CDK2 In Complex Research Articles

Aurora Kinase Inhibition As an Anticancer Strategy

Article Tools UNDERSTANDING THE PATHWAY Article Tools OPTIONS & TOOLS Export Citation Track Citation Add To Favorites Rights & Permissions COMPANION ARTICLES No companion articles ARTICLE CITATION DOI: 10.1200/JCO.2013.50.7988 Journal of Clinical Oncology - published online before print September 16, 2013 PMID: 24043748 Aurora Kinase Inhibition As an Anticancer Strategy John F. HiltonxJohn F. HiltonSearch for articles by this author , Geoffrey I. ShapiroxGeoffrey I. ShapiroSearch for articles by this author Show More Dana-Farber Cancer Institute, Brigham and Women's Hospital, and Harvard Medical School, Boston, MA https://doi.org/10.1200/JCO.2013.50.7988 First Page Full Text PDF Figures and Tables © 2013 by American Society of Clinical Oncology

CDK4 T172 Phosphorylation Is Central in a CDK7-Dependent Bidirectional CDK4/CDK2 Interplay Mediated by p21 Phosphorylation at the Restriction Point

Cell cycle progression, including genome duplication, is orchestrated by cyclin-dependent kinases (CDKs). CDK activation depends on phosphorylation of their T-loop by a CDK–activating kinase (CAK). In animals, the only known CAK for CDK2 and CDK1 is cyclin H-CDK7, which is constitutively active. Therefore, the critical activation step is dephosphorylation of inhibitory sites by Cdc25 phosphatases rather than unrestricted T-loop phosphorylation. Homologous CDK4 and CDK6 bound to cyclins D are master integrators of mitogenic/oncogenic signaling cascades by initiating the inactivation of the central oncosuppressor pRb and cell cycle commitment at the restriction point. Unlike the situation in CDK1 and CDK2 cyclin complexes, and in contrast to the weak but constitutive T177 phosphorylation of CDK6, we have identified the T-loop phosphorylation at T172 as the highly regulated step determining CDK4 activity. Whether both CDK4 and CDK6 phosphorylations are catalyzed by CDK7 remains unclear. To answer this question, we took a chemical-genetics approach by using analogue-sensitive CDK7(as/as) mutant HCT116 cells, in which CDK7 can be specifically inhibited by bulky adenine analogs. Intriguingly, CDK7 inhibition prevented activating phosphorylations of CDK4/6, but for CDK4 this was at least partly dependent on its binding to p21cip1. In response to CDK7 inhibition, p21-binding to CDK4 increased concomitantly with disappearance of the most abundant phosphorylation of p21, which we localized at S130 and found to be catalyzed by both CDK4 and CDK2. The S130A mutation of p21 prevented the activating CDK4 phosphorylation, and inhibition of CDK4/6 and CDK2 impaired phosphorylations of both p21 and p21-bound CDK4. Therefore, specific CDK7 inhibition revealed the following: a crucial but partly indirect CDK7 involvement in phosphorylation/activation of CDK4 and CDK6; existence of CDK4-activating kinase(s) other than CDK7; and novel CDK7-dependent positive feedbacks mediated by p21 phosphorylation by CDK4 and CDK2 to sustain CDK4 activation, pRb inactivation, and restriction point passage.

Assembly, Activation, and Substrate Specificity of Cyclin D1/Cdk2 Complexes

Previous studies have shown conflicting data regarding cyclin D1/cyclin-dependent kinase 2 (Cdk2) complexes, and considering the widespread overexpression of cyclin D1 in cancer, it is important to fully understand their relevance. While many have shown that cyclin D1 and Cdk2 form active complexes, others have failed to show activity or association. Here, using a novel p21-PCNA fusion protein as well as p21 mutant proteins, we show that p21 is a required scaffolding protein, with cyclin D1 and Cdk2 failing to complex in its absence. These p21/cyclin D1/Cdk2 complexes are active and also bind the trimeric PCNA complex, with each trimer capable of independently binding distinct cyclin/Cdk complexes. We also show that increased p21 levels due to treatment with chemotherapeutic agents result in increased formation and kinase activity of cyclin D1/Cdk2 complexes, and that cyclin D1/Cdk2 complexes are able to phosphorylate a number of substrates in addition to Rb. Nucleophosmin and Cdh1, two proteins important for centrosome replication and implicated in the chromosomal instability of cancer, are shown to be phosphorylated by cyclin D1/Cdk2 complexes. Additionally, polypyrimidine tract binding protein-associated splicing factor (PSF) is identified as a novel Cdk2 substrate, being phosphorylated by Cdk2 complexed with either cyclin E or cyclin D1, and given the many functions of PSF, it could have important implications on cellular activity.

The ClC-3 chloride channel protein is a downstream target of cyclin D1 in nasopharyngeal carcinoma cells

It has been demonstrated previously by us that cyclin D1 and ClC-3 play important roles in regulation of the cell cycle in nasopharyngeal carcinoma cells. The action of cyclin D1 on the functional activities and expression of chloride channels were investigated in nasopharyngeal carcinoma CNE-2Z cells in this study. The results indicated that enhanced cyclin D1 expression increased the activation of volume-activated chloride currents and promoted the expression of ClC-3 chloride channel proteins. The fluorescence resonance energy transfer (FRET) experiments demonstrated that the distance between cyclin D1 and ClC-3 was less than 10nm, and there existed interaction between the two proteins. ClC-3 was partially colocalized with cyclin D1 and CDK4/6. Dialyzing CDK4 antibodies into cells via recording pipettes activated a chloride current, but dialysis of CDK6 antibodies inhibited basal and volume-activated Cl− currents. The CDK4/6 inhibitor fascaplysin chloride hydrate (highly selective for CDK4/cyclin D1 with IC50=0.35μM and less selective for CDK6/D1 with IC50=3.4μM) activated a chloride current in low concentration, but did not show significantly facilitative effects on the current in high concentration. In conclusion, our data suggest that the ClC-3 chloride channel is an important target of cyclin D1. Cyclin D1 may regulate the functional activities of the chloride channel via CDK4 and CDK6, and/or the expression of the chloride channel. Cyclin D1–CDK4 complexes may phosphorylate chloride channels resulting in inhibition or inactivation of the channels, and cyclin D1–CDK6 complexes may facilitate the activation of chloride channels.

Molecular dynamics simulations and statistical coupling analysis reveal functional coevolution network of oncogenic mutations in the CDKN2A–CDK6 complex

Coevolution between proteins is crucial for understanding protein–protein interaction. Simultaneous changes allow a protein complex to maintain its overall structural–functional integrity. In this study, we combined statistical coupling analysis (SCA) and molecular dynamics simulations on the CDK6–CDKN2A protein complex to evaluate coevolution between proteins. We reconstructed an inter-protein residue coevolution network, consisting of 37 residues and 37 interactions. It shows that most of the coevolved residue pairs are spatially proximal. When the mutations happened, the stable local structures were broken up and thus the protein interaction was decreased or inhibited, with a following increased risk of melanoma. The identification of inter-protein coevolved residues in the CDK6–CDKN2A complex can be helpful for designing protein engineering experiments.

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

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

The Male Gamete Is Not a Somatic Cell—the Possible Meaning of Varying Sperm RNA Levels

Antioxidants & Redox SignalingVol. 18, No. 2 Letter to the EditorThe Male Gamete Is Not a Somatic Cell—the Possible Meaning of Varying Sperm RNA LevelsAlexandra Amaral and João Ramalho-SantosAlexandra AmaralBiology of Reproduction and Human Fertility Research Group, Centre for Neuroscience and Cell Biology, University of Coimbra, Largo Marquês de Pombal, Coimbra, Portugal.Human Genetics Research Group, IDIBAPS, Faculty of Medicine, University of Barcelona, Barcelona, Spain.Biochemistry and Molecular Genetics Service, Clinic Hospital, Barcelona, Spain.Search for more papers by this author and João Ramalho-SantosBiology of Reproduction and Human Fertility Research Group, Centre for Neuroscience and Cell Biology, University of Coimbra, Largo Marquês de Pombal, Coimbra, Portugal.Department of Life Sciences, University of Coimbra, Coimbra, Portugal.Search for more papers by this authorPublished Online:30 Nov 2012https://doi.org/10.1089/ars.2012.4715AboutSectionsView articleView Full TextPDF/EPUB ToolsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View articleFiguresReferencesRelatedDetailsCited ByEnergy metabolism in mammalian sperm motility9 June 2022 | WIREs Mechanisms of Disease, Vol. 14, No. 5Cyclin–CDK Complexes are Key Controllers of Capacitation-Dependent Actin Dynamics in Mammalian Spermatozoa29 August 2019 | International Journal of Molecular Sciences, Vol. 20, No. 17The combined human sperm proteome: cellular pathways and implications for basic and clinical science29 September 2013 | Human Reproduction Update, Vol. 20, No. 1 Volume 18Issue 2Jan 2013 InformationCopyright 2013, Mary Ann Liebert, Inc.To cite this article:Alexandra Amaral and João Ramalho-Santos.The Male Gamete Is Not a Somatic Cell—the Possible Meaning of Varying Sperm RNA Levels.Antioxidants & Redox Signaling.Jan 2013.179-185.http://doi.org/10.1089/ars.2012.4715Published in Volume: 18 Issue 2: November 30, 2012Online Ahead of Print:October 11, 2012Online Ahead of Editing: June 15, 2012PDF download

FASCAPLYSIN as a Specific Inhibitor for CDK4: Insights from Molecular Modelling

Cyclin-dependent kinases (CDKs) play a key role in the cell cycle and are important anti-cancer drug targets. The natural product fascaplysin inhibits CDK4 with surprising selectivity (IC50 = 0.4 µM) compared to the close homolog CDK2 (IC50 = 500 µM). Free energy calculations of the positively charged fascaplysin and an uncharged iso-electronic derivative in the CDK2 and CDK4 inhibitor complexes indicate that the positive charge of fascaplysin is crucial for selectivity. This finding will guide further improvements in the design of fascaplysin-based selective inhibitors for CDK4.

Improved antibody immobilization using aniline‐catalyzed aldehyde‐hydrazide chemistry for the study of protein‐protein interactions

Covalent attachment of an antibody to a beaded support before immunoprecipitation prevents antibody contamination upon antigen elution and allows for the detection of antigens with similar molecular weights as antibody heavy and light chains. Common methods of antibody immobilization include direct coupling of antibody amino groups to aldehyde supports followed by reduction and indirect coupling of antibodies to Protein A/G‐coated beads followed by amine‐reactive crosslinking. Drawbacks of these methods include the loss of antibody functionality due to chemical modification or obstruction of the antigen binding site. An alternative approach for the covalent attachment of antibody to a beaded support utilizes oxidization of sugars in the antibody Fc region to aldehydes, which react with a hydrazide‐activated support. This method allows for unobstructed and oriented antibody conjugation without adverse modifications to antigen binding sites. However, compared to amine‐reactive antibody crosslinking, aldehyde‐hydrazide coupling often has lower efficiency and requires significantly longer incubation times. In order to improve this conjugation chemistry, we developed new aldehyde‐hydrazide immobilization and immunoprecipitation kits. These kits include aniline as a catalyst in a buffer with optimized pH for increased antibody coupling efficiency. The addition of aniline results in an increase of coupling efficiency from 50% to 90% for most antibody subtypes, and shortens reaction times to less than 4 hours. In addition, we demonstrate improved immunoprecipitation of Epitheleal Growth Factor Receptor (EGFR) and Signal Transducers and Activator of Transcription 1 Protein (STAT1), as well as the co‐immunoprecipitation of endogenous Cyclin B1/Cyclin Dependant Kinase 1 (Cdk1) complex from cell lysates using 2–10μg of immobilized antibody.

PAX7-FKHR fusion gene inhibits myogenic differentiation via NF-kappaB upregulation

Alveolar rhabdomyosarcomas (ARMS) are characterised by a PAX3/7-FKHR translocation, which is presumed to promote a differentiation arrest in the myogenic lineage, in which setting secondary genetic events occur, resulting in sarcomagenesis. The aim of this study was to identify the mechanism by which PAX3/7-FKHR expression results in a myogenic differentiation block, as discrete from the secondary genetic events that complete the sarcomagenic process. We performed a novel differential gene expression analysis comparing normal mesenchymal stem cells with previously generated non-tumorigenic mesenchymal stem cells expressing the PAX7-FKHR fusion gene, as well as with a known tumorigenic, PAX7-FKHR-expressing ARMS cell line, CW9019. This novel analysis uncovered the upregulation of the NF-kappaB pathway as a function of PAX3/7-FKHR expression, but distinct from the secondary sarcomagenic process; thus implicating NF-kappaB as a mediator of the PAX3/7-FKHR differentiation block. We further show that NF-kappaB activity is upregulated in PAX7-FKHR cells when compared to parental MSCs due to upregulation of the PI3K/AKT pathway. In addition we show that NF-kappaB inhibits myogenesis via activation of cyclinD1/ cdk4 complexes, which sequester MyoD1, a key myogenic transcription factor. Our results highlight the importance of the NF-kappaB pathway in myogenesis and sarcomagenesis and suggest that this pathway may be one of the potential therapeutic targets in the treatment of ARMS.

Among B-Type Cyclins Only CLB5 and CLB6 Promote Premeiotic S Phase in Saccharomyces cerevisiae

The Saccharomyces cerevisiae cyclin Clb5 is required for premeiotic S phase, meiotic recombination, and successful progression through meiosis. Clb5 is not essential for mitotic proliferation because Clb1-Clb4 can support DNA replication in clb5 clb6 mutants. Clb1, Clb3, and Clb4 accumulate in clb5 clb6 cells during meiotic differentiation yet fail to promote premeiotic DNA replication. When expressed under the regulation of the CLB5 promoter, Clb1 and Clb3 accumulate and are active in the early stages of meiotic differentiation but cannot induce premeiotic DNA replication, suggesting that they do not target Cdk1 to the necessary substrates. The Clb5 hydrophobic patch (HP) residues are important for Clb5 function but this motif alone does not provide the specificity required for Clb5 to induce premeiotic S phase. Domain exchange experiments demonstrated that the amino terminus of Clb5 when fused to Clb3 confers upon Clb3 the ability to induce premeiotic S phase. Chimeric cyclins containing smaller regions of the Clb5 amino terminus displayed reduced ability to activate premeiotic DNA replication despite being more abundant and having greater associated histone H1 kinase activity than endogenous Clb5. These observations suggest that Clb5 has a unique ability to trigger premeiotic S phase and that the amino-terminal region of Clb5 contributes to its specificity and regulates the functions performed by the cyclin-Cdk complex.

The Cell Cycle is a Limit Cycle

Progression along the successive phases of the mammalian cell cycle is driven by a network of cyclin-dependent kinases (Cdks). This network is regulated by a variety of negative and positive feedback loops. We previously proposed a detailed, 39-variable model for the Cdk network and showed that it is capable of temporal self-organization in the form of sustained oscillations, which correspond to the repetitive, transient, sequential activation of the cyclin- Cdk complexes that govern the successive phases of the cell cycle [Gerard and Goldbeter (2009) Proc Natl Acad Sci 106, 21643-8]. Here we compare the dynamical behavior of three models of different complexity for the Cdk network driving the mammalian cell cycle. The first is the detailed model that counts 39 variables and is based on Michaelis-Menten kinetics for the enzymatic steps. From this detailed model, we build a version based only on mass-action kinetics, which counts 80 variables. In this version we do not need to assume that enzymes are present in much smaller amounts that their substrates, which is not necessarily the case in the cell cycle. We show that these two versions of the model for the Cdk network yield similar results. In particular they predict sustained oscillations of the limit cycle type. We show that the model for the Cdk network can be reduced to a version containing only 5 variables, which is more amenable to stochastic simulations. This skeleton version retains the dynamic properties of the more complex versions of the model for the Cdk network in regard to Cdk oscillations. The regulatory wiring of the Cdk network therefore governs its dynamic behavior, regardless of the degree of molecular detail. We discuss the relative advantages of each version of the model, all of which support the view that the mammalian cell cycle behaves as a limit cycle oscillator.

Berberine Inhibits Growth and Induces G1 Arrest and Apoptosis in Human Cholangiocarcinoma QBC939 Cells

The chemotherapeutic approach using non-toxic natural products may be one of the strategies for the management of the cholangiocarcinoma. Here we report that in vitro treatment of human cholangiocarcinoma QBC939 cells with berberine, a naturally occurring isoquinoline alkaloid, decreased cell viability and induced cell death in a dose-dependent manner, which was associated with an increase in G1 arrest. Our western blot analysis showed that berberine-induced G1 cell cycle arrest was mediated through the increased expression of cyclin-dependent kinase inhibitors (Cdki) proteins (Cip1/p21 and Kip1/p27); a simultaneous decrease in Cdk2 and Cdk4 and cyclins D1, and reduced activity of the Cyclins–Cdk complex. In additional studies, treatment of QBC939 cells with different concentrations (10, 40, 80 μM) of berberine for 48 h resulted in a significant dose-dependent increase in apoptosis compared to the non-berberine–treated control, which was associated with an increased expression of pro-apoptotic protein Bax and decreased expression of anti-apoptotic proteins Bcl-2 and Bcl-xL. Together, this study for the first time identified berberine as a chemotherapeutic agent against human cholangiocarcinoma cells QBC939 cells in vitro. Further in vivo studies are required to determine whether berberine could be an effective chemotherapeutic agent for the management of cholangiocarcinoma.

Molecular Dynamic Behavior and Binding Affinity of Flavonoid Analogues to the Cyclin Dependent Kinase 6/cyclin D Complex

The cyclin dependent kinases (CDKs), each with their respective regulatory partner cyclin that are involved in the regulation of the cell cycle, apoptosis, and transcription, are potentially interesting targets for cancer therapy. The CDK6 complex with cyclin D (CDK6/cycD) drives cellular proliferation by phosphorylation of specific key target proteins. To understand the flavonoids that inhibit the CDK6/cycD functions, molecular dynamics simulations (MDSs) were performed on three inhibitors, fisetin (FST), apigenin (AGN), and chrysin (CHS), complexed with CDK6/cycD, including the two different binding orientations of CHS: FST-like (CHS_A) and deschloro-flavopiridol-like (CHS_B). For all three inhibitors, including both CHS orientations, the conserved interaction between the 4-keto group of the flavonoid and the backbone V101 nitrogen of CDK6 was strongly detected. The 3'- and 4'-OH groups on the flavonoid phenyl ring and the 3-OH group on the benzopyranone ring of inhibitor were found to significantly increase the binding and inhibitory efficiency. Besides the electrostatic interactions, especially through hydrogen bond formation, the van der Waals (vdW) interactions with the I19, V27, F98, H100, and L152 residues of CDK6 are also important factors in the binding efficiency of flavonoids against the CDK6/cycD complex. On the basis of the docking calculation and MM-PBSA method, the order of the predicted inhibitory affinities of these three inhibitors toward the CDK6/cycD was FST > AGN > CHS, which is in good agreement with the experimental data. In addition, CHS preferentially binds to the active CDK6 in a different orientation to FST and AGN but similar to its related analog, deschloro-flavopiridol. The obtained results are useful as the basic information for the further design of potent anticancer drugs specifically targeting the CDK6 enzyme.

P5-03-03: Identification of New Substrates for Breast Tumor Specific Low-Molecular-Weight Cyclin E Cyclin-Dependent-Kinase 2.

Abstract Background: Cyclin E overexpression occurs in 25% of breast cancer tumors and is linked to poor prognosis. In tumor cells full length cyclin E (FL-E) is processed by an elastase-like protease into low-molecular weight isoforms (LMW-E) that are biochemically hyperactive. When overexpressed in breast cancer cells, LMW-E induced genomic instability and resistance to p21, p27, and antiestrogens. LMW-E are tumor specific and are associated with the aggressive triple negative breast cancer (TNBC). Transgenic mice overexpressing LMW-E had increased incidence of mammary tumors and distant metastasis when compared to FL-E. Furthermore, when the role of CDK2 in LMW-E mediated mammary tumorigenesis was interrogated by crossing LMW-E transgenic mice with CDK2 knock out mice, we found that the mice are protected against mammary tumor formation. Additionally, treatment of LMW-E transgenic mice with roscovitine (a CDK2 inhibitor) delays mammary tumor formation. These results indicate that kinase inhibition may have therapeutic activity in LMW-E overexpressing tumors as shown in this preclinical model. These results lead us to hypothesize that the biological and biochemical differences between FL-E and LMW-E may be due to the phosphorylation of a distinct set of substrates when complexed with CDK2. Our goal is to identify distinct LMW-E/CDK2 substrates on a proteome-wide scale that could serve as novel therapeutic targets for the treatment of the aggressive LMW-E expressing TNBC. Material and Methods: We used two different approaches to identify LMW-E/CDK2 substrates. 1) We generated an analog sensitive CDK2 kinase, (F80A or F80G)-CDK2 to specifically radiolabel its substrates in cell extracts followed by their identification by mass spectroscopy. 2) We incubated ProtoArrayMicroarrays spotted with 3000 GST-tagged human protein on high density glass slides either with recombinant active FL-E/CDK2 or LMW-E/CDK2. Results: In the first approach, we expressed and purified wild-type CDK2 in complex with FL-E or LMW-E and CDK2 (F80A) and CDK2 (F80G) from insect cells. Although all 3 CDK2 kinases can use ATP to phosphorylate GST-Rb protein, only the F80G mutant can use PE-ATP-γ-S. In the second approach, we incubated protein arrays either with recombinant active FL-E/CDK2 or LMW-E/CDK2 at a concentration of 50 nM. Our first screen identified a total of 122 potential substrates to both FL-E/CDK2 or LMW-E/CDK2 kinase complexes. We only identified 1 protein that is phosphorylated by FL-E/CDK2 significantly more than by LMW-E/CDK2 as compared to the 32 potential substrates specific to LMW-E/CDK2 suggesting that by losing the N-terminal portion, the LMW-E/CDK2 kinase complex is able to specifically interact and phosphorylate novel proteins. Discussion: The identification of new physiological LMW-E/CDK2 substrates will lead to the development of novel targets for therapeutics and the identification of the biological function for the treatment of the aggressive LMW-E expressing TNBC. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-03-03.

The Role of p53 and MDM2 in Head and Neck Cancer

Head and neck cancer is a complex disorder that includes mostly squamous cell carcinomas that can develop in the throat, larynx, nose, sinuses, and mouth. Etiopathogenesis is due to tobacco and alcohol consumption and to infection by human papillomavirus (HPV) type 16/18. Tumors often develop within preneoplastic fields of genetically altered cells. Most head and neck cancers result from multistep accumulation of genetic alterationsm resulting in clonal outgrowth of transformed cells. These DNA changes are caused by a variety of mechanisms like endogenous mutations and exogenous mutations. Dysregulated molecular pathway includes alterations of critical inhibitor of cyclin CDK complexes, inactivating mutations of p53 gene, and activation of oncogenes and growth factors. This paper attempts to review the role of p53 and MDM2 genetic aberrations and pathways in head and neck cancer.

The Cyclin K/Cdk12 complex maintains genomic stability via regulation of expression of DNA damage response genes.

Various cyclin-dependent kinase (Cdk) complexes have been implicated in the regulation of transcription. In this study, we identified a 70-kDa Cyclin K (CycK) that binds Cdk12 and Cdk13 to form two different complexes (CycK/Cdk12 or CycK/Cdk13) in human cells. The CycK/Cdk12 complex regulates phosphorylation of Ser2 in the C-terminal domain of RNA polymerase II and expression of a small subset of human genes, as revealed in expression microarrays. Depletion of CycK/Cdk12 results in decreased expression of predominantly long genes with high numbers of exons. The most prominent group of down-regulated genes are the DNA damage response genes, including the critical regulators of genomic stability: BRCA1 (breast and ovarian cancer type 1 susceptibility protein 1), ATR (ataxia telangiectasia and Rad3-related), FANCI, and FANCD2. We show that CycK/Cdk12, rather than CycK/Cdk13, is necessary for their expression. Nuclear run-on assays and chromatin immunoprecipitations with RNA polymerase II on the BRCA1 and FANCI genes suggest a transcriptional defect in the absence of CycK/Cdk12. Consistent with these findings, cells without CycK/Cdk12 induce spontaneous DNA damage and are sensitive to a variety of DNA damage agents. We conclude that through regulation of expression of DNA damage response genes, CycK/Cdk12 protects cells from genomic instability. The essential role of CycK for organisms in vivo is further supported by the result that genetic inactivation of CycK in mice causes early embryonic lethality.

Unraveling interactions of cell cycle‐regulating proteins Sic1 and B‐type cyclins in living yeast cells: a FLIM‐FRET approach

Sic1, cyclin-dependent kinase inhibitor of budding yeast, is synthesized in anaphase and largely degraded at the S-phase onset to regulate timing of DNA synthesis. Sic1 interacts with phase-specific B-type cyclin (Clb)-kinase (Cdk1) complexes, central regulators in cell cycle control. Its appearance is timed to mediate reduction in kinase activities at appropriate stages. Clbs are unstable proteins with extremely short half-lives. Interactions of Sic1 with Clbs have been detected both in vitro and in vivo by high-throughput genome-wide screenings. Furthermore, we have recently shown that Sic1 regulates waves of Clbs, acting as a timer in their appearance, thus controlling Cdk1-Clbs activation. The molecular mechanism is not yet fully understood but is hypothesized to occur via stoichiometric binding of Sic1 to Cdk1-Clb complexes. Using Förster resonance energy transfer (FRET) via fluorescence lifetime imaging microscopy (FLIM), we showed association of Sic1 to Clb cyclins in living yeast cells. This finding is consistent with the notion that inhibition of kinase activity can occur over the whole cell cycle progression despite variable Sic1 levels. Specifically, Sic1/Clb3 interaction was observed for the first time, and Sic1/Clb2 and Sic1/Clb5 pairs were confirmed, but no Sic1/Clb4 interaction was found, which suggests that, despite high functional homology between Clbs, only some of them can target Sic1 function in vivo.

Cascades of multisite phosphorylation control Sic1 destruction at the onset of S phase

Multisite phosphorylation of proteins has been proposed to transform a graded protein kinase signal into an ultrasensitive switch-like response. Although many multiphosphorylated targets have been identified, the dynamics and sequence of individual phosphorylation events within the multisite phosphorylation process have never been thoroughly studied. In Saccharomyces cerevisiae, the initiation of S phase is thought to be governed by complexes of Cdk1 and Cln cyclins that phosphorylate six or more sites on the Clb5-Cdk1 inhibitor Sic1, directing it to SCF-mediated destruction. The resulting Sic1-free Clb5-Cdk1 complex triggers S phase. Here, we demonstrate that Sic1 destruction depends on a more complex process in which both Cln2-Cdk1 and Clb5-Cdk1 act in processive multiphosphorylation cascades leading to the phosphorylation of a small number of specific phosphodegrons. The routes of these phosphorylation cascades are shaped by precisely oriented docking interactions mediated by cyclin-specific docking motifs in Sic1 and by Cks1, the phospho-adaptor subunit of Cdk1. Our results indicate that Clb5-Cdk1-dependent phosphorylation generates positive feedback that is required for switch-like Sic1 destruction. Our evidence for a docking network within clusters of phosphorylation sites uncovers a new level of complexity in Cdk1-dependent regulation of cell cycle transitions, and has general implications for the regulation of cellular processes by multisite phosphorylation.

Cyclin-Specific Docking Motifs Promote Phosphorylation of Yeast Signaling Proteins by G1/S Cdk Complexes

The eukaryotic cell cycle begins with a burst of cyclin-dependent kinase (Cdk) phosphorylation. In budding yeast, several Cdk substrates are preferentially phosphorylated at the G1/S transition rather than later in the cell cycle when Cdk activity levels are high. These early Cdk substrates include signaling proteins in the pheromone response pathway. Two such proteins, Ste5 and Ste20, are phosphorylated only when Cdk is associated with the G1/S cyclins Cln1 and Cln2 and not G1, S, or M cyclins. The basis of this cyclin specificity is unknown. Here we show that Ste5 and Ste20 have recognition sequences, or "docking" sites, for the G1/S cyclins. These docking sites, which are distinct from Clb5/cyclin A-binding "RXL" motifs, bind preferentially to Cln2. They strongly enhance Cln2-driven phosphorylation of each substrate invivo and function largely independent of position and distance to the Cdk sites. We exploited this functional independence to rewire a Cdk regulatory circuit in a way that changes the target of Cdk inhibition in the pheromone response pathway. Furthermore, we uncover functionally active Cln2 docking motifs inseveral other Cdk substrates. The docking motifs drive cyclin-specific phosphorylation, and the cyclin preference can be switched by using a distinct motif. Our findings indicate that some Cdk substrates are intrinsically capable of being phosphorylated by several different cyclin-Cdk forms, but they are inefficiently phosphorylated invivo without a cyclin-specific docking site. Docking interactions may play a prevalent but previously unappreciated role in driving phosphorylation of select Cdk substrates preferentially at the G1/S transition.