dependent kinase, cdk1, forming a complex which initiates the mitotic program through phosphorylation of select proteins. Cyclin B regulates the activation, subcellular localization, and substrate specificity of cdk1, and destruction of cyclin B is necessary for mitotic exit. Overexpression of human cyclin B1 has been found in numerous cancers and has been associated with tumor aggressiveness. Here we report the crystal structure of human cyclin B1 to 2.9 A. Comparison of the structure with cyclin A and cyclin E reveals remarkably similar N-terminal cyclin box motifs but significant differences among the C-terminal cyclin box lobes. Divergence in sequence gives rise to unique interaction surfaces at the proposed cyclin B/cdk1 interface as well as the 'RxL' motif substrate binding site on cyclin B. Examination of the structure provides insight into the molecular basis for differential affinities of protein based cyclin/cdk inhibitors such as p27, substrate recognition, and cdk interaction.