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Sample records for cdk inhibitor r-roscovitine

  1. Pharmacological cdk inhibitor R-Roscovitine suppresses JC virus proliferation

    SciTech Connect

    Orba, Yasuko; Sunden, Yuji; Suzuki, Tadaki; Nagashima, Kazuo; Kimura, Takashi; Tanaka, Shinya; Sawa, Hirofumi

    2008-01-05

    The human Polyomavirus JC virus (JCV) utilizes cellular proteins for viral replication and transcription in the host cell nucleus. These cellular proteins represent potential targets for antiviral drugs against the JCV. In this study, we examined the antiviral effects of the pharmacological cyclin-dependent kinase (cdk) inhibitor R-Roscovitine, which has been shown to have antiviral activity against other viruses. We found that Roscovitine significantly inhibited the viral production and cytopathic effects of the JCV in a JCV-infected cell line. Roscovitine attenuated the transcriptional activity of JCV late genes, but not early genes, and also prevented viral replication via inhibiting phosphorylation of the viral early protein, large T antigen. These data suggest that the JCV requires cdks to transcribe late genes and to replicate its own DNA. That Roscovitine exhibited antiviral activity in JCV-infected cells suggests that Roscovitine might have therapeutic utility in the treatment of progressive multifocal leukoencephalopathy (PML)

  2. (R)-roscovitine, a cyclin-dependent kinase inhibitor, enhances tonic GABA inhibition in rat hippocampus.

    PubMed

    Ivanov, A; Tyzio, R; Zilberter, Y; Ben-Ari, Yehezkel

    2008-10-02

    Pharmacological agents that mediate a persistent GABAergic conductance are of considerable interest for treatment of epilepsy. (R)-roscovitine is a membrane permeable cyclin-dependent kinase inhibitor, designed to block cell division. It is currently undergoing a phase II clinical trial as an anticancer drug. We show that (R)-roscovitine increases a tonic GABA-mediated current in rat hippocampal neurons. This enhanced tonic current appears independent of synaptic GABA release and requires functional transmembrane GABA transport. The effect of (R)-roscovitine is associated with neither modification of GABAA receptors nor protein kinase activity, but is associated with a significant increase in intracellular GABA concentration in hippocampal GABAergic neurons. (R)-roscovitine-induced tonic inhibition significantly suppresses spontaneous spiking activity of hippocampal pyramidal cells. Therefore, (R)-roscovitine is a potent modulator of neuronal activity in rat hippocampus and may provide a tool for preventing paroxysmal activity.

  3. R-roscovitine reduces lung inflammation induced by lipoteichoic acid and Streptococcus pneumoniae.

    PubMed

    Hoogendijk, Arie J; Roelofs, Joris J T H; Duitman, Janwillem; van Lieshout, Miriam H P; Blok, Dana C; van der Poll, Tom; Wieland, Catharina W

    2012-09-25

    Bacterial pneumonia remains associated with high morbidity and mortality. The gram-positive pathogen Streptococcus pneumoniae is the most common cause of community-acquired pneumonia. Lipoteichoic acid (LTA) is an important proinflammatory component of the gram-positive bacterial cell wall. R-roscovitine, a purine analog, is a potent cyclin-dependent kinase (CDK)-1, -2, -5 and -7 inhibitor that has the ability to inhibit the cell cycle and to induce polymorphonuclear cell (PMN) apoptosis. We sought to investigate the effect of R-roscovitine on LTA-induced activation of cell lines with relevance for lung inflammation in vitro and on lung inflammation elicited by either LTA or viable S. pneumoniae in vivo. In vitro R-roscovitine enhanced apoptosis in PMNs and reduced tumor necrosis factor (TNF)-α and keratinocyte chemoattractant (KC) production in MH-S (alveolar macrophage) and MLE-12/MLE-15 (respiratory epithelial) cell lines. In vivo R-roscovitine treatment reduced PMN numbers in bronchoalveolar lavage fluid during LTA-induced lung inflammation; this effect was reversed by inhibiting apoptosis. Postponed treatment with R-roscovitine (24 and 72 h) diminished PMN numbers in lung tissue during gram-positive pneumonia; this step was associated with a transient increase in pulmonary bacterial loads. R-roscovitine inhibits proinflammatory responses induced by the gram-positive stimuli LTA and S. pneumoniae. R-roscovitine reduces PMN numbers in lungs upon LTA administration by enhancing apoptosis. The reduction in PMN numbers caused by R-roscovitine during S. pneumoniae pneumonia may hamper antibacterial defense.

  4. A phase I trial of the selective oral cyclin-dependent kinase inhibitor seliciclib (CYC202; R-Roscovitine), administered twice daily for 7 days every 21 days

    PubMed Central

    Benson, C; White, J; Bono, J De; O'Donnell, A; Raynaud, F; Cruickshank, C; McGrath, H; Walton, M; Workman, P; Kaye, S; Cassidy, J; Gianella-Borradori, A; Judson, I; Twelves, C

    2006-01-01

    Seliciclib (CYC202; R-roscovitine) is the first selective, orally bioavailable inhibitor of cyclin-dependent kinases 1, 2, 7 and 9 to enter clinical trial. Preclinical studies showed antitumour activity in a broad range of human tumour xenografts. A phase I trial was performed with a 7-day b.i.d. p.o. schedule. Twenty-one patients (median age 62 years, range: 39–73 years) were treated with doses of 100, 200 and 800 b.i.d. Dose-limiting toxicities were seen at 800 mg b.i.d.; grade 3 fatigue, grade 3 skin rash, grade 3 hyponatraemia and grade 4 hypokalaemia. Other toxicities included reversible raised creatinine (grade 2), reversible grade 3 abnormal liver function and grade 2 emesis. An 800 mg portion was investigated further in 12 patients, three of whom had MAG3 renograms. One patient with a rapid increase in creatinine on day 3 had a reversible fall in renal perfusion, with full recovery by day 14, and no changes suggestive of renal tubular damage. Further dose escalation was precluded by hypokalaemia. Seliciclib reached peak plasma concentrations between 1 and 4 h and elimination half-life was 2–5 h. Inhibition of retinoblastoma protein phosphorylation was not demonstrated in peripheral blood mononuclear cells. No objective tumour responses were noted, but disease stabilisation was recorded in eight patients; this lasted for a total of six courses (18 weeks) in a patient with ovarian cancer. PMID:17179992

  5. Modulating innate and adaptative immunity by (R)-roscovitine: potential therapeutic opportunity in cystic fibrosis

    PubMed Central

    MEIJER, Laurent; NELSON, Deborah; RIAZANSKI, Vladimir; GABDOULKHAKOVA, Aida G.; HERY-ARNAUD, Geneviève; LE BERRE, Rozenn; LOAËC, Nadège; OUMATA, Nassima; GALONS, Hervé; NOWAK, Emmanuel; GUEGANTON, Laetitia; DOROTHEE, Guillaume; PROCHAZKOVA, Michaela; HALL, Bradford; KULKARNI, Ashok B.; GRAY, Robert D.; ROSSI, Adriano G.; WITKO-SARSAT, Véronique; NOREZ, Caroline; BECQ, Frédéric; RAVEL, Denis; MOTTIER, Dominique; RAULT, Gilles

    2016-01-01

    (R)-Roscovitine, a pharmacological inhibitor of kinases, is currently in phase II clinical trial as a drug candidate for the treatment of cancers, Cushing disease and rheumatoid arthritis. We here review the data that support investigation of (R)-roscovitine as a potential therapeutic agent for the treatment of cystic fibrosis (CF). (R)-Roscovitine displays four independent properties that may favourably combine against CF: (1) it partially protects F508del-CFTR from proteolytic degradation and favours its trafficking to the plasma membrane, (2) by increasing membrane targeting of the TRPC6 ion channel, it rescues acidification in phagolysosomes of CF alveolar macrophages (which show abnormally high pH) and consequently restores their bactericidal activity, (3) its effects on neutrophils (induction of apoptosis), eosinophils (inhibition of degranulation, induction of apoptosis) and lymphocytes (modification of the Th17/Treg balance in favor of the differentiation of anti-inflammatory lymphocytes and reduced production of various interleukins, notably IL-17A) contribute to the resolution of inflammation and restoration of innate immunity, (4) roscovitine displays analgesic properties in animal pain models. The fact that (R)-roscovitine has undergone extensive preclinical safety/pharmacology studies, phase I clinical and phase II clinical trials in cancer patients encourage its repurposing as a CF drug candidate. PMID:26987072

  6. Novel R-roscovitine NO-donor hybrid compounds as potential pro-resolution of inflammation agents

    PubMed Central

    Montanaro, Gabriele; Bertinaria, Massimo; Rolando, Barbara; Fruttero, Roberta; Lucas, Christopher D.; Dorward, David A.; Rossi, Adriano G.; Megson, Ian L.; Gasco, Alberto

    2013-01-01

    Neutrophils play a pivotal role in the pathophysiology of multiple human inflammatory diseases. Novel pharmacological strategies which drive neutrophils to undergo programmed cell death (apoptosis) have been shown to facilitate the resolution of inflammation. Both the cyclin-dependent kinase inhibitor (CDKi) R-roscovitine and nitric oxide (NO) have been shown to enhance apoptosis of neutrophils and possess pro-resolution of inflammation properties. In order to search for new multi-target pro-resolution derivatives, here we describe the design, synthesis and investigation of the biological potential of a small series of hybrid compounds obtained by conjugating R-roscovitine with two different NO-donor moieties (compounds 2, 9a, 9c). The synthesized compounds were tested as potential pro-resolution agents, with their ability to promote human neutrophil apoptosis evaluated. Both compound 9a and 9c showed an increased pro-apoptotic activity when compared with either R-roscovitine or structurally related compounds devoid of the ability to release NO (des-NO analogues). Inhibition of either NO-synthase or soluble guanylate cyclase did not affect the induction of apoptosis by the R-roscovitine derivatives, similar to that reported for other classes of NO-donors. In contrast the NO scavenger PTIO prevented the enhanced apoptosis seen with compound 9a over R-roscovitine. These data show that novel compounds such as CDKi–NO-donor hybrids may have additive pro-resolution of inflammation effects. PMID:23394865

  7. Multiple CDK inhibitor dinaciclib suppresses neuroblastoma growth via inhibiting CDK2 and CDK9 activity

    PubMed Central

    Chen, Zhenghu; Wang, Zhenyu; Pang, Jonathan C.; Yu, Yang; Bieerkehazhi, Shayahati; Lu, Jiaxiong; Hu, Ting; Zhao, Yanling; Xu, Xin; Zhang, Hong; Yi, Joanna S.; Liu, Shangfeng; Yang, Jianhua

    2016-01-01

    Neuroblastoma (NB), the most common extracranial solid tumor of childhood, is responsible for approximately 15% of cancer-related mortality in children. Aberrant activation of cyclin-dependent kinases (CDKs) has been shown to contribute to tumor cell progression in many cancers including NB. Therefore, small molecule inhibitors of CDKs comprise a strategic option in cancer therapy. Here we show that a novel multiple-CDK inhibitor, dinaciclib (SCH727965, MK-7965), exhibits potent anti-proliferative effects on a panel of NB cell lines by blocking the activity of CDK2 and CDK9. Dinaciclib also significantly sensitized NB cell lines to the treatment of chemotherapeutic agents such as doxorubicin (Dox) and etoposide (VP-16). Furthermore, dinaciclib revealed in vivo antitumor efficacy in an orthotopic xenograft mouse model of two NB cell lines and blocked tumor development in the TH-MYCN transgenic NB mouse model. Taken together, this study suggests that CDK2 and CDK9 are potential therapeutic targets in NB and that abrogating CDK2 and CDK9 activity by small molecules like dinaciclib is a promising strategy and a treatment option for NB patients. PMID:27378523

  8. In Search of Novel CDK8 Inhibitors by Virtual Screening.

    PubMed

    Kumarasiri, Malika; Teo, Theodosia; Yu, Mingfeng; Philip, Stephen; Basnet, Sunita K C; Albrecht, Hugo; Sykes, Matthew J; Wang, Peng; Wang, Shudong

    2017-03-27

    Aberrant activity of cyclin-dependent kinase (CDK) 8 is implicated in various cancers. While CDK8-targeting anticancer drugs are highly sought-after, no CDK8 inhibitor has yet reached clinical trials. Herein a large library of drug-like molecules was computationally screened using two complementary cascades to identify potential CDK8 inhibitors. Thirty-three hits were identified to inhibit CDK8 and seven of them were active against colorectal cancer cell lines. Finally, the primary target was confirmed using three promising hits.

  9. Highlights of the Latest Advances in Research on CDK Inhibitors.

    PubMed

    Cicenas, Jonas; Kalyan, Karthik; Sorokinas, Aleksandras; Jatulyte, Asta; Valiunas, Deividas; Kaupinis, Algirdas; Valius, Mindaugas

    2014-10-27

    Uncontrolled proliferation is the hallmark of cancer and other proliferative disorders and abnormal cell cycle regulation is, therefore, common in these diseases. Cyclin-dependent kinases (CDKs) play a crucial role in the control of the cell cycle and proliferation. These kinases are frequently deregulated in various cancers, viral infections, neurodegenerative diseases, ischemia and some proliferative disorders. This led to a rigorous pursuit for small-molecule CDK inhibitors for therapeutic uses. Early efforts to block CDKs with nonselective CDK inhibitors led to little specificity and efficacy but apparent toxicity, but the recent advance of selective CDK inhibitors allowed the first successful efforts to target these kinases for the therapies of several diseases. Major ongoing efforts are to develop CDK inhibitors as monotherapies and rational combinations with chemotherapy and other targeted drugs.

  10. Understanding and modulating cyclin-dependent kinase inhibitor specificity: molecular modeling and biochemical evaluation of pyrazolopyrimidinones as CDK2/cyclin A and CDK4/cyclin D1 inhibitors

    NASA Astrophysics Data System (ADS)

    Rossi, Karen A.; Markwalder, Jay A.; Seitz, Steven P.; Chang, Chong-Hwan; Cox, Sarah; Boisclair, Michael D.; Brizuela, Leonardo; Brenner, Stephen L.; Stouten, Pieter F. W.

    2005-02-01

    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.

  11. First CDK 4/6 Inhibitor Heads to Market.

    PubMed

    2015-04-01

    The FDA granted accelerated approval to palbociclib for the treatment of estrogen receptor-positive, HER2-negative metastatic breast cancer in postmenopausal women who have not yet received endocrine-based therapy. Palbociclib is the first cell cycle-targeting CDK 4/6 inhibitor to reach the market.

  12. Structure and inhibitor specificity of the PCTAIRE-family kinase CDK16

    PubMed Central

    Dixon-Clarke, Sarah E.; Shehata, Saifeldin N.; Krojer, Tobias; Sharpe, Timothy D.; vonDelft, Frank; Sakamoto, Kei

    2017-01-01

    CDK16 (also known as PCTAIRE1 or PCTK1) is an atypical member of the cyclin-dependent kinase (CDK) family that has emerged as a key regulator of neurite outgrowth, vesicle trafficking and cancer cell proliferation. CDK16 is activated through binding to cyclin Y via a phosphorylation-dependent 14-3-3 interaction and has a unique consensus substrate phosphorylation motif compared with conventional CDKs. To elucidate the structure and inhibitor-binding properties of this atypical CDK, we screened the CDK16 kinase domain against different inhibitor libraries and determined the co-structures of identified hits. We discovered that the ATP-binding pocket of CDK16 can accommodate both type I and type II kinase inhibitors. The most potent CDK16 inhibitors revealed by cell-free and cell-based assays were the multitargeted cancer drugs dabrafenib and rebastinib. An inactive DFG-out binding conformation was confirmed by the first crystal structures of CDK16 in separate complexes with the inhibitors indirubin E804 and rebastinib, respectively. The structures revealed considerable conformational plasticity, suggesting that the isolated CDK16 kinase domain was relatively unstable in the absence of a cyclin partner. The unusual structural features and chemical scaffolds identified here hold promise for the development of more selective CDK16 inhibitors and provide opportunity to better characterise the role of CDK16 and its related CDK family members in various physiological and pathological contexts. PMID:28057719

  13. Structure and inhibitor specificity of the PCTAIRE-family kinase CDK16.

    PubMed

    Dixon-Clarke, Sarah E; Shehata, Saifeldin N; Krojer, Tobias; Sharpe, Timothy D; von Delft, Frank; Sakamoto, Kei; Bullock, Alex N

    2017-02-20

    CDK16 (also known as PCTAIRE1 or PCTK1) is an atypical member of the cyclin-dependent kinase (CDK) family that has emerged as a key regulator of neurite outgrowth, vesicle trafficking and cancer cell proliferation. CDK16 is activated through binding to cyclin Y via a phosphorylation-dependent 14-3-3 interaction and has a unique consensus substrate phosphorylation motif compared with conventional CDKs. To elucidate the structure and inhibitor-binding properties of this atypical CDK, we screened the CDK16 kinase domain against different inhibitor libraries and determined the co-structures of identified hits. We discovered that the ATP-binding pocket of CDK16 can accommodate both type I and type II kinase inhibitors. The most potent CDK16 inhibitors revealed by cell-free and cell-based assays were the multitargeted cancer drugs dabrafenib and rebastinib. An inactive DFG-out binding conformation was confirmed by the first crystal structures of CDK16 in separate complexes with the inhibitors indirubin E804 and rebastinib, respectively. The structures revealed considerable conformational plasticity, suggesting that the isolated CDK16 kinase domain was relatively unstable in the absence of a cyclin partner. The unusual structural features and chemical scaffolds identified here hold promise for the development of more selective CDK16 inhibitors and provide opportunity to better characterise the role of CDK16 and its related CDK family members in various physiological and pathological contexts.

  14. Development of mice without Cip/Kip CDK inhibitors

    SciTech Connect

    Tateishi, Yuki; Matsumoto, Akinobu; Kanie, Tomoharu; Hara, Eiji; Nakayama, Keiko; Nakayama, Keiichi I.

    2012-10-19

    Highlights: Black-Right-Pointing-Pointer Mice lacking Cip/Kip CKIs (p21, p27, and p57) survive until embryonic day 13.5. Black-Right-Pointing-Pointer Proliferation of MEFs lacking all three Cip/Kip CKIs appears unexpectedly normal. Black-Right-Pointing-Pointer CDK2 kinase activity of the triple mutant MEFs is increased in G0 phase. -- Abstract: Timely exit of cells from the cell cycle is essential for proper cell differentiation during embryogenesis. Cyclin-dependent kinase (CDK) inhibitors (CKIs) of the Cip/Kip family (p21, p27, and p57) are negative regulators of cell cycle progression and are thought to be essential for development. However, the extent of functional redundancy among Cip/Kip family members has remained largely unknown. We have now generated mice that lack all three Cip/Kip CKIs (TKO mice) and compared them with those lacking each possible pair of these proteins (DKO mice). We found that the TKO embryos develop normally until midgestation but die around embryonic day (E) 13.5, slightly earlier than p27/p57 DKO embryos. The TKO embryos manifested morphological abnormalities as well as increased rates of cell proliferation and apoptosis in the placenta and lens that were essentially indistinguishable from those of p27/p57 DKO mice. Unexpectedly, the proliferation rate and cell cycle profile of mouse embryonic fibroblasts (MEFs) lacking all three Cip/Kip CKIs did not differ substantially from those of control MEFs. The abundance and kinase activity of CDK2 were markedly increased, whereas CDK4 activity and cyclin D1 abundance were decreased, in both p27/p57 DKO and TKO MEFs during progression from G{sub 0} to S phase compared with those in control MEFs. The extents of the increase in CDK2 activity and the decrease in CDK4 activity and cyclin D1 abundance were greater in TKO MEFs than in p27/p57 DKO MEFs. These results suggest that p27 and p57 play an essential role in mouse development after midgestation, and that p21 plays only an auxiliary role in

  15. Do CDK4/6 inhibitors have potential as targeted therapeutics for squamous cell cancers?

    PubMed

    Kalu, Nene N; Johnson, Faye M

    2017-02-01

    Introduction Dysregulation of cell cycle progression has an established link to neoplasia and cancer progression. Components of the cyclin D-CDK4/6-INK4-Rb pathway are frequently altered in squamous cell carcinomas (SCCs) by diverse mechanisms, including viral oncogene-induced degradation, mutation, deletion, and amplification. Activation of the CDK4/6 pathway may predict response to CDK4/6 inhibitors and provide clinical biomarkers. Recently, the CDK4/6 inhibitor palbociclib showed clinical efficacy in combination with cetuximab in HNSCC patients. Areas covered This review focuses on the current research on the use of CDK4/6 inhibitors, comprising preclinical animal studies through phase II clinical trials across all SCCs. Expert opinion CDK4/6 inhibitors have a proven clinical benefit in breast cancer, but data on SCCs are sparse. Although frequent dysregulation of the cyclin D-CDK4/6-INK4-Rb pathway in SCCs suggests that targeting CDK4/6 may hold promise for improved clinical outcomes, single-agent activity has been modest in preclinical studies and absent in clinical studies. Combinations with immunotherapy or inhibitors of the PI3 K/mTOR or EGFR pathway may be effective. Given that SCCs caused by human papillomavirus have high levels of p16 and low levels of Rb, the CDK4/6 inhibitors are predicted to be ineffective in these cancers.

  16. Novel, selective CDK9 inhibitors for the treatment of HIV infection.

    PubMed

    Németh, G; Varga, Z; Greff, Z; Bencze, G; Sipos, A; Szántai-Kis, C; Baska, F; Gyuris, A; Kelemenics, K; Szathmáry, Z; Minárovits, J; Kéri, G; Orfi, L

    2011-01-01

    Cyclin Dependent Kinases (CDKs) are important regulators of cell cycle and gene expression. Since an up-to-date review about the pharmacological inhibitors of CDK family (CDK1-10) is not available; therefore in the present paper we briefly summarize the most relevant inhibitors and point out the low number of selective inhibitors. Among CDKs, CDK9 is a validated pathological target in HIV infection, inflammation and cardiac hypertrophy; however selective CDK9 inhibitors are still not available. We present a selective inhibitor family of CDK9 based on the 4-phenylamino-6- phenylpyrimidine nucleus. We show a convenient synthetic method to prepare a useful intermediate and its derivatisation resulting in novel compounds. The CDK9 inhibitory activity of the derivatives was measured in specific kinase assay and the CDK inhibitory profile of the best ones (IC(50) < 100 nM) was determined. The most selective compounds had high selectivity over CDK1, 2, 3, 5, 6, 7 and showed at least one order of magnitude higher inhibitory activity over CDK4 inhibition. The most selective molecules were examined in cytotoxicity assays and their ability to inhibit HIV-1 replication was determined in cellular assays.

  17. Modulating the interaction between CDK2 and cyclin A with a quinoline-based inhibitor.

    PubMed

    Deng, Yongqi; Shipps, Gerald W; Zhao, Lianyun; Siddiqui, M Arshad; Popovici-Muller, Janeta; Curran, Patrick J; Duca, Jose S; Hruza, Alan W; Fischmann, Thierry O; Madison, Vincent S; Zhang, Rumin; McNemar, Charles W; Mayhood, Todd W; Syto, Rosalinda; Annis, Allen; Kirschmeier, Paul; Lees, Emma M; Parry, David A; Windsor, William T

    2014-01-01

    A new class of quinoline-based kinase inhibitors has been discovered that both disrupt cyclin dependent 2 (CDK2) interaction with its cyclin A subunit and act as ATP competitive inhibitors. The key strategy for discovering this class of protein-protein disrupter compounds was to screen the monomer CDK2 in an affinity-selection/mass spectrometry-based technique and to perform secondary assays that identified compounds that bound only to the inactive CDK2 monomer and not the active CDK2/cyclin A heterodimer. Through a series of chemical modifications the affinity (Kd) of the original hit improved from 1 to 0.005μM.

  18. Revisiting CDK Inhibitors for Treatment of Glioblastoma Multiforme.

    PubMed

    Lubanska, Dorota; Porter, Lisa

    2017-03-21

    Despite extensive efforts and continual progress in research and medicine, outcomes for patients with high-grade glioma remain exceptionally poor. Over the past decade, research has revealed a great deal about the complex biology behind glioma development, and has brought to light some of the major barriers preventing successful treatment. Glioblastoma multiforme (GBM) (stage 4 astrocytoma) is a highly dynamic tumour and one of the most extreme examples of intratumoural heterogeneity, making targeting with specific therapeutics an inefficient and highly unpredictable goal. The cancer stem cell hypothesis offers a new view on the possible mechanisms dictating the heterogeneous nature of this disease and contributes to our understanding of glioma resistance and recurrence. Revealing cell division characteristics of initiating cell populations within GBM may represent novel treatment targets and/or the effective repurposing of existing therapies. In this review, we discuss the potential role of targeting the cyclin-dependent kinases (CDKs) driving this specific population. We also describe developments using multi-omic approaches that may aid in stratifying patient populations for CDK inhibitor therapy.

  19. Identification of a neuronal Cdk5 activator-binding protein as Cdk5 inhibitor.

    PubMed

    Ching, Yick-Pang; Pang, Andy S H; Lam, Wing-Ho; Qi, Robert Z; Wang, Jerry H

    2002-05-03

    Neuronal Cdc2-like kinase (Nclk) plays an important role in a variety of cellular processes, including neuronal cell differentiation, apoptosis, neuron migration, and formation of neuromuscular junction. The active kinase consists of a catalytic subunit, Cdk5, and an essential regulatory subunit, neuronal Cdk5 activator (p35(nck5a) or p25(nck5a)), which is expressed primarily in neurons of central nervous tissue. In our previous study using the yeast two-hybrid screening method, three novel p35(nck5a)-associated proteins were isolated. Here we show that one of these proteins, called C42, specifically inhibits the activation of Cdk5 by Nck5a. Co-immunoprecipitation data suggested that C42 and p35(nck5a) could form a complex within cultured mammalian cells. Deletion analysis has mapped the inhibitory domain of C42 to a region of 135 amino acids, which is conserved in Pho81, a yeast protein that inhibits the yeast cyclin-dependent protein kinase Pho85. The Pho85.Pho80 kinase complex has been shown to be the yeast functional homologue of the mammalian Cdk5/p35(nck5a) kinase.

  20. A possible usage of a CDK4 inhibitor for breast cancer stem cell-targeted therapy

    SciTech Connect

    Han, Yu Kyeong; Lee, Jae Ho; Park, Ga-Young; Chun, Sung Hak; Han, Jeong Yun; Kim, Sung Dae; Lee, Janet; Lee, Chang-Woo; Yang, Kwangmo; Lee, Chang Geun

    2013-01-25

    Highlights: ► A CDK4 inhibitor may be used for breast cancer stem cell-targeted therapy. ► The CDK4 inhibitor differentiated the cancer stem cell population (CD24{sup −}/CD44{sup +}) of MDA-MB-231. ► The differentiation of the cancer stem cells by the CDK4 inhibitor radiosensitized MDA-MB-231. -- Abstract: Cancer stem cells (CSCs) are one of the main reasons behind cancer recurrence due to their resistance to conventional anti-cancer therapies. Thus, many efforts are being devoted to developing CSC-targeted therapies to overcome the resistance of CSCs to conventional anti-cancer therapies and decrease cancer recurrence. Differentiation therapy is one potential approach to achieve CSC-targeted therapies. This method involves inducing immature cancer cells with stem cell characteristics into more mature or differentiated cancer cells. In this study, we found that a CDK4 inhibitor sensitized MDA-MB-231 cells but not MCF7 cells to irradiation. This difference appeared to be associated with the relative percentage of CSC-population between the two breast cancer cells. The CDK4 inhibitor induced differentiation and reduced the cancer stem cell activity of MDA-MB-231 cells, which are shown by multiple marker or phenotypes of CSCs. Thus, these results suggest that radiosensitization effects may be caused by reducing the CSC-population of MDA-MB-231 through the use of the CDK4 inhibitor. Thus, further investigations into the possible application of the CDK4 inhibitor for CSC-targeted therapy should be performed to enhance the efficacy of radiotherapy for breast cancer.

  1. A novel Cdk9 inhibitor preferentially targets tumor cells and synergizes with fludarabine

    PubMed Central

    Walsby, Elisabeth; Pratt, Guy; Shao, Hao; Abbas, Abdullah Y.; Fischer, Peter M.; Bradshaw, Tracey D.; Brennan, Paul; Fegan, Chris; Wang, Shudong; Pepper, Chris

    2014-01-01

    Cdk9 is a key elongation factor for RNA transcription and functions by phosphorylating the C-terminal domain of RNA polymerase II. Here we present direct evidence that cdk9 is important for cancer cell survival and describe the characterization of the potent cdk9 inhibitor CDKI-73 in primary human leukemia cells. CDKI-73 induced caspase-dependent apoptosis that was preceded by dephosphorylation of cdk9 and serine 2 of RNA polymerase II. CDKI-73 was more potent than the pan-cdk inhibitor flavopiridol and showed >200-fold selectivity against primary leukemia cells when compared with normal CD34+ cells. Furthermore, CDKI-73 was equipotent in poor prognostic sub-groups of leukemia patients and showed cytotoxic synergy with the nucleoside analog fludarabine. The Mechanism of synergy was associated with CDKI-73-mediated transcriptional inhibition of MCL1 and XIAP that was maintained when used in combination with fludarabine. Our data present a strong rationale for the development of cdk9 inhibitors such as CDKI-73 as anticancer therapeutics. PMID:24495868

  2. Evaluating the Effects of CDK Inhibitors in Ischemia-Reperfusion Injury Models.

    PubMed

    Guevara, Tatiana

    2016-01-01

    CDK inhibitors have been used to induce protection in various experimental models. Kidney ischemia-reperfusion (I/R) is a form of acute kidney injury resulting in a cascade of cellular events prompting rapid cellular damage and suppression of kidney function. I/R injury, an inevitable impairment during renal transplant surgery, remains one of the major causes of acute kidney injury and represents the most prominent factor leading to delayed graft function after transplantation. Understanding the molecular events responsible for tubule damage and recovery would help to develop new strategies for organ preservation. This chapter describes procedures to study the effect of CDK inhibitors in the cellular I/R model developed from an epithelial cell line deriving from pig kidney proximal tubule cells (LLC-PK1). We briefly describe methods for determining the protective effect of CDK inhibitors such as activation of caspase 3/7, western blot analysis, gene silencing, and immunoprecipitation.

  3. Covalent targeting of remote cysteine residues to develop CDK12 and 13 inhibitors

    PubMed Central

    Zhang, Tinghu; Kwiatkowski, Nicholas; Olson, Calla M; Dixon-Clarke, Sarah E; Abraham, Brian J; Greifenberg, Ann K; Ficarro, Scott B; Elkins, Jonathan M; Liang, Yanke; Hannett, Nancy M; Manz, Theresa; Hao, Mingfeng; Bartkowiak, Bartlomiej; Greenleaf, Arno L; Marto, Jarrod A; Geyer, Matthias; Bullock, Alex N; Young, Richard A; Gray, Nathanael S

    2016-01-01

    Cyclin-dependent kinases 12 and 13 (CDK12 and 13) play critical roles in the regulation of gene transcription. However, the absence of CDK12 and 13 inhibitors has hindered the ability to investigate the consequences of their inhibition in healthy cells and cancer cells. Here we describe the rational design of a first-in-class CDK12 and 13 covalent inhibitor, THZ531. Co-crystallization with CDK12-cyclin K indicates that THZ531 irreversibly targets a cysteine located outside the kinase domain. THZ531 causes a loss of gene expression with concurrent loss of elongating and hyperphosphorylated RNA polymerase II. In particular, THZ531 substantially decreases the expression of DNA damage response genes and key super–enhancer–associated transcription factor genes. Coincident with transcriptional perturbation, THZ531 dramatically induced apoptotic cell death. Small molecules capable of specifically targeting CDK12 and 13 may thus help identify cancer subtypes that are particularly dependent on their kinase activities. PMID:27571479

  4. Role of p53 in cdk Inhibitor VMY-1-103-induced Apoptosis in Prostate Cancer

    DTIC Science & Technology

    2013-11-01

    JA, Uren A. Arsenic trioxide inhibits human cancer cell growth and tumor development in mice by blocking Hedgehog /GLI pathway. J Clin Invest. 2011...induced apoptosis in prostate cancer PRINCIPAL INVESTIGATOR: Lymor Ringer...2013 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Role of p53 in cdk inhibitor VMY-1-103-induced apoptosis in prostate cancer 5b. GRANT NUMBER

  5. In silico study of porphyrin-anthraquinone hybrids as CDK2 inhibitor.

    PubMed

    Arba, Muhammad; Ihsan, Sunandar; Ramadhan, La Ode Ahmad Nur; Tjahjono, Daryono Hadi

    2017-04-01

    Cyclin-Dependent Kinases (CDKs) are known to play crucial roles in controlling cell cycle progression of eukaryotic cell and inhibition of their activity has long been considered as potential strategy in anti-cancer drug research. In the present work, a series of porphyrin-anthraquinone hybrids bearing meso-substituents, i.e. either pyridine or pyrazole rings were designed and computationally evaluated for their Cyclin Dependent Kinase-2 (CDK2) inhibitory activity using molecular docking, molecular dynamics simulation, and binding free energy calculation. The molecular docking simulation revealed that all six porphyrin hybrids were able to bind to ATP-binding site of CDK2 and interacted with key residues constituted the active cavity of CDK2, while molecular dynamics simulation indicated that all porphyrins bound to CDK2 were stable for 6ns. The binding free energies predicted by MM-PBSA method showed that most compounds exhibited higher affinity than that of native ligand (4-anilinoquinazoline, DTQ) and the affinity of mono-H2PyP-AQ was about three times better than that of DTQ, indicating its potential to be advanced as a new CDK2 inhibitor.

  6. Potential Clinical Uses of CDK Inhibitors: Lessons from Synthetic Lethality Screens.

    PubMed

    Vymětalová, Ladislava; Kryštof, Vladimír

    2015-11-01

    Developments in genetic and genomic technology have produced vast quantities of data that are gradually yielding new insights into fundamental cellular and molecular processes. In particular, they have revealed some differences between normal and transformed cells that could potentially be exploited to develop targeted, personalized cancer therapies with unprecedented efficiencies. This review summarizes recent findings from synthetic lethality (SL) screens against cyclin-dependent kinases (CDKs) that can be targeted with small molecule kinase inhibitors. SL screens can be used to identify cancers sensitive to CDK inhibitors. Several SL partners of specific CDKs have been identified, including MYC, K-Ras, VHL, PI3K, and PARP, all of which are discussed in the review. CDK inhibitors have been in clinical trials for nearly 20 years and it has become clear that effective therapy using these compounds will require careful selection of patients with respect to the specific molecular phenotype of their disease.

  7. CDK4/6 inhibitors in HER2-positive breast cancer.

    PubMed

    Corona, Silvia Paola; Ravelli, Andrea; Cretella, Daniele; Cappelletti, Maria Rosa; Zanotti, Laura; Dester, Martina; Gobbi, Angela; Petronini, Pier Giorgio; Generali, Daniele

    2017-04-01

    Notwithstanding the continuous progress made in cancer treatment in the last 20 years, and the availability of new targeted therapies, metastatic Breast Cancer (BC) is still incurable. Targeting the cell cycle machinery has emerged as an attractive strategy to tackle cancer progression, showing very promising results in the preclinical and clinical settings. The first selective inhibitors of CDK4/6 received breakthrough status and FDA approval in combination with letrozole (February 2015) and fulvestrant (February 2016) as first-line therapy in ER-positive advanced and metastatic BC. Considering the success of this family of compounds in hormone-positive BC, new possible applications are being investigated in other molecular subtypes. This review summarizes the latest findings on the use of CDK4/6 inhibitors in HER2 positive BC.

  8. The Cdk5 inhibitor Roscovitine increases LTP induction in corticostriatal synapses

    PubMed Central

    Miranda-Barrientos, Jorge; Nieto-Mendoza, Elizabeth; Hernández-Echeagaray, Elizabeth

    2014-01-01

    In corticostriatal synapses, LTD (long-term depression) and LTP (long-term potentiation) are modulated by the activation of DA (dopamine) receptors, with LTD being the most common type of long-term plasticity induced using the standard stimulation protocols. In particular, activation of the D1 signaling pathway increases cAMP/PKA (protein kinase A) phosphorylation activity and promotes an increase in the amplitude of glutamatergic corticostriatal synapses. However, if the Cdk5 (cyclin-dependent kinase 5) phosphorylates the DARPP-32 (dopamine and cAMP-regulated phosphoprotein of 32 kDa) at Thr75, DARPP-32 becomes a strong inhibitor of PKA activity. Roscovitine is a potent Cdk5 inhibitor; it has been previously shown that acute application of Roscovitine increases striatal transmission via Cdk5/DARPP-32. Since DARPP-32 controls long-term plasticity in the striatum, we wondered whether switching off CdK5 activity with Roscovitine contributes to the induction of LTP in corticostriatal synapses. For this purpose, excitatory population spikes and whole cell EPSC (excitatory postsynaptic currents) were recorded in striatal slices from C57/BL6 mice. Experiments were carried out in the presence of Roscovitine (20 μM) in the recording bath. Roscovitine increased the amplitude of excitatory population spikes and the percentage of population spikes that exhibited LTP after HFS (high-frequency stimulation; 100Hz). Results obtained showed that the mechanisms responsible for LTP induction after Cdk5 inhibition involved the PKA pathway, DA and NMDA (N-methyl-D-aspartate) receptor activation, L-type calcium channels activation and the presynaptic modulation of neurotransmitter release. PMID:24555476

  9. Flavonoids as CDK1 Inhibitors: Insights in Their Binding Orientations and Structure-Activity Relationship.

    PubMed

    Navarro-Retamal, Carlos; Caballero, Julio

    2016-01-01

    In the last years, the interactions of flavonoids with protein kinases (PKs) have been described by using crystallographic experiments. Interestingly, different orientations have been found for one flavonoid inside different PKs and different chemical substitutions lead to different orientations of the flavonoid scaffold inside one PK. Accordingly, orientation predictions of novel analogues could help to the design of flavonoids with high PK inhibitory activities. With this in mind, we studied the binding modes of 37 flavonoids (flavones and chalcones) inside the cyclin-dependent PK CDK1 using docking experiments. We found that the compounds under study adopted two different orientations into the active site of CDK1 (orientations I and II in the manuscript). In addition, quantitative structure-activity relationship (QSAR) models using CoMFA and CoMSIA methodologies were constructed to explain the trend of the CDK1 inhibitory activities for the studied flavonoids. Template-based and docking-based alignments were used. Models developed starting from docking-based alignment were applied for describing the whole dataset and compounds with orientation I. Adequate R2 and Q2 values were obtained by each method; interestingly, only hydrophobic and hydrogen bond donor fields describe the differential potency of the flavonoids as CDK1 inhibitors for both defined alignments and subsets. Our current application of docking and QSAR together reveals important elements to be drawn for the design of novel flavonoids with increased PK inhibitory activities.

  10. Flavonoids as CDK1 Inhibitors: Insights in Their Binding Orientations and Structure-Activity Relationship

    PubMed Central

    Navarro-Retamal, Carlos

    2016-01-01

    In the last years, the interactions of flavonoids with protein kinases (PKs) have been described by using crystallographic experiments. Interestingly, different orientations have been found for one flavonoid inside different PKs and different chemical substitutions lead to different orientations of the flavonoid scaffold inside one PK. Accordingly, orientation predictions of novel analogues could help to the design of flavonoids with high PK inhibitory activities. With this in mind, we studied the binding modes of 37 flavonoids (flavones and chalcones) inside the cyclin-dependent PK CDK1 using docking experiments. We found that the compounds under study adopted two different orientations into the active site of CDK1 (orientations I and II in the manuscript). In addition, quantitative structure–activity relationship (QSAR) models using CoMFA and CoMSIA methodologies were constructed to explain the trend of the CDK1 inhibitory activities for the studied flavonoids. Template-based and docking-based alignments were used. Models developed starting from docking-based alignment were applied for describing the whole dataset and compounds with orientation I. Adequate R2 and Q2 values were obtained by each method; interestingly, only hydrophobic and hydrogen bond donor fields describe the differential potency of the flavonoids as CDK1 inhibitors for both defined alignments and subsets. Our current application of docking and QSAR together reveals important elements to be drawn for the design of novel flavonoids with increased PK inhibitory activities. PMID:27517610

  11. Characterization of molecular and cellular functions of the cyclin-dependent kinase CDK9 using a novel specific inhibitor

    PubMed Central

    Albert, T K; Rigault, C; Eickhoff, J; Baumgart, K; Antrecht, C; Klebl, B; Mittler, G; Meisterernst, M

    2014-01-01

    BACKGROUND AND PURPOSE The cyclin-dependent kinase CDK9 is an important therapeutic target but currently available inhibitors exhibit low specificity and/or narrow therapeutic windows. Here we have used a new highly specific CDK9 inhibitor, LDC000067 to interrogate gene control mechanisms mediated by CDK9. EXPERIMENTAL APPROACH The selectivity of LDC000067 was established in functional kinase assays. Functions of CDK9 in gene expression were assessed with in vitro transcription experiments, single gene analyses and genome-wide expression profiling. Cultures of mouse embryonic stem cells, HeLa cells, several cancer cell lines, along with cells from patients with acute myelogenous leukaemia were also used to investigate cellular responses to LDC000067. KEY RESULTS The selectivity of LDC000067 for CDK9 over other CDKs exceeded that of the known inhibitors flavopiridol and DRB. LDC000067 inhibited in vitro transcription in an ATP-competitive and dose-dependent manner. Gene expression profiling of cells treated with LDC000067 demonstrated a selective reduction of short-lived mRNAs, including important regulators of proliferation and apoptosis. Analysis of de novo RNA synthesis suggested a wide ranging positive role of CDK9. At the molecular and cellular level, LDC000067 reproduced effects characteristic of CDK9 inhibition such as enhanced pausing of RNA polymerase II on genes and, most importantly, induction of apoptosis in cancer cells. CONCLUSIONS AND IMPLICATIONS Our study provides a framework for the mechanistic understanding of cellular responses to CDK9 inhibition. LDC000067 represents a promising lead for the development of clinically useful, highly specific CDK9 inhibitors. PMID:24102143

  12. Structure-activity relationship study of 2,4-diaminothiazoles as cdk5/p25 kinase inhibitors

    PubMed Central

    Laha, Joydev K.; Zhang, Xuemei; Qiao, Lixin; Liu, Min; Chatterjee, Snigdha; Robinson, Shaughnessy; Kosik, Kenneth S.; Cuny, Gregory D.

    2011-01-01

    Cdk5/p25 has emerged as a principle therapeutic target for numerous acute and chronic neurodegenerative diseases, including Alzheimer’s disease. A structure-activity relationship study of 2,4-diaminothiazole inhibitors revealed that increased Cdk5/p25 inhibitory activity could be accomplished by incorporating pyridines on the 2-amino group and addition of substituents to the 2- or 3-position of the phenyl ketone moiety. Interpretation of the SAR results for many of the analogs was aided through in silico docking with Cdk5/p25 and calculating protein hydrations sites using WaterMap. Finally, improved in vitro mouse microsomal stability was also achieved. PMID:21353545

  13. Analysing the Effect of Mutation on Protein Function and Discovering Potential Inhibitors of CDK4: Molecular Modelling and Dynamics Studies

    PubMed Central

    N, Nagasundaram; Zhu, Hailong; Liu, Jiming; V, Karthick; C, George Priya Doss; Chakraborty, Chiranjib; Chen, Luonan

    2015-01-01

    The cyclin-dependent kinase 4 (CDK4)-cyclin D1 complex plays a crucial role in the transition from the G1 phase to S phase of the cell cycle. Among the CDKs, CDK4 is one of the genes most frequently affected by somatic genetic variations that are associated with various forms of cancer. Thus, because the abnormal function of the CDK4-cyclin D1 protein complex might play a vital role in causing cancer, CDK4 can be considered a genetically validated therapeutic target. In this study, we used a systematic, integrated computational approach to identify deleterious nsSNPs and predict their effects on protein-protein (CDK4-cyclin D1) and protein-ligand (CDK4-flavopiridol) interactions. This analysis resulted in the identification of possible inhibitors of mutant CDK4 proteins that bind the conformations induced by deleterious nsSNPs. Using computational prediction methods, we identified five nsSNPs as highly deleterious: R24C, Y180H, A205T, R210P, and R246C. From molecular docking and molecular dynamic studies, we observed that these deleterious nsSNPs affected CDK4-cyclin D1 and CDK4-flavopiridol interactions. Furthermore, in a virtual screening approach, the drug 5_7_DIHYDROXY_ 2_ (3_4_5_TRI HYDROXYPHENYL) _4H_CHROMEN_ 4_ONE displayed good binding affinity for proteins with the mutations R24C or R246C, the drug diosmin displayed good binding affinity for the protein with the mutation Y180H, and the drug rutin displayed good binding affinity for proteins with the mutations A205T and R210P. Overall, this computational investigation of the CDK4 gene highlights the link between genetic variation and biological phenomena in human cancer and aids in the discovery of molecularly targeted therapies for personalized treatment. PMID:26252490

  14. Analysing the Effect of Mutation on Protein Function and Discovering Potential Inhibitors of CDK4: Molecular Modelling and Dynamics Studies.

    PubMed

    N, Nagasundaram; Zhu, Hailong; Liu, Jiming; V, Karthick; C, George Priya Doss; Chakraborty, Chiranjib; Chen, Luonan

    2015-01-01

    The cyclin-dependent kinase 4 (CDK4)-cyclin D1 complex plays a crucial role in the transition from the G1 phase to S phase of the cell cycle. Among the CDKs, CDK4 is one of the genes most frequently affected by somatic genetic variations that are associated with various forms of cancer. Thus, because the abnormal function of the CDK4-cyclin D1 protein complex might play a vital role in causing cancer, CDK4 can be considered a genetically validated therapeutic target. In this study, we used a systematic, integrated computational approach to identify deleterious nsSNPs and predict their effects on protein-protein (CDK4-cyclin D1) and protein-ligand (CDK4-flavopiridol) interactions. This analysis resulted in the identification of possible inhibitors of mutant CDK4 proteins that bind the conformations induced by deleterious nsSNPs. Using computational prediction methods, we identified five nsSNPs as highly deleterious: R24C, Y180H, A205T, R210P, and R246C. From molecular docking and molecular dynamic studies, we observed that these deleterious nsSNPs affected CDK4-cyclin D1 and CDK4-flavopiridol interactions. Furthermore, in a virtual screening approach, the drug 5_7_DIHYDROXY_ 2_ (3_4_5_TRI HYDROXYPHENYL) _4H_CHROMEN_ 4_ONE displayed good binding affinity for proteins with the mutations R24C or R246C, the drug diosmin displayed good binding affinity for the protein with the mutation Y180H, and the drug rutin displayed good binding affinity for proteins with the mutations A205T and R210P. Overall, this computational investigation of the CDK4 gene highlights the link between genetic variation and biological phenomena in human cancer and aids in the discovery of molecularly targeted therapies for personalized treatment.

  15. Expression of an S phase-stabilized version of the CDK inhibitor Dacapo can alter endoreplication

    PubMed Central

    Swanson, Christina I.; Meserve, Joy H.; McCarter, Patrick C.; Thieme, Alexis; Mathew, Tony; Elston, Timothy C.; Duronio, Robert J.

    2015-01-01

    In developing organisms, divergence from the canonical cell division cycle is often necessary to ensure the proper growth, differentiation, and physiological function of a variety of tissues. An important example is endoreplication, in which endocycling cells alternate between G and S phase without intervening mitosis or cytokinesis, resulting in polyploidy. Although significantly different from the canonical cell cycle, endocycles use regulatory pathways that also function in diploid cells, particularly those involved in S phase entry and progression. A key S phase regulator is the Cyclin E-Cdk2 kinase, which must alternate between periods of high (S phase) and low (G phase) activity in order for endocycling cells to achieve repeated rounds of S phase and polyploidy. The mechanisms that drive these oscillations of Cyclin E-Cdk2 activity are not fully understood. Here, we show that the Drosophila Cyclin E-Cdk2 inhibitor Dacapo (Dap) is targeted for destruction during S phase via a PIP degron, contributing to oscillations of Dap protein accumulation during both mitotic cycles and endocycles. Expression of a PIP degron mutant Dap attenuates endocycle progression but does not obviously affect proliferating diploid cells. A mathematical model of the endocycle predicts that the rate of destruction of Dap during S phase modulates the endocycle by regulating the length of G phase. We propose from this model and our in vivo data that endo S phase-coupled destruction of Dap reduces the threshold of Cyclin E-Cdk2 activity necessary to trigger the subsequent G-S transition, thereby influencing endocycle oscillation frequency and the extent of polyploidy. PMID:26493402

  16. Fragment based discovery of Arginine isosteres through REPLACE: towards non-ATP competitive CDK inhibitors

    PubMed Central

    Premnath, Padmavathy Nandha; Liu, Shu; Perkins, Tracy; Abbott, Jennifer; Anderson, Erin; McInnes, Campbell

    2013-01-01

    In order to develop non-ATP competitive CDK2/cyclin A inhibitors, the REPLACE strategy has been applied to generate fragment alternatives for the N-terminal tetrapeptide of the cyclin binding motif (HAKRRLIF) involved in substrate recruitment prior to phosphotransfer. The docking approach used for the prediction of small molecule mimics for peptide determinants was validated through reproduction of experimental binding modes of known inhibitors and provides useful information for evaluating binding to protein-protein interaction sites. Further to this, potential arginine isosteres predicted using the validated LigandFit docking method were ligated to the truncated C-terminal peptide, RLIF using solid phase synthesis and evaluated in a competitive binding assay. After testing, identified fragments were shown to represent not only appropriate mimics for a critical arginine residue but also to interact effectively with a minor hydrophobic pocket present in the binding groove. Further evaluation of binding modes was undertaken to optimize the potency of these compounds. Through further application of the REPLACE strategy in this study, peptide-small molecule hybrid CDK2 inhibitors were identified that are more drug-like and suitable for further optimization as anti-tumor therapeutics. PMID:24286762

  17. Targeting Transcriptional Addictions In Small Cell Lung Cancer With a Covalent CDK7 Inhibitor

    PubMed Central

    Christensen, Camilla L.; Kwiatkowski, Nicholas; Abraham, Brian J.; Carretero, Julian; Al-shahrour, Fatima; Zhang, Tinghu; Chipumuro, Edmond; Herter-Sprie, Grit S.; Akbay, Esra A.; Altabef, Abigail; Zhang, Jianming; Shimamura, Takeshi; Capelletti, Marzia; Reibel, Jakob B.; Cavanaugh, Jillian; Gao, Peng; Liu, Yan; Michaelsen, Signe R.; Poulsen, Hans S.; Aref, Amir R.; Barbie, David A.; Bradner, James E.; George, Rani; Gray, Nathanael S.; Young, Richard A.; Wong, Kwok-Kin

    2014-01-01

    SUMMARY Small cell lung cancer (SCLC) is an aggressive disease with high mortality. The identification of effective pharmacological strategies to target SCLC biology represents an urgent need. Using a high-throughput cellular screen of a diverse chemical library we observe that SCLC is sensitive to transcription-targeting drugs, and in particular to THZ1, a recent identified covalent inhibitor of cyclin-dependent kinase 7 (CDK7). We find that expression of super-enhancer associated transcription factor genes including MYC family proto-oncogenes and neuroendocrine lineage-specific factors are highly vulnerability to THZ1 treatment. We propose that downregulation of these transcription factors contributes, in part, to SCLC sensitivity to transcriptional inhibitors and that THZ1 represents a prototype drug for tailored SCLC therapy. PMID:25490451

  18. Proteolysis of Xenopus Cip-type CDK inhibitor, p16Xic2, is regulated by PCNA binding and CDK2 phosphorylation

    PubMed Central

    2013-01-01

    Background Cell division is positively regulated by cyclin-dependent kinases (CDKs) partnered with cyclins and negatively regulated by CDK inhibitors. In the frog, Xenopus laevis, three types of CDK inhibitors have been described: p27Xic1 (Xic1) which shares sequence homology with both p21Cip1 and p27Kip1 from mammals, p16Xic2 (Xic2) which shares sequence homology with p21Cip1, and p17Xic3 (Xic3) which shares sequence homology with p27Kip1. While past studies have demonstrated that during DNA polymerase switching, Xic1 is targeted for protein turnover dependent upon DNA, Proliferating Cell Nuclear Antigen (PCNA), and the ubiquitin ligase CRL4Cdt2, little is known about the processes that regulate Xic2 or Xic3. Methods We used the Xenopus interphase egg extract as a model system to examine the regulation of Xic2 by proteolysis and phosphorylation. Results Our studies indicated that following primer synthesis during the initiation of DNA replication, Xic2 is targeted for DNA- and PCNA-dependent ubiquitin-mediated proteolysis and that Cdt2 can promote Xic2 turnover. Additionally, during interphase, Xic2 is phosphorylated by CDK2 at Ser-98 and Ser-131 in a DNA-independent manner, inhibiting Xic2 turnover. In the presence of double-stranded DNA ends, Xic2 is also phosphorylated at Ser-78 and Ser-81 by a caffeine-sensitive kinase, but this phosphorylation does not alter Xic2 turnover. Conversely, in the presence or absence of DNA, Xic3 was stable in the Xenopus interphase egg extract and did not exhibit a shift indicative of phosphorylation. Conclusions During interphase, Xic2 is targeted for DNA- and PCNA-dependent proteolysis that is negatively regulated by CDK2 phosphorylation. During a response to DNA damage, Xic2 may be alternatively regulated by phosphorylation by a caffeine-sensitive kinase. Our studies suggest that the three types of Xenopus CDK inhibitors, Xic1, Xic2, and Xic3 appear to be uniquely regulated which may reflect their specialized roles during cell

  19. Association of CDK4 germline and BRAF somatic mutations in a patient with multiple primary melanomas and BRAF inhibitor resistance.

    PubMed

    Governa, Maurizio; Caprarella, Evelina; Dalla Pozza, Edoardo; Vigato, Enrico; Maritan, Monia; Caputo, Glenda G; Zannoni, Marina; Rosina, Paolo; Elefanti, Lisa; Stagni, Camilla; Menin, Chiara

    2015-10-01

    Many genetic alterations, including predisposing or somatic mutations, may contribute toward the development of melanoma. Although CDKN2A and CDK4 are high-penetrance genes for melanoma, MC1R is a low-penetrance gene that has been associated most consistently with the disease. Moreover, BRAF is the most frequently somatically altered oncogene and is a validated therapeutic target in melanoma. This paper reports a case of multiple primary melanoma with germline CDK4 mutation, MC1R variant, and somatic BRAF mutation in nine out of 10 melanomas, indicating that a common pathogenesis, because of a predisposing genetic background, may be shared among distinct subsequent melanomas of probable clonal origin. After 3 months of targeted therapy with BRAF inhibitor, our patient developed resistance with rapid progression of the disease leading to death. This is the first case in which early resistance to BRAF inhibitor has been reported in a patient with CDK4 germline mutation.

  20. A Novel CDK7 Inhibitor of the Pyrazolotriazine Class Exerts Broad-Spectrum Antiviral Activity at Nanomolar Concentrations

    PubMed Central

    Hutterer, Corina; Eickhoff, Jan; Milbradt, Jens; Korn, Klaus; Zeitträger, Isabel; Bahsi, Hanife; Wagner, Sabrina; Zischinsky, Gunther; Wolf, Alexander; Degenhart, Carsten; Unger, Anke; Baumann, Matthias; Klebl, Bert

    2015-01-01

    Protein kinases represent central and multifunctional regulators of a balanced virus-host interaction. Cyclin-dependent protein kinase 7 (CDK7) plays crucial regulatory roles in cell cycle and transcription, both connected with the replication of many viruses. Previously, we developed a CDK7 inhibitor, LDC4297, that inhibits CDK7 in vitro in the nano-picomolar range. Novel data from a kinome-wide evaluation (>330 kinases profiled in vitro) demonstrate a kinase selectivity. Importantly, we provide first evidence for the antiviral potential of the CDK7 inhibitor LDC4297, i.e., in exerting a block of the replication of human cytomegalovirus (HCMV) in primary human fibroblasts at nanomolar concentrations (50% effective concentration, 24.5 ± 1.3 nM). As a unique feature compared to approved antiherpesviral drugs, inhibition occurred already at the immediate-early level of HCMV gene expression. The mode of antiviral action was considered multifaceted since CDK7-regulated cellular factors that are supportive of HCMV replication were substantially affected by the inhibitors. An effect of LDC4297 was identified in the interference with HCMV-driven inactivation of retinoblastoma protein (Rb), a regulatory step generally considered a hallmark of herpesviral replication. In line with this finding, a broad inhibitory activity of the drug could be demonstrated against a selection of human and animal herpesviruses and adenoviruses, whereas other viruses only showed intermediate drug sensitivity. Summarized, the CDK7 inhibitor LDC4297 is a promising candidate for further antiviral drug development, possibly offering new options for a comprehensive approach to antiviral therapy. PMID:25624324

  1. Optimization of non-ATP competitive CDK/cyclin groove Inhibitors through REPLACE mediated Fragment Assembly

    PubMed Central

    Liu, Shu; Premnath, Padmavathy Nandha; Bolger, Joshua K.; Perkins, Tracy; Kirkland, Lindsay O.; Kontopidis, George; McInnes, Campbell

    2013-01-01

    A major challenge in drug discovery is to develop and improve methods for targeting protein-protein interactions. Further exemplification of the REPLACE strategy for generating inhibitors of protein-protein interactions demonstrated that it can be used to optimize fragment alternatives of key determinants, to combine these in an effective way and was achieved for compounds targeting the CDK2 substrate recruitment site on the cyclin regulatory subunit. Phenylheterocyclic isosteres replacing a critical charge-charge interaction provided new structural insights for binding to the cyclin groove. In particular, these results shed light onto the key contributions of a H-bond observed in crystal structures of N-terminally capped peptides. Furthermore the structure-activity relationship of a bisarylether C-terminal capping group mimicking dipeptide interactions, was probed through ring substitutions, allowing increased complementarity with the primary hydrophobic pocket. This study further validates REPLACE as an effective strategy for converting peptidic compounds to more pharmaceutically relevant compounds. PMID:23323521

  2. Structural basis for specificity and potency of a flavonoid inhibitor of human CDK2, a cell cycle kinase

    SciTech Connect

    Filgueira de Azevedo, W. Jr.; Mueller-Dieckmann, H.J.; Schulze-Gahmen, U.

    1996-04-02

    The central role of cyclin-dependent kinases (CDKs) in cell cycle regulation makes them a promising target for studying inhibitory molecules that can modify the degree of cell proliferation. The discovery of specific inhibitors of CDKs such as polyhydroxylated flavones has opened the way to investigation and design of antimitotic compounds. A novel flavone, (-)-cis-5,7-dihydroxyphenyl-8-[4-(3-hydroxy-1-methyl)piperidinyl]-4H-1-benzopyran-4-one hydrochloride hemihydrate (L868276), is a potent inhibitor of CDKs. A chlorinated form, flavopiridol, is currently in phase I clinical trials as a drug against breast tumors. We determined the crystal structure of a complex between CDK2 and L868276 at 2.33-{Angstrom} resolution and refined to an R{sub factor} of 20.3%. The aromatic portion of the inhibitor binds to the adenine-binding pocket of CDK2, and the position of the phenyl group of the inhibitor enables the inhibitor to make contacts with the enzyme not observed in the ATP complex structure. The analysis of the position of this phenyl ring not only explains the great differences of kinase inhibition among the flavonoid inhibitors but also explains the specificity of L868276 to inhibit CDK2 and CDC2. 36 refs., 4 figs., 2 tabs.

  3. An in silico exploration of the interaction mechanism of pyrazolo[1,5-a]pyrimidine type CDK2 inhibitors.

    PubMed

    Li, Yan; Gao, Weimin; Li, Feng; Wang, Jinghui; Zhang, Jingxiao; Yang, Yinfeng; Zhang, Shuwei; Yang, Ling

    2013-09-01

    CDK2, which interacts with cyclin A and cyclin E, is an important member of the CDK family. Having been proved to be associated with many diseases for its vital role in cell cycle, CDK2 is a promising target of anti-cancer drugs dealing with cell cycle disorders. In the present work, a total of 111 pyrazolo[1,5-a]pyrimidines (PHTPPs) as CDK2/cyclin A inhibitors were studied to conduct three-dimensional quantitative structure-activity (3D-QSAR) analyses. The optimal comparative molecular similarity indices analysis (CoMSIA) model shows that Q(2) = 0.516, Rncv(2) = 0.912, Rpre(2) = 0.914, Rm(2) = 0.843, SEP = 0.812, SEE = 0.347 with 10 components using steric, hydrophobic and H-bond donor field descriptors, indicating its effective internal and external predictive capacity. The contour maps further indicate that (1) bulky substituents in R1 are beneficial while H-bond donor groups at this position are detrimental; (2) hydrophobic contributions in the R2 area are favorable; (3) large and hydrophilic groups are well tolerated at the R3 position (a close H-bond donor moiety is favorable while a distal H-bond donor moiety in this area is disfavored); (4) bulky and hydrophobic features in the R4 region are beneficial for the biological activities and (5) the 7-N-aryl substitution is crucial to boost the inhibitory activities of the PHTPP inhibitors. Finally, docking and MD simulations demostrate that PHTPP derivatives are stabilized in a 'flying bat' conformation mainly through the H-bond interactions and hydrophobic contacts. Comparative studies indicate that PHTPP derivatives fit well within the ATP binding cleft in CDK2, with the core heterocyclic ring overlapping significantly with the adenine group of ATP despite a small deflection. In comparison to numerous other inhibitors binding to the ATP pocket, PHTPP analogues follow the binding fashion of purine inhibitors of this kinase. It is anticipated that the binding mechanism and structural features of PHTPP inhibitors

  4. Imbalance between pSmad3 and Notch induces CDK inhibitors in old muscle stem cells.

    PubMed

    Carlson, Morgan E; Hsu, Michael; Conboy, Irina M

    2008-07-24

    Adult skeletal muscle robustly regenerates throughout an organism's life, but as the muscle ages, its ability to repair diminishes and eventually fails. Previous work suggests that the regenerative potential of muscle stem cells (satellite cells) is not triggered in the old muscle because of a decline in Notch activation, and that it can be rejuvenated by forced local activation of Notch. Here we report that, in addition to the loss of Notch activation, old muscle produces excessive transforming growth factor (TGF)-beta (but not myostatin), which induces unusually high levels of TGF-beta pSmad3 in resident satellite cells and interferes with their regenerative capacity. Importantly, endogenous Notch and pSmad3 antagonize each other in the control of satellite-cell proliferation, such that activation of Notch blocks the TGF-beta-dependent upregulation of the cyclin-dependent kinase (CDK) inhibitors p15, p16, p21 and p27, whereas inhibition of Notch induces them. Furthermore, in muscle stem cells, Notch activity determines the binding of pSmad3 to the promoters of these negative regulators of cell-cycle progression. Attenuation of TGF-beta/pSmad3 in old, injured muscle restores regeneration to satellite cells in vivo. Thus a balance between endogenous pSmad3 and active Notch controls the regenerative competence of muscle stem cells, and deregulation of this balance in the old muscle microniche interferes with regeneration.

  5. Azolium analogues as CDK4 inhibitors: Pharmacophore modeling, 3D QSAR study and new lead drug discovery

    NASA Astrophysics Data System (ADS)

    Rondla, Rohini; Padma Rao, Lavanya Souda; Ramatenki, Vishwanath; Vadija, Rajender; Mukkera, Thirupathi; Potlapally, Sarita Rajender; Vuruputuri, Uma

    2017-04-01

    The cyclin-dependent kinase 4 (CDK4) enzyme is a key regulator in cell cycle G1 phase progression. It is often overexpressed in variety of cancer cells, which makes it an attractive therapeutic target for cancer treatment. A number of chemical scaffolds have been reported as CDK4 inhibitors in the literature, and in particular azolium scaffolds as potential inhibitors. Here, a ligand based pharmacophore modeling and an atom based 3D-QSAR analyses for a series of azolium based CDK4 inhibitors are presented. A five point pharmacophore hypothesis, i.e. APRRR with one H-bond acceptor (A), one positive cationic feature (P) and three ring aromatic sites (R) is developed, which yielded an atom based 3D-QSAR model that shows an excellent correlation coefficient value- R2 = 0.93, fisher ratio- F = 207, along with good predictive ability- Q2 = 0.79, and Pearson R value = 0.89. The visual inspection of the 3D-QSAR model, with the most active and the least active ligands, demonstrates the favorable and unfavorable structural regions for the activity towards CDK4. The roles of positively charged nitrogen, the steric effect, ligand flexibility, and the substituents on the activity are in good agreement with the previously reported experimental results. The generated 3D QSAR model is further applied as query for a 3D database screening, which identifies 23 lead drug candidates with good predicted activities and diverse scaffolds. The ADME analysis reveals that, the pharmacokinetic parameters of all the identified new leads are within the acceptable range.

  6. Metabolism and pharmacokinetics of 8-hydroxypiperidinylmethyl-baicalein (BA-j) as a novel selective CDK1 inhibitor in monkey.

    PubMed

    Guo, Hong-Min; Sun, Yu-Ming; Zhang, Shi-Xuan; Ju, Xiu-Lan; Xie, Ai-Yun; Li, Jing; Zou, Liang; Sun, Xiao-Dan; Li, Hai-Liang; Zheng, Yang

    2015-12-01

    Cyclin-dependent kinase 1 (CDK1) is the only necessary CDK in the cell proliferation process and a new target in the research and development of anti-cancer drugs. 8-Hydroxypiperidinemethyl-baicalein (BA-j) is a Mannich base derivative of baicalein (BA) isolated from Scutellaria baicalensis, as a novel selective CDK1 inhibitor. 12 metabolites of BA-j in the monkey urine were identified by LC-MS-MS and (1)H NMR. The major metabolic pathways of BA-j, by capturing oxygen free radicals ((.)O2(-)) and releasing peroxides (H2O2), are degraded into active intermediate metabolite dihydroflavonol, then into main metabolite M179 by Shiff reaction, second metabolite M264 by sulfation, trace amount of metabolite M559 by glucuronidation UGT1A9, and without metabolism by CYP3A4. The metabolic process of BA-j by regulating intracellular reactive oxygen species (ROS) was related with BA-j selectively inducing apoptosis in cancer cells. Pharmacokinetics of 10mg/kg oral BA-j in monkey by HPLC-UV was best fitted to a two-compartment open model, with t1/2(β) of 4.2h, Cmax 25.4μM at 2h, and Vd 12.6L, meaning the drug distributing widely in body fluids with no special selectivity to certain tissues, and being able to permeate through the blood-brain barrier. The protein binding rate of BA-j was 91.8%. BA-j has excellent druggability for oral administration or injection, and it may be developed into a novel anti-cancer drug as a selective CDK1 inhibitor.

  7. Cdk5 inhibitor roscovitine alleviates neuropathic pain in the dorsal root ganglia by downregulating N-methyl-D-aspartate receptor subunit 2A.

    PubMed

    Yang, Lei; Gu, Xiaoping; Zhang, Wei; Zhang, Juan; Ma, Zhengliang

    2014-09-01

    Cyclin-dependent kinase 5 (Cdk5) is a member of the small proline-directed serine/threonine kinase family. Cdk5 is not involved in cell cycle regulation, but is implicated in neurodegenerative disorders. However, the role of Cdk5 in neuropathic pain remains unclear. This study aimed to evaluate the possibility that Cdk5 is involved in neuropathic pain in the dorsal root ganglia (DRG). We injected intrathecally Cdk5 inhibitor roscovitine in rat model of chronic compression of dorsal root ganglion and examined pain behaviors and the expression of N-methyl-d-aspartate receptor subunit 2A (NR2A) but not NR2B or NR1 in DRG. We found that roscovitine alleviated neuropathic pain, causing decline in paw withdrawal mechanical threshold and paw withdrawal thermal latency. Furthermore, roscovitine inhibited NR2A expression in DRG. These data suggest that Cdk5-NR2A pathway regulates neuropathic pain in DRG, and intrathecal injection of roscovitine could alleviate neuropathic pain. Our findings provide new insight into the analgesic effects of Roscovitine and identify Cdk5-NR2A pathway as a potential target for effective treatment of neuropathic pain.

  8. THZ1 targeting CDK7 suppresses STAT transcriptional activity and sensitizes T-cell lymphomas to BCL2 inhibitors

    PubMed Central

    Cayrol, Florencia; Praditsuktavorn, Pannee; Fernando, Tharu M.; Kwiatkowski, Nicholas; Marullo, Rosella; Calvo-Vidal, M. Nieves; Phillip, Jude; Pera, Benet; Yang, Shao Ning; Takpradit, Kaipol; Roman, Lidia; Gaudiano, Marcello; Crescenzo, Ramona; Ruan, Jia; Inghirami, Giorgio; Zhang, Tinghu; Cremaschi, Graciela; Gray, Nathanael S.; Cerchietti, Leandro

    2017-01-01

    Peripheral T-cell lymphomas (PTCL) are aggressive diseases with poor response to chemotherapy and dismal survival. Identification of effective strategies to target PTCL biology represents an urgent need. Here we report that PTCL are sensitive to transcription-targeting drugs, and, in particular, to THZ1, a covalent inhibitor of cyclin-dependent kinase 7 (CDK7). The STAT-signalling pathway is highly vulnerable to THZ1 even in PTCL cells that carry the activating STAT3 mutation Y640F. In mutant cells, CDK7 inhibition decreases STAT3 chromatin binding and expression of highly transcribed target genes like MYC, PIM1, MCL1, CD30, IL2RA, CDC25A and IL4R. In surviving cells, THZ1 decreases the expression of STAT-regulated anti-apoptotic BH3 family members MCL1 and BCL-XL sensitizing PTCL cells to BH3 mimetic drugs. Accordingly, the combination of THZ1 and the BH3 mimetic obatoclax improves lymphoma growth control in a primary PTCL ex vivo culture and in two STAT3-mutant PTCL xenografts, delineating a potential targeted agent-based therapeutic option for these patients. PMID:28134252

  9. First BRET-based screening assay performed in budding yeast leads to the discovery of CDK5/p25 interaction inhibitors.

    PubMed

    Corbel, Caroline; Wang, Qian; Bousserouel, Hadjira; Hamdi, Amel; Zhang, Bing; Lozach, Olivier; Ferandin, Yoan; Tan, Vincent B C; Guéritte, Françoise; Colas, Pierre; Couturier, Cyril; Bach, Stéphane

    2011-07-01

    The protein kinase CDK5 (cyclin-dependent kinase 5) is activated through its association with a cyclin-like protein p35 or p39. In pathological conditions (such as Alzheimer's disease and various other neuropathies), truncation of p35 leads to the appearance of the p25 protein. The interaction of p25 with CDK5 up-regulates the kinase activity and modifies the substrate specificity. ATP-mimetic inhibitors of CDK5 have already been developed. However, the lack of selectivity of such inhibitors is often a matter of concern. An alternative approach can be used to identify highly specific inhibitors that disrupt protein interactions involving protein kinases. We have developed a bioluminescence resonance energy transfer (BRET)-based screening assay in yeast to discover protein-protein interaction inhibitors (P2I2). Here, we present the first use of BRET in yeast for the screening of small molecule libraries. This screening campaign led to the discovery of one molecule that prevents the interaction between CDK5 and p25, thus inhibiting the protein kinase activity. This molecule may give rise to high-specificity drug candidates.

  10. Antitumor action of CDK inhibitor LS-007 as a single agent and in combination with ABT-199 against human acute leukemia cells

    PubMed Central

    Xie, Shao; Jiang, Hui; Zhai, Xiao-wen; Wei, Fan; Wang, Shu-dong; Ding, Jian; Chen, Yi

    2016-01-01

    Aim: LS-007 is a CDK inhibitor, which exhibits potent antitumor activity against chronic lymphocytic leukemia and ovarian cancer cells. In this study, we further evaluated the antitumor activity of LS-007 alone and in combination with a Bcl-2 inhibitor ABT-199 in acute leukemia (AL) cells. Methods: Cell viability was detected using resazurin assay, and cell apoptosis was examined using Annexin V/PI double staining and flow cytometry. The inhibition of LS-007 on kinases was evaluated with the mobility shift assay or ELISA. The expression of relevant signaling molecules was assessed using Western blotting and RT-PCR. Primary lymphocytes from patients with acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) were separated using Ficoll-Paque PLUS. Results: LS-007 inhibited the proliferation of 6 AL cell lines with IC50 values of 100–200 nmol/L, and decreased the survival of ALL and AML patient-derived lymphocytes with mean LD50 value of 67 and 102 nmol/L, respectively. In kinase assays in vitro, LS-007 was more selective for the CDK family, inhibiting CDK2, CDK9, CDK1 and CDK4 at low nanomolar concentrations. In HL-60 and CCRF-CEM cells, LS-007 (0.1–0.4 μmol/L) dose-dependently induced cell apoptosis predominantly through CDK9 inhibition-related dephosphorylation at the ser2 residue of RNA pol II and the corresponding depletion of anti-apoptotic proteins, especially Mcl-1 and XIAP. LS-007 (0.2 and 0.4 μmol/L) also induced cell apoptosis in the patient-derived lymphocytes. In HL-60, CCRF-CEM and Molt-4 cells, combined application of LS-007 with ABT-199 (1 or 2 μmol/L) markedly increased cell apoptosis with a maximal decrease in the XIAP levels as compared with either drug used alone. Conclusion: CDK inhibitor LS-007 potently inhibits the established human AL cell lines and primary AL blasts, and it also shows remarkable synergy with Bcl-2 inhibitor ABT-199. PMID:27569395

  11. CDK inhibitors, p21{sup Cip1} and p27{sup Kip1}, participate in cell cycle exit of mammalian cardiomyocytes

    SciTech Connect

    Tane, Shoji; Ikenishi, Aiko; Okayama, Hitomi; Iwamoto, Noriko; Nakayama, Keiichi I.; Takeuchi, Takashi

    2014-01-17

    Highlights: •Expression of p21 and p27 in the hearts showed a peak during postnatal stages. •p21 and p27 bound to cyclin E, cyclin A and CDK2 in the hearts at postnatal stages. •Cardiomyocytes in both KO mice showed failure in the cell cycle exit at G1-phase. •These data show the first apparent phenotypes in the hearts of Cip/Kip KO mice. -- Abstract: Mammalian cardiomyocytes actively proliferate during embryonic stages, following which cardiomyocytes exit their cell cycle after birth. The irreversible cell cycle exit inhibits cardiac regeneration by the proliferation of pre-existing cardiomyocytes. Exactly how the cell cycle exit occurs remains largely unknown. Previously, we showed that cyclin E- and cyclin A-CDK activities are inhibited before the CDKs levels decrease in postnatal stages. This result suggests that factors such as CDK inhibitors (CKIs) inhibit CDK activities, and contribute to the cell cycle exit. In the present study, we focused on a Cip/Kip family, which can inhibit cyclin E- and cyclin A-CDK activities. Expression of p21{sup Cip1} and p27{sup Kip1} but not p57{sup Kip2} showed a peak around postnatal day 5, when cyclin E- and cyclin A-CDK activities start to decrease. p21{sup Cip1} and p27{sup Kip1} bound to cyclin E, cyclin A and CDK2 at postnatal stages. Cell cycle distribution patterns of postnatal cardiomyocytes in p21{sup Cip1} and p27{sup Kip1} knockout mice showed failure in the cell cycle exit at G1-phase, and endoreplication. These results indicate that p21{sup Cip1} and p27{sup Kip} play important roles in the cell cycle exit of postnatal cardiomyocytes.

  12. TARGETING CDK4 AND CDK6: FROM DISCOVERY TO THERAPY

    PubMed Central

    Sherr, Charles J.; Beach, David; Shapiro, Geoffrey I.

    2015-01-01

    Biochemical and genetic characterization of D-type cyclins, their cyclin D-dependent kinases (CDK4 and CDK6), and the polypeptide CDK4/6 inhibitor p16INK4 over two decades ago revealed how mammalian cells regulate entry into the DNA synthetic (S) phase of the cell division cycle in a retinoblastoma protein (RB)-dependent manner. These investigations provided proof-of-principle that CDK4/6 inhibitors, particularly when combined with co-inhibition of allied mitogen-dependent signal transduction pathways, might prove valuable in cancer therapy. FDA-approval of the CDK4/6 inhibitor palbociclib used with the aromatase inhibitor letrozole for breast cancer treatment highlights long sought success. The newest findings herald clinical trials targeting other cancers. PMID:26658964

  13. M-phase regulation of the recruitment of mRNAs onto polysomes using the CDK1/cyclin B inhibitor aminopurvalanol.

    PubMed

    Le Breton, Magali; Bellé, Robert; Cormier, Patrick; Mulner-Lorillon, Odile; Morales, Julia

    2003-07-11

    Translation under the control of the universal cell cycle regulator CDK1/cyclin B was investigated during the first cell cycle in sea urchin embryos. The CDK1/cyclin B inhibitor aminopurvalanol arrested embryos at the G2/M transition. Polysomal mRNAs were purified from control and arrested embryos, and screened for specific mRNA recruitment or release at M-phase by subtractive hybridization. The polysomal repartition of clones issued from this screen was analyzed. Three specific mRNAs were selectively recruited onto polysomes at M-phase. Conversely, two other specific mRNAs were released from polysomes. The isolation of these translationally regulated mRNAs gives now important tools for insights into the regulation of protein synthesis by the cell cycle regulator CDK1-cyclin B.

  14. Jumping the nuclear envelop barrier: Improving polyplex-mediated gene transfection efficiency by a selective CDK1 inhibitor RO-3306.

    PubMed

    Zhou, Xuefei; Liu, Xiangrui; Zhao, Bingxiang; Liu, Xin; Zhu, Dingcheng; Qiu, Nasha; Zhou, Quan; Piao, Ying; Zhou, Zhuxian; Tang, Jianbin; Shen, Youqing

    2016-07-28

    Successful transfection of plasmid DNA (pDNA) requires intranuclear internalization of pDNA effectively and the nuclear envelope appears to be one of the critical intracellular barriers for polymer mediated pDNA delivery. Polyethylenimine (PEI), as the classic cationic polymer, compact the negatively charged pDNA tightly and make up stable polyplexes. The polyplexes are too large to enter the nuclear through nuclear pores and it is believed that the nuclear envelope breakdown in mitosis could facilitate the nuclear entry of polyplexes. To jump the nuclear envelope barrier, we used a selective and reversible CDK1 inhibitor RO-3306 to control the G2/M transition of the cell cycle and increased the proportion of mitotic cells which have disappeared nuclear envelope during transfection. Herein, we show that RO-3306 remarkably increases the transfection efficiency of PEI polyplexes through enhanced nuclear localization of PEI and pDNA. However, RO-3306 is less effective to the charge-reversal polymer poly[(2-acryloyl)ethyl(p-boronic acid benzyl)diethylammonium bromide] (B-PDEAEA) which responses to cellular stimuli and releases free pDNA in cytoplasm. Our findings not only offer new opportunities for improving non-viral based gene delivery but also provide theoretical support for the rational design of novel functional polymers for gene delivery. We also report current data showing that RO-3306 synergizes TRAIL gene induced apoptosis in cancer cells.

  15. AZD5438, an Inhibitor of Cdk1, 2, and 9, Enhances the Radiosensitivity of Non-Small Cell Lung Carcinoma Cells

    SciTech Connect

    Raghavan, Pavithra; Tumati, Vasu; Yu Lan; Chan, Norman; Tomimatsu, Nozomi; Burma, Sandeep; Bristow, Robert G.; Saha, Debabrata

    2012-11-15

    Purpose: Radiation therapy (RT) is one of the primary modalities for treatment of non-small cell lung cancer (NSCLC). However, due to the intrinsic radiation resistance of these tumors, many patients experience RT failure, which leads to considerable tumor progression including regional lymph node and distant metastasis. This preclinical study evaluated the efficacy of a new-generation cyclin-dependent kinase (Cdk) inhibitor, AZD5438, as a radiosensitizer in several NSCLC models that are specifically resistant to conventional fractionated RT. Methods and Materials: The combined effect of ionizing radiation and AZD5438, a highly specific inhibitor of Cdk1, 2, and 9, was determined in vitro by surviving fraction, cell cycle distribution, apoptosis, DNA double-strand break (DSB) repair, and homologous recombination (HR) assays in 3 NSCLC cell lines (A549, H1299, and H460). For in vivo studies, human xenograft animal models in athymic nude mice were used. Results: Treatment of NSCLC cells with AZD5438 significantly augmented cellular radiosensitivity (dose enhancement ratio rangeing from 1.4 to 1.75). The degree of radiosensitization by AZD5438 was greater in radioresistant cell lines (A549 and H1299). Radiosensitivity was enhanced specifically through inhibition of Cdk1, prolonged G{sub 2}-M arrest, inhibition of HR, delayed DNA DSB repair, and increased apoptosis. Combined treatment with AZD5438 and irradiation also enhanced tumor growth delay, with an enhancement factor ranging from 1.2-1.7. Conclusions: This study supports the evaluation of newer generation Cdk inhibitors, such as AZD5438, as potent radiosensitizers in NSCLC models, especially in tumors that demonstrate variable intrinsic radiation responses.

  16. Cooperation between the Cdk inhibitors p27KIP1 and p57KIP2 in the control of tissue growth and development

    PubMed Central

    Zhang, Pumin; Wong, Calvin; DePinho, Ronald A.; Harper, J. Wade; Elledge, Stephen J.

    1998-01-01

    Cell cycle exit is required for terminal differentiation of many cell types. The retinoblastoma protein Rb has been implicated both in cell cycle exit and differentiation in several tissues. Rb is negatively regulated by cyclin-dependent kinases (Cdks). The main effectors that down-regulate Cdk activity to activate Rb are not known in the lens or other tissues. In this study, using multiple mutant mice, we show that the Cdk inhibitors p27KIP1 and p57KIP2 function redundantly to control cell cycle exit and differentiation of lens fiber cells and placental trophoblasts. These studies demonstrate that p27KIP1 and p57KIP2 are critical terminal effectors of signal transduction pathways that control cell differentiation. PMID:9784491

  17. The cyclin-dependent kinase (CDK) inhibitor flavopiridol inhibits glycogen phosphorylase.

    PubMed

    Kaiser, A; Nishi, K; Gorin, F A; Walsh, D A; Bradbury, E M; Schnier, J B

    2001-02-15

    Flavopiridol has been shown to induce cell cycle arrest and apoptosis in various tumor cells in vitro and in vivo. Using immobilized flavopiridol, we identified glycogen phosphorylases (GP) from liver and brain as flavopiridol binding proteins from HeLa cell extract. Purified rabbit muscle GP also bound to the flavopiridol affinity column. GP is the rate-limiting enzyme in intracellular glycogen breakdown. Flavopiridol significantly inhibited the AMP-activated GP-b form of the purified rabbit muscle isoenzyme (IC50 of 1 microM at 0.8 mM AMP), but was less inhibitory to the active phosphorylated form of GP, GP-a (IC50 of 2.5 microM). The AMP-bound GP-a form was poorly inhibited by flavopiridol (40% at 10 microM). Increasing concentrations of the allosteric effector AMP resulted in a linear decrease in the GP-inhibitory activity of flavopiridol suggesting interference between flavopiridol and AMP. In contrast the GP inhibitor caffeine had no effect on the relative GP inhibition by flavopiridol, suggesting an additive effect of caffeine. Flavopiridol also inhibited the phosphorylase kinase-catalyzed phosphorylation of GP-b by inhibiting the kinase in vitro. Flavopiridol thus is able to interfere with both activating modifications of GP-b, AMP activation and phosphorylation. In A549 NSCLC cells flavopiridol treatment caused glycogen accumulation despite of an increase in GP activity, suggesting direct GP inhibition in vivo rather than inhibition of GP activation by phosphorylase kinase. These results suggest that the cyclin-dependent kinase inhibitor flavopiridol interferes with glycogen degradation, which may be responsible for flavopiridol's cytotoxicity and explain its resistance in some cell lines.

  18. Conformation and recognition of DNA damaged by antitumor cis-dichlorido platinum(II) complex of CDK inhibitor bohemine.

    PubMed

    Novakova, Olga; Liskova, Barbora; Vystrcilova, Jana; Suchankova, Tereza; Vrana, Oldrich; Starha, Pavel; Travnicek, Zdenek; Brabec, Viktor

    2014-05-06

    A substitution of the ammine ligands of cisplatin, cis-[Pt(NH3)2Cl2], for cyclin dependent kinase (CDK) inhibitor bohemine (boh), [2-(3-hydroxypropylamino)-6-benzylamino-9-isopropylpurine], results in a compound, cis-[Pt(boh)2Cl2] (C1), with the unique anticancer profile which may be associated with some features of the damaged DNA and/or its cellular processing (Travnicek Z et al. (2003) J Inorg Biochem94, 307-316; Liskova B (2012) Chem Res Toxicol25, 500-509). A combination of biochemical and molecular biology techniques was used to establish mechanistic differences between cisplatin and C1 with respect to the DNA damage they produce and their interactions with critical DNA-binding proteins, DNA-processing enzymes and glutathione. The results show that replacement of the NH3 groups in cisplatin by bohemine modulates some aspects of the mechanism of action of C1. More specifically, the results of the present work are consistent with the thesis that, in comparison with cisplatin, effects of other factors, such as: (i) slower rate of initial binding of C1 to DNA; (ii) the lower efficiency of C1 to form bifunctional adducts; (iii) the reduced bend of longitudinal DNA axis induced by the major 1,2-GG intrastrand cross-link of C1; (iv) the reduced affinity of HMG domain proteins to the major adduct of C1; (v) the enhanced efficiency of the DNA adducts of C1 to block DNA polymerization and to inhibit transcription activity of human RNA pol II and RNA transcription; (vi) slower rate of the reaction of C1 with glutathione, may partially contribute to the unique activity of C1.

  19. PP2A inhibitors arrest G2/M transition through JNK/Sp1- dependent down-regulation of CDK1 and autophagy-dependent up-regulation of p21.

    PubMed

    Gong, Fei-Ran; Wu, Meng-Yao; Shen, Meng; Zhi, Qiaoming; Xu, Ze-Kuan; Wang, Rong; Wang, Wen-Jie; Zong, Yang; Li, Zeng-Liang; Wu, Yadi; Zhou, Binhua P; Chen, Kai; Tao, Min; Li, Wei

    2015-07-30

    Protein phosphatase 2A (PP2A) plays an important role in the control of the cell cycle. We previously reported that the PP2A inhibitors, cantharidin and okadaic acid (OA), efficiently repressed the growth of cancer cells. In the present study, we found that PP2A inhibitors arrested the cell cycle at the G2 phase through a mechanism that was dependent on the JNK pathway. Microarrays further showed that PP2A inhibitors induced expression changes in multiple genes that participate in cell cycle transition. To verify whether these expression changes were executed in a PP2A-dependent manner, we targeted the PP2A catalytic subunit (PP2Ac) using siRNA and evaluated gene expression with a microarray. After the cross comparison of these microarray data, we identified that CDK1 was potentially the same target when treated with either PP2A inhibitors or PP2Ac siRNA. In addition, we found that the down-regulation of CDK1 occurred in a JNK-dependent manner. Luciferase reporter gene assays demonstrated that repression of the transcription of CDK1 was executed through the JNK-dependent activation of the Sp1 transcription factor. By constructing deletion mutants of the CDK1 promoter and by using ChIP assays, we identified an element in the CDK1 promoter that responded to the JNK/Sp1 pathway after stimulation with PP2A inhibitors. Cantharidin and OA also up-regulated the expression of p21, an inhibitor of CDK1, via autophagy rather than PP2A/JNK pathway. Thus, this present study found that the PP2A/JNK/Sp1/CDK1 pathway and the autophagy/p21 pathway participated in G2/M cell cycle arrest triggered by PP2A inhibitors.

  20. Metal-based paullones as putative CDK inhibitors for antitumor chemotherapy.

    PubMed

    Schmid, Wolfgang F; John, Roland O; Mühlgassner, Gerhard; Heffeter, Petra; Jakupec, Michael A; Galanski, Markus; Berger, Walter; Arion, Vladimir B; Keppler, Bernhard K

    2007-12-13

    Paullones constitute a class of potent cyclin-dependent kinase inhibitors. To overcome the insufficient solubility and bioavailability, which hamper their potential medical application, we aim at the development of metal-based derivatives. Two types of paullone ligands, L (1) - L (3) and L (4) , with different locations of metal-binding sites, were prepared. They were found to form organometallic complexes of the general formula [M (II)Cl(eta (6)- p-cymene)L]Cl ( 1- 4, L = L (1) - L (4) ; a, M = Ru; b, M = Os). The complexes were characterized by X-ray crystallography, one- and two-dimensional NMR spectroscopy and other physical methods. Complexes 1- 3, with a coordinated amidine unit, were found to undergo E/ Z isomerization in solution. The reaction was studied by NMR spectroscopy, and activation parameters Delta H (double dagger) and Delta S (double dagger) were determined. Antiproliferative activity in the low micromolar range was observed in vitro in three human cancer cell lines by means of MTT assays. (3)H-Thymidine incorporation assays revealed the compounds to lower the rate of DNA synthesis, and flow cytometric analyses showed cell cycle arrest mainly in G 0/ G 1 phase.

  1. The CDK9 Inhibitor Dinaciclib Exerts Potent Apoptotic and Antitumor Effects in Preclinical Models of MLL-Rearranged Acute Myeloid Leukemia.

    PubMed

    Baker, Adele; Gregory, Gareth P; Verbrugge, Inge; Kats, Lev; Hilton, Joshua J; Vidacs, Eva; Lee, Erwin M; Lock, Richard B; Zuber, Johannes; Shortt, Jake; Johnstone, Ricky W

    2016-03-01

    Translocations of the mixed lineage leukemia (MLL) gene occur in 60% to 80% of all infant acute leukemias and are markers of poor prognosis. MLL-AF9 and other MLL fusion proteins aberrantly recruit epigenetic regulatory proteins, including histone deacetylases (HDAC), histone methyltransferases, bromodomain-containing proteins, and transcription elongation factors to mediate chromatin remodeling and regulate tumorigenic gene expression programs. We conducted a small-molecule inhibitor screen to test the ability of candidate pharmacologic agents targeting epigenetic and transcriptional regulatory proteins to induce apoptosis in leukemic cells derived from genetically engineered mouse models of MLL-AF9-driven acute myeloid leukemia (AML). We found that the CDK inhibitor dinaciclib and HDAC inhibitor panobinostat were the most potent inducers of apoptosis in short-term in vitro assays. Treatment of MLL-rearranged leukemic cells with dinaciclib resulted in rapidly decreased expression of the prosurvival protein Mcl-1, and accordingly, overexpression of Mcl-1 protected AML cells from dinaciclib-induced apoptosis. Administration of dinaciclib to mice bearing MLL-AF9-driven human and mouse leukemias elicited potent antitumor responses and significantly prolonged survival. Collectively, these studies highlight a new therapeutic approach to potentially overcome the resistance of MLL-rearranged AML to conventional chemotherapies and prompt further clinical evaluation of CDK inhibitors in AML patients harboring MLL fusion proteins.

  2. Studies of CDK 8/19 inhibitors: Discovery of novel and selective CDK8/19 dual inhibitors and elimination of their CYP3A4 time-dependent inhibition potential.

    PubMed

    Fujimoto, Jun; Hirayama, Takaharu; Hirata, Yasuhiro; Hikichi, Yukiko; Murai, Saomi; Hasegawa, Maki; Hasegawa, Yuka; Yonemori, Kazuko; Hata, Akito; Aoyama, Kazunobu; Cary, Douglas R

    2017-03-30

    In this article, synthetic studies around a pyridylacrylamide-based hit compound (1), utilizing structure-based drug design guided by CDK8 docking models, is discussed. Modification of the pendant 4-fluorophenyl group to various heteroaromatic rings was conducted aiming an interaction with the proximal amino acids, and then replacement of the morpholine ring was targeted for decreasing potential of time-dependent CYP3A4 inhibition. These efforts led to the compound 4k, with enhanced CDK8 inhibitory activity and no apparent potential for time-dependent CYP3A4 inhibition (CDK8 IC50: 2.5nM; CYP3A4 TDI: 99% compound remaining). Compound 4k was found to possess a highly selective kinase inhibition profile, and also showed favorable pharmacokinetic profile. Oral administration of 4k (15mg/kg, bid. for 2weeks) suppressed tumor growth (T/C 29%) in an RPMI8226 mouse xenograft model.

  3. CDK1 structures reveal conserved and unique features of the essential cell cycle CDK

    PubMed Central

    Brown, Nicholas R.; Korolchuk, Svitlana; Martin, Mathew P.; Stanley, Will A.; Moukhametzianov, Rouslan; Noble, Martin E. M.; Endicott, Jane A.

    2015-01-01

    CDK1 is the only essential cell cycle CDK in human cells and is required for successful completion of M-phase. It is the founding member of the CDK family and is conserved across all eukaryotes. Here we report the crystal structures of complexes of CDK1–Cks1 and CDK1–cyclin B–Cks2. These structures confirm the conserved nature of the inactive monomeric CDK fold and its ability to be remodelled by cyclin binding. Relative to CDK2–cyclin A, CDK1–cyclin B is less thermally stable, has a smaller interfacial surface, is more susceptible to activation segment dephosphorylation and shows differences in the substrate sequence features that determine activity. Both CDK1 and CDK2 are potential cancer targets for which selective compounds are required. We also describe the first structure of CDK1 bound to a potent ATP-competitive inhibitor and identify aspects of CDK1 structure and plasticity that might be exploited to develop CDK1-selective inhibitors. PMID:25864384

  4. CDK1 structures reveal conserved and unique features of the essential cell cycle CDK

    NASA Astrophysics Data System (ADS)

    Brown, Nicholas R.; Korolchuk, Svitlana; Martin, Mathew P.; Stanley, Will A.; Moukhametzianov, Rouslan; Noble, Martin E. M.; Endicott, Jane A.

    2015-04-01

    CDK1 is the only essential cell cycle CDK in human cells and is required for successful completion of M-phase. It is the founding member of the CDK family and is conserved across all eukaryotes. Here we report the crystal structures of complexes of CDK1-Cks1 and CDK1-cyclin B-Cks2. These structures confirm the conserved nature of the inactive monomeric CDK fold and its ability to be remodelled by cyclin binding. Relative to CDK2-cyclin A, CDK1-cyclin B is less thermally stable, has a smaller interfacial surface, is more susceptible to activation segment dephosphorylation and shows differences in the substrate sequence features that determine activity. Both CDK1 and CDK2 are potential cancer targets for which selective compounds are required. We also describe the first structure of CDK1 bound to a potent ATP-competitive inhibitor and identify aspects of CDK1 structure and plasticity that might be exploited to develop CDK1-selective inhibitors.

  5. A Novel High-Throughput 3D Screening System for EMT Inhibitors: A Pilot Screening Discovered the EMT Inhibitory Activity of CDK2 Inhibitor SU9516

    PubMed Central

    Eguchi, Takanori; Rahman, M. Mamunur; Sakamoto, Ruriko; Masuda, Norio; Nakatsura, Tetsuya; Calderwood, Stuart K.; Kozaki, Ken-ichi; Itoh, Manabu

    2016-01-01

    Epithelial-mesenchymal transition (EMT) is a crucial pathological event in cancer, particularly in tumor cell budding and metastasis. Therefore, control of EMT can represent a novel therapeutic strategy in cancer. Here, we introduce an innovative three-dimensional (3D) high-throughput screening (HTS) system that leads to an identification of EMT inhibitors. For the establishment of the novel 3D-HTS system, we chose NanoCulture Plates (NCP) that provided a gel-free micro-patterned scaffold for cells and were independent of other spheroid formation systems using soft-agar. In the NCP-based 3D cell culture system, A549 lung cancer cells migrated, gathered, and then formed multiple spheroids within 7 days. Live cell imaging experiments showed that an established EMT-inducer TGF-β promoted peripheral cells around the core of spheroids to acquire mesenchymal spindle shapes, loss of intercellular adhesion, and migration from the spheroids. Along with such morphological change, EMT-related gene expression signatures were altered, particularly alteration of mRNA levels of ECAD/CDH1, NCAD/CDH2, VIM and ZEB1/TCF8. These EMT-related phenotypic changes were blocked by SB431542, a TGF-βreceptor I (TGFβR1) inhibitor. Inside of the spheroids were highly hypoxic; in contrast, spheroid-derived peripheral migrating cells were normoxic, revealed by visualization and quantification using Hypoxia Probe. Thus, TGF-β-triggered EMT caused spheroid hypoplasia and loss of hypoxia. Spheroid EMT inhibitory (SEMTIN) activity of SB431542 was calculated from fluorescence intensities of the Hypoxia Probe, and then was utilized in a drug screening of EMT-inhibitory small molecule compounds. In a pilot screening, 9 of 1,330 compounds were above the thresholds of the SEMTIN activity and cell viability. Finally, two compounds SB-525334 and SU9516 showed SEMTIN activities in a dose dependent manner. SB-525334 was a known TGFβR1 inhibitor. SU9516 was a cyclin-dependent kinase 2 (CDK2) inhibitor

  6. Regulation of the G1 phase of the cell cycle by periodic stabilization and degradation of the p25rum1 CDK inhibitor.

    PubMed Central

    Benito, J; Martín-Castellanos, C; Moreno, S

    1998-01-01

    In fission yeast, the cyclin-dependent kinase (CDK) inhibitor p25(rum1) is a key regulator of progression through the G1 phase of the cell cycle. We show here that p25(rum1) protein levels are sharply periodic. p25(rum1) begins to accumulate at anaphase, persists in G1 and is destroyed during S phase. p25(rum1 )is stabilized and polyubiquitinated in a mutant defective in the 26S proteasome, suggesting that its degradation normally occurs through the ubiquitin-dependent 26S proteasome pathway. Phosphorylation of p25(rum1 )by cdc2-cyclin complexes at residues T58 and T62 is important to target the protein for degradation. Mutation of one or both of these residues to alanine causes stabilization of p25(rum1) and induces a cell cycle delay in G1 and polyploidization due to occasional re-initiation of DNA replication before mitosis. The CDK-cyclin complex cdc2-cig1, which is insensitive to p25(rum1 )inhibition, seems to be the main kinase that phosphorylates p25(rum1). Phosphorylation of p25(rum1) in S phase and G2 serves as the trigger for p25(rum1) proteolysis. Thus, periodic accumulation and degradation of the CDK inhibitor p25(rum1 )in G1 plays a role in setting a threshold of cyclin levels important in determining the length of the pre-Start G1 phase and in ensuring the correct order of cell cycle events. PMID:9430640

  7. Mitigation of acute kidney injury by cell-cycle inhibitors that suppress both CDK4/6 and OCT2 functions.

    PubMed

    Pabla, Navjotsingh; Gibson, Alice A; Buege, Mike; Ong, Su Sien; Li, Lie; Hu, Shuiying; Du, Guoqing; Sprowl, Jason A; Vasilyeva, Aksana; Janke, Laura J; Schlatter, Eberhard; Chen, Taosheng; Ciarimboli, Giuliano; Sparreboom, Alex

    2015-04-21

    Acute kidney injury (AKI) is a potentially fatal syndrome characterized by a rapid decline in kidney function caused by ischemic or toxic injury to renal tubular cells. The widely used chemotherapy drug cisplatin accumulates preferentially in the renal tubular cells and is a frequent cause of drug-induced AKI. During the development of AKI the quiescent tubular cells reenter the cell cycle. Strategies that block cell-cycle progression ameliorate kidney injury, possibly by averting cell division in the presence of extensive DNA damage. However, the early signaling events that lead to cell-cycle activation during AKI are not known. In the current study, using mouse models of cisplatin nephrotoxicity, we show that the G1/S-regulating cyclin-dependent kinase 4/6 (CDK4/6) pathway is activated in parallel with renal cell-cycle entry but before the development of AKI. Targeted inhibition of CDK4/6 pathway by small-molecule inhibitors palbociclib (PD-0332991) and ribociclib (LEE011) resulted in inhibition of cell-cycle progression, amelioration of kidney injury, and improved overall survival. Of additional significance, these compounds were found to be potent inhibitors of organic cation transporter 2 (OCT2), which contributes to the cellular accumulation of cisplatin and subsequent kidney injury. The unique cell-cycle and OCT2-targeting activities of palbociclib and LEE011, combined with their potential for clinical translation, support their further exploration as therapeutic candidates for prevention of AKI.

  8. A computational study of the protein-ligand interactions in CDK2 inhibitors: using quantum mechanics/molecular mechanics interaction energy as a predictor of the biological activity.

    PubMed

    Alzate-Morales, Jans H; Contreras, Renato; Soriano, Alejandro; Tuñon, Iñaki; Silla, Estanislao

    2007-01-15

    We report a combined quantum mechanics/molecular mechanics (QM/MM) study to determine the protein-ligand interaction energy between CDK2 (cyclin-dependent kinase 2) and five inhibitors with the N(2)-substituted 6-cyclohexyl-methoxy-purine scaffold. The computational results in this work show that the QM/MM interaction energy is strongly correlated to the biological activity and can be used as a predictor, at least within a family of substrates. A detailed analysis of the protein-ligand structures obtained from molecular dynamics simulations shows specific interactions within the active site that, in some cases, have not been reported before to our knowledge. The computed interaction energy gauges the strength of protein-ligand interactions. Finally, energy decomposition and multiple regression analyses were performed to check the contribution of the electrostatic and van der Waals energies to the total interaction energy and to show the capabilities of the computational model to identify new potent inhibitors.

  9. Marine steroids as potential anticancer drug candidates: In silico investigation in search of inhibitors of Bcl-2 and CDK-4/Cyclin D1.

    PubMed

    Saikia, Surovi; Kolita, Bhaskor; Dutta, Partha P; Dutta, Deep J; Neipihoi; Nath, Shyamalendu; Bordoloi, Manobjyoti; Quan, Pham Minh; Thuy, Tran Thu; Phuong, Doan Lan; Long, Pham Quoc

    2015-10-01

    Star fishes (Asteroidea) are rich in polar steroids with diverse structural characteristics. The structural modifications of star fish steroids occur at 3β, 4β, 5α, 6α (or β), 7α (or β), 8, 15α (or β) and 16β positions of the steroidal nucleus and in the side chain. Widely found polar steroids in starfishes include polyhydroxysteroids, steroidal sulfates, glycosides, steroid oligoglycosides etc. Bioactivity of these steroids is less studied; only a few reports like antibacterial, cytotoxic activity etc. are available. In continuation of our search for bioactive molecules from natural sources, we undertook in silico screening of steroids from star fishes against Bcl-2 and CDK-4/Cyclin D1 - two important targets of progression and proliferation of cancer cells. We have screened 182 natural steroids from star fishes occurring in different parts of the world and their 282 soft-derivatives by in silico methods. Their physico-chemical properties, drug-likeliness, binding potential with the selected targets, ADMET (absorption, distribution, metabolism, toxicity) were predicted. Further, the results were compared with those of existing steroidal and non steroidal drugs and inhibitors of Bcl-2 and CDK-4/Cyclin D1. The results are promising and unveil that some of these steroids can be potent leads for cancer treatments.

  10. Short Communication: The Broad-Spectrum Histone Deacetylase Inhibitors Vorinostat and Panobinostat Activate Latent HIV in CD4+ T Cells In Part Through Phosphorylation of the T-Loop of the CDK9 Subunit of P-TEFb

    PubMed Central

    Jamaluddin, Md Saha; Hu, Pei-Wen; Jan, Yih; Siwak, Edward B.

    2016-01-01

    Abstract Cessation of highly active antiretroviral therapy (HAART) in HIV-infected individual leads to a rebound of viral replication due to reactivation of a viral reservoir composed largely of latently infected memory CD4+ T cells. Efforts to deplete this reservoir have focused on reactivation of transcriptionally silent latent proviruses. HIV provirus transcription depends critically on the positive transcription elongation factor b (P-TEFb), whose core components are cyclin-dependent kinase 9 (CDK9) and cyclin T1. In resting CD4+ cells, the functional levels of P-TEFb are extremely low. Cellular activation upregulates cyclin T1 protein levels and CDK9 T-loop (T186) phosphorylation. The broad-spectrum histone deacetylase inhibitors (HDACis) vorinostat and panobinostat have been shown to reactivate latent virus in vivo in HAART-treated individuals. In this study, we have found that vorinostat and panobinostat activate P-TEFb in resting primary CD4+ T cells through induction of CDK9 T-loop phosphorylation. In contrast, tacedinaline and romidepsin, HDAC 1 and 2 inhibitors, were unable to activate CDK9 T-loop phosphorylation. We used a CCL19 primary CD4+ T-cell model HIV latency to assess the correlation between induction of CDK9 T-loop phosphorylation and reactivation of latent HIV virus by HDACis. Vorinostat and panobinostat treatment of cells harboring latent HIV increased CDK9 T-loop phosphorylation and reactivation of latent virus, whereas tacedinaline and romidepsin failed to induce T-loop phosphorylation or reactivate latent virus. We conclude that the ability of vorinostat and panobinostat to induce latent HIV is, in part, likely due to the ability of the broad-spectrum HDACis to upregulate P-TEFb through increased CDK9 T-loop phosphorylation. PMID:26727990

  11. Loss of CDKN2A expression is a frequent event in primary invasive melanoma and correlates with sensitivity to the CDK4/6 inhibitor PD0332991 in melanoma cell lines.

    PubMed

    Young, Richard J; Waldeck, Kelly; Martin, Claire; Foo, Jung H; Cameron, Donald P; Kirby, Laura; Do, Hongdo; Mitchell, Catherine; Cullinane, Carleen; Liu, Wendy; Fox, Stephen B; Dutton-Regester, Ken; Hayward, Nicholas K; Jene, Nicholas; Dobrovic, Alexander; Pearson, Richard B; Christensen, James G; Randolph, Sophia; McArthur, Grant A; Sheppard, Karen E

    2014-07-01

    We have investigated the potential for the p16-cyclin D-CDK4/6-retinoblastoma protein pathway to be exploited as a therapeutic target in melanoma. In a cohort of 143 patients with primary invasive melanoma, we used fluorescence in situ hybridization to detect gene copy number variations (CNVs) in CDK4, CCND1, and CDKN2A and immunohistochemistry to determine protein expression. CNVs were common in melanoma, with gain of CDK4 or CCND1 in 37 and 18% of cases, respectively, and hemizygous or homozygous loss of CDKN2A in 56%. Three-quarters of all patients demonstrated a CNV in at least one of the three genes. The combination of CCND1 gain with either a gain of CDK4 and/or loss of CDKN2A was associated with poorer melanoma-specific survival. In 47 melanoma cell lines homozygous loss, methylation or mutation of CDKN2A gene or loss of protein (p16(INK) (4A) ) predicted sensitivity to the CDK4/6 inhibitor PD0332991, while RB1 loss predicted resistance.

  12. FoxF1 and FoxF2 transcription factors synergistically promote Rhabdomyosarcoma carcinogenesis by repressing transcription of p21Cip1 CDK inhibitor

    PubMed Central

    Cai, Yuqi; Le, Tien; Turpin, Brian; Kalinichenko, Vladimir V.; Kalin, Tanya V.

    2016-01-01

    The role of Forkhead Box F1 (FoxF1) transcription factor in carcinogenesis is not well characterized. Depending on tissue and histological type of cancer, FoxF1 was shown to be either oncogene or tumor suppressor. Alveolar rhabdomyosarcoma (RMS) is the most aggressive pediatric soft tissue sarcoma. While FoxF1 is highly expressed in alveolar RMS, the functional role of FoxF1 in RMS is unknown. The present study demonstrates that expression of FoxF1 and its closely related transcription factor FoxF2 are essential for rhabdomyosarcoma tumor growth. Depletion of FoxF1 or FoxF2 in rhabdomyosarcoma cells decreased tumor growth in orthotopic mouse models of RMS. The decreased tumorigenesis was associated with the reduced tumor cell proliferation. Cell cycle regulatory proteins Cdk2, Cdk4/6, Cyclin D1 and Cyclin E2 were decreased in FoxF1- and FoxF2-deficient RMS tumors. Depletion of either FoxF1 or FoxF2 delayed G1-S cell cycle progression, decreased levels of phosphorylated Rb and increased protein levels of the CDK inhibitors, p21Cip1 and p27Kip1. Depletion of both FoxF1 and FoxF2 in tumor cells completely abrogated RMS tumor growth in mice. Overexpression of either FoxF1 or FoxF2 in tumor cells was sufficient to increase carcinogenesis in orthotopic RMS mouse model. FoxF1 and FoxF2 directly bound to and repressed transcriptional activity of p21Cip1 promoter through −556/−545 bp region, but did not affect p27Kip1 transcription. Knockdown of p21Cip1 restored cell cycle progression in the FoxF1- or FoxF2-deficient tumor cells. Altogether, FoxF1 and FoxF2 promoted RMS tumorigenesis by inducing tumor cell proliferation via transcriptional repression of p21Cip1 gene promoter. Due to robust oncogenic activity in RMS tumors, FoxF1 and FoxF2 may represent promising targets for anti-tumor therapy. PMID:27425595

  13. Dual Targeting of CDK4 and ARK5 Using a Novel Kinase Inhibitor ON123300 Exerts Potent Anticancer Activity against Multiple Myeloma.

    PubMed

    Perumal, Deepak; Kuo, Pei-Yu; Leshchenko, Violetta V; Jiang, Zewei; Divakar, Sai Krishna Athaluri; Cho, Hearn Jay; Chari, Ajai; Brody, Joshua; Reddy, M V Ramana; Zhang, Weijia; Reddy, E Premkumar; Jagannath, Sundar; Parekh, Samir

    2016-03-01

    Multiple myeloma is a fatal plasma cell neoplasm accounting for over 10,000 deaths in the United States each year. Despite new therapies, multiple myeloma remains incurable, and patients ultimately develop drug resistance and succumb to the disease. The response to selective CDK4/6 inhibitors has been modest in multiple myeloma, potentially because of incomplete targeting of other critical myeloma oncogenic kinases. As a substantial number of multiple myeloma cell lines and primary samples were found to express AMPK-related protein kinase 5(ARK5), a member of the AMPK family associated with tumor growth and invasion, we examined whether dual inhibition of CDK4 and ARK5 kinases using ON123300 results in a better therapeutic outcome. Treatment of multiple myeloma cell lines and primary samples with ON123300 in vitro resulted in rapid induction of cell-cycle arrest followed by apoptosis. ON123300-mediated ARK5 inhibition or ARK5-specific siRNAs resulted in the inhibition of the mTOR/S6K pathway and upregulation of the AMPK kinase cascade. AMPK upregulation resulted in increased SIRT1 levels and destabilization of steady-state MYC protein. Furthermore, ON123300 was very effective in inhibiting tumor growth in mouse xenograft assays. In addition, multiple myeloma cells sensitive to ON123300 were found to have a unique genomic signature that can guide the clinical development of ON123300. Our study provides preclinical evidence that ON123300 is unique in simultaneously inhibiting key oncogenic pathways in multiple myeloma and supports further development of ARK5 inhibition as a therapeutic approach in multiple myeloma.

  14. Cdk6 contributes to cytoskeletal stability in erythroid cells.

    PubMed

    Uras, Iris Z; Scheicher, Ruth M; Kollmann, Karoline; Glösmann, Martin; Prchal-Murphy, Michaela; Tigan, Anca S; Fux, Daniela A; Altamura, Sandro; Neves, Joana; Muckenthaler, Martina; Bennett, Keiryn L; Kubicek, Stefan; Hinds, Philip W; von Lindern, Marieke; Sexl, Veronika

    2017-03-02

    Mice lacking Cdk6 kinase activity suffer from mild anemia accompanied by elevated numbers of Ter119+ cells in the bone marrow. The animals show hardly any alterations in erythroid development, indicating that Cdk6 is not required for proliferation and maturation of erythroid cells. There is also no difference in stress erythropoiesis following hemolysis in vivo. However, Cdk6-/- erythrocytes have a shortened lifespan and are more sensitive to mechanical stress in vitro, suggesting differences in the cytoskeletal architecture. Erythroblasts contain both Cdk4 and Cdk6, while mature erythrocytes apparently lack Cdk4 and their Cdk6 is partly associated with the cytoskeleton. We used mass spectrometry to show that Cdk6 interacts with a number of proteins involved in cytoskeletal organization. Cdk6-/- erythroblasts show impaired F-actin formation and lower levels of gelsolin, which interacts with Cdk6. We show further that Cdk6 regulates the transcription of a panel of genes involved in actin (de-) polymerization. Cdk6-deficient cells are sensitive to drugs that interfere with the cytoskeleton, suggesting that our findings are relevant to the treatment of patients with anemia and may be relevant to cancer patients treated with the new generation of CDK6 inhibitors.

  15. VMY-1-103 is a novel CDK inhibitor that disrupts chromosome organization and delays metaphase progression in medulloblastoma cells.

    PubMed

    Ringer, Lymor; Sirajuddin, Paul; Heckler, Mary; Ghosh, Anup; Suprynowicz, Frank; Yenugonda, Venkata M; Brown, Milton L; Toretsky, Jeffrey A; Uren, Aykut; Lee, YiChien; MacDonald, Tobey J; Rodriguez, Olga; Glazer, Robert I; Schlegel, Richard; Albanese, Chris

    2011-11-01

    Medulloblastoma is the most prevalent of childhood brain malignancies, constituting 25% of childhood brain tumors. Craniospinal radiotherapy is a standard of care, followed by a 12mo regimen of multi-agent chemotherapy. For children less than 3 y of age, irradiation is avoided due to its destructive effects on the developing nervous system. Long-term prognosis is worst for these youngest children and more effective treatment strategies with a better therapeutic index are needed. VMY-1-103, a novel dansylated analog of purvalanol B, was previously shown to inhibit cell cycle progression and proliferation in prostate and breast cancer cells more effectively than purvalanol B. In the current study, we have identified new mechanisms of action by which VMY-1-103 affected cellular proliferation in medulloblastoma cells. VMY-1-103, but not purvalanol B, significantly decreased the proportion of cells in S phase and increased the proportion of cells in G(2)/M. VMY-1-103 increased the sub G(1) fraction of apoptotic cells, induced PARP and caspase-3 cleavage and increased the levels of the Death Receptors DR4 and DR5, Bax and Bad while decreasing the number of viable cells, all supporting apoptosis as a mechanism of cell death. p21(CIP1/WAF1) levels were greatly suppressed. Importantly, we found that while both VMY and flavopiridol inhibited intracellular CDK1 catalytic activity, VMY-1-103 was unique in its ability to severely disrupt the mitotic spindle apparatus significantly delaying metaphase and disrupting mitosis. Our data suggest that VMY-1-103 possesses unique antiproliferative capabilities and that this compound may form the basis of a new candidate drug to treat medulloblastoma.

  16. Rho/ROCK pathway inhibition by the CDK inhibitor p27(kip1) participates in the onset of macrophage 3D-mesenchymal migration.

    PubMed

    Gui, Philippe; Labrousse, Arnaud; Van Goethem, Emeline; Besson, Arnaud; Maridonneau-Parini, Isabelle; Le Cabec, Véronique

    2014-09-15

    Infiltration of macrophages into tissue can promote tumour development. Depending on the extracellular matrix architecture, macrophages can adopt two migration modes: amoeboid migration--common to all leukocytes, and mesenchymal migration--restricted to macrophages and certain tumour cells. Here, we investigate the initiating mechanisms involved in macrophage mesenchymal migration. We show that a single macrophage is able to use both migration modes. Macrophage mesenchymal migration is correlated with decreased activity of Rho/Rho-associated protein kinase (ROCK) and is potentiated when ROCK is inhibited, suggesting that amoeboid inhibition participates in mechanisms that initiate mesenchymal migration. We identify the cyclin-dependent kinase (CDK) inhibitor p27(kip1) (also known as CDKN1B) as a new effector of macrophage 3D-migration. By using p27(kip1) mutant mice and small interfering RNA targeting p27(kip1), we show that p27(kip1) promotes mesenchymal migration and hinders amoeboid migration upstream of the Rho/ROCK pathway, a process associated with a relocation of the protein from the nucleus to the cytoplasm. Finally, we observe that cytoplasmic p27(kip1) is required for in vivo infiltration of macrophages within induced tumours in mice. This study provides the first evidence that silencing of amoeboid migration through inhibition of the Rho/ROCK pathway by p27(kip1) participates in the onset of macrophage mesenchymal migration.

  17. Neuroprotective Mechanisms Mediated by CDK5 Inhibition

    PubMed Central

    Mushtaq, Gohar; Greig, Nigel H.; Anwar, Firoz; Al-Abbasi, Fahad A.; Zamzami, Mazin A.; Al-Talhi, Hasan A.; Kamal, Mohammad A.

    2016-01-01

    Cyclin-dependent kinase 5 (CDK5) is a proline-directed serine/threonine kinase belonging to the family of cyclin-dependent kinases. In addition to maintaining the neuronal architecture, CDK5 plays an important role in the regulation of synaptic plasticity, neurotransmitter release, neuron migration and neurite outgrowth. Although various reports have shown links between neurodegeneration and deregulation of cyclin-dependent kinases, the specific role of CDK5 inhibition in causing neuroprotection in cases of neuronal insult or in neurodegenerative diseases is not well-understood. This article discusses current evidence for the involvement of CDK5 deregulation in neurodegenerative disorders and neurodegeneration associated with stroke through various mechanisms. These include upregulation of cyclin D1 and overactivation of CDK5 mediated neuronal cell death pathways, aberrant hyperphosphorylation of human tau proteins and/or neurofilament proteins, formation of neurofibrillary lesions, excitotoxicity, cytoskeletal disruption, motor neuron death (due to abnormally high levels of CDK5/p25) and colchicine-induced apoptosis in cerebellar granule neurons. A better understanding of the role of CDK5 inhibition in neuroprotective mechanisms will help scientists and researchers to develop selective, safe and efficacious pharmacological inhibitors of CDK5 for therapeutic use against human neurodegenerative disorders, such as Alzheimer’s disease, amyotrophic lateral sclerosis and neuronal loss associated with stroke. PMID:26601962

  18. Molecular modeling studies of 4,5-dihydro-1H-pyrazolo[4,3-h] quinazoline derivatives as potent CDK2/Cyclin a inhibitors using 3D-QSAR and docking.

    PubMed

    Ai, Yong; Wang, Shao-Teng; Sun, Ping-Hua; Song, Fa-Jun

    2010-09-28

    CDK2/cyclin A has appeared as an attractive drug targets over the years with diverse therapeutic potentials. A computational strategy based on comparative molecular fields analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) followed by molecular docking studies were performed on a series of 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline derivatives as potent CDK2/cyclin A inhibitors. The CoMFA and CoMSIA models, using 38 molecules in the training set, gave r(2) (cv) values of 0.747 and 0.518 and r(2) values of 0.970 and 0.934, respectively. 3D contour maps generated by the CoMFA and CoMSIA models were used to identify the key structural requirements responsible for the biological activity. Molecular docking was applied to explore the binding mode between the ligands and the receptor. The information obtained from molecular modeling studies may be helpful to design novel inhibitors of CDK2/cyclin A with desired activity.

  19. In Silico Identification and In Vitro and In Vivo Validation of Anti-Psychotic Drug Fluspirilene as a Potential CDK2 Inhibitor and a Candidate Anti-Cancer Drug

    PubMed Central

    Yao, Hong; Liu, Xu; Li, Ling; Leung, Kwong-Sak; Kung, Hsiang-fu; Lu, Di; Wong, Man-Hon; Lin, Marie Chia-mi

    2015-01-01

    Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide. Surgical resection and conventional chemotherapy and radiotherapy ultimately fail due to tumor recurrence and HCC’s resistance. The development of novel therapies against HCC is thus urgently required. The cyclin-dependent kinase (CDK) pathways are important and well-established targets for cancer treatment. In particular, CDK2 is a key factor regulating the cell cycle G1 to S transition and a hallmark for cancers. In this study, we utilized our free and open-source protein-ligand docking software, idock, prospectively to identify potential CDK2 inhibitors from 4,311 FDA-approved small molecule drugs using a repurposing strategy and an ensemble docking methodology. Sorted by average idock score, nine compounds were purchased and tested in vitro. Among them, the anti-psychotic drug fluspirilene exhibited the highest anti-proliferative effect in human hepatocellular carcinoma HepG2 and Huh7 cells. We demonstrated for the first time that fluspirilene treatment significantly increased the percentage of cells in G1 phase, and decreased the expressions of CDK2, cyclin E and Rb, as well as the phosphorylations of CDK2 on Thr160 and Rb on Ser795. We also examined the anti-cancer effect of fluspirilene in vivo in BALB/C nude mice subcutaneously xenografted with human hepatocellular carcinoma Huh7 cells. Our results showed that oral fluspirilene treatment significantly inhibited tumor growth. Fluspirilene (15 mg/kg) exhibited strong anti-tumor activity, comparable to that of the leading cancer drug 5-fluorouracil (10 mg/kg). Moreover, the cocktail treatment with fluspirilene and 5-fluorouracil exhibited the highest therapeutic effect. These results suggested for the first time that fluspirilene is a potential CDK2 inhibitor and a candidate anti-cancer drug for the treatment of human hepatocellular carcinoma. In view of the fact that fluspirilene has a long history of safe human

  20. Fission yeast LAMMER kinase Lkh1 regulates the cell cycle by phosphorylating the CDK-inhibitor Rum1

    SciTech Connect

    Yu, Eun-Young; Lee, Ju-Hee; Kang, Won-Hwa; Park, Yun-Hee; Kim, Lila; Park, Hee-Moon

    2013-03-01

    Highlights: ► Deletion of lkh1{sup +} made cells pass the G1/S phase faster than the wild type. ► Lkh1 can interact with a cyclin-dependent kinase inhibitor (CKI) Rum1. ► Lkh1 can phosphorylate Rum1 to activate its CKI activity. ► Thr110 was confirmed as the Lkh1-dependent phosphorylation site of Rum1. ► Positive acting mechanism for the Rum1 activation is reported for the first time. - Abstract: In eukaryotes, LAMMER kinases are involved in various cellular events, including the cell cycle. However, no attempt has been made to investigate the mechanisms that underlie the involvement of LAMMER kinase. In this study, we performed a functional analysis of LAMMER kinase using the fission yeast, Schizosaccharomyces pombe. FACS analyses revealed that deletion of the gene that encodes the LAMMER kinase Lkh1 made mutant cells pass through the G1/S phase faster than their wild-type counterparts. Co-immunoprecipitation and an in vitro kinase assay also revealed that Lkh1 can interact with and phosphorylate Rum1 to activate this molecule as a cyclin-dependent kinase inhibitor, which blocks cell cycle progression from the G1 phase to the S phase. Peptide mass fingerprinting and kinase assay with Rum1{sup T110A} confirmed T110 as the Lkh1-dependent phosphorylation residue. In this report we present for the first time a positive acting mechanism that is responsible for the CKI activity of Rum1, in which the LAMMER kinase-mediated phosphorylation of Rum1 is involved.

  1. CDK inhibitors (p16/p19/p21) induce senescence and autophagy in cancer-associated fibroblasts, “fueling” tumor growth via paracrine interactions, without an increase in neo-angiogenesis

    PubMed Central

    Capparelli, Claudia; Chiavarina, Barbara; Whitaker-Menezes, Diana; Pestell, Timothy G.; Pestell, Richard G.; Hulit, James; Andò, Sebastiano; Howell, Anthony; Martinez-Outschoorn, Ubaldo E.; Sotgia, Federica; Lisanti, Michael P.

    2012-01-01

    Here, we investigated the compartment-specific role of cell cycle arrest and senescence in breast cancer tumor growth. For this purpose, we generated a number of hTERT-immortalized senescent fibroblast cell lines overexpressing CDK inhibitors, such as p16(INK4A), p19(ARF) or p21(WAF1/CIP1). Interestingly, all these senescent fibroblast cell lines showed evidence of increased susceptibility toward the induction of autophagy (either at baseline or after starvation), as well as significant mitochondrial dysfunction. Most importantly, these senescent fibroblasts also dramatically promoted tumor growth (up to ~2-fold), without any comparable increases in tumor angiogenesis. Conversely, we generated human breast cancer cells (MDA-MB-231 cells) overexpressing CDK inhibitors, namely p16(INK4A) or p21(WAF1/CIP1). Senescent MDA-MB-231 cells also showed increased expression of markers of cell cycle arrest and autophagy, including β-galactosidase, as predicted. Senescent MDA-MB-231 cells had retarded tumor growth, with up to a near 2-fold reduction in tumor volume. Thus, the effects of CDK inhibitors are compartment-specific and are related to their metabolic effects, which results in the induction of autophagy and mitochondrial dysfunction. Finally, induction of cell cycle arrest with specific inhibitors (PD0332991) or cellular stressors [hydrogen peroxide (H₂O₂) or starvation] indicated that the onset of autophagy and senescence are inextricably linked biological processes. The compartment-specific induction of senescence (and hence autophagy) may be a new therapeutic target that could be exploited for the successful treatment of human breast cancer patients. PMID:22935696

  2. Enhanced malignant tumorigenesis in Cdk4 transgenic mice.

    PubMed

    Miliani de Marval, Paula L; Macias, Everardo; Conti, Claudio J; Rodriguez-Puebla, Marcelo L

    2004-03-11

    In a previous study, we reported that overexpression of cyclin-dependent kinase-4 (CDK4) in mouse epidermis results in epidermal hyperplasia, hypertrophy and severe dermal fibrosis. In this study, we have investigated the susceptibility to skin tumor formation by forced expression of CDK4. Skin tumors from transgenic mice showed a dramatic increase in the rate of malignant progression to squamous cell carcinomas (SCC) in an initiation-promotion protocol. Histopathological analysis of papillomas from transgenic mice showed an elevated number of premalignant lesions characterized by dysplasia and marked atypia. Interestingly, transgenic mice also developed tumors in initiated but not promoted skin, demonstrating that CDK4 replaced the action of tumor promoters. These results suggest that expression of cyclin D1 upon ras activation synergizes with CDK4 overexpression. However, cyclin D1 transgenic mice and double transgenic mice for cyclin D1 and CDK4 did not show increased malignant progression in comparison to CDK4 transgenic mice. Biochemical analysis of tumors showed that CDK4 sequesters the CDK2 inhibitors p27Kip1 and p21Cip1, suggesting that indirect activation of CDK2 plays an important role in tumor development. These results indicate that, contrary to the general assumption, the catalytic subunit, CDK4, has higher oncogenic activity than cyclin D1, revealing a potential use of CDK4 as therapeutic target.

  3. CDK9 inhibitors selectively target estrogen receptor-positive breast cancer cells through combined inhibition of MYB and MCL-1 expression

    PubMed Central

    Mitra, Partha; Yang, Ren-Ming; Sutton, James; Ramsay, Robert G.; Gonda, Thomas J.

    2016-01-01

    Our previous studies showed that MYB is required for proliferation of, and confers protection against apoptosis on, estrogen receptor-positive (ER+ve) breast cancer cells, which are almost invariably also MYB+ve. We have also shown that MYB expression in ER+ve breast cancer cells is regulated at the level of transcriptional elongation and as such, is suppressed by CDK9i. Here we examined the effects of CDK9i on breast cancer cells and the involvement of MYB in these effects. ER+ve breast cancer cell lines including MCF-7 were much more sensitive (> 10 times) to killing by CDK9i than ER−ve/MYB−ve cells. Moreover, surviving cells showed a block at the G2/M phase of the cell cycle. Importantly, ectopic MYB expression conferred resistance to apoptosis induction, cell killing and G2/M accumulation. Expression of relevant MYB target genes including BCL2 and CCNB1 was suppressed by CDK9 inhibition, and this too was reversed by ectopic MYB expression. Nevertheless, inhibition of BCL2 alone either by MYB knockdown or by ABT-199 treatment was insufficient for significant induction of apoptosis. Further studies implied that suppression of MCL-1, a well-documented target of CDK9 inhibition, was additionally required for apoptosis induction, while maximal levels of apoptosis induced by CDK9i are likely to also involve inhibition of BCL2L1 expression. Taken together these data suggest that MYB regulation of BCL2 underlies the heightened sensitivity of ER+ve compared to ER−ve breast cancer cells to CDK9 inhibition, and that these compounds represent a potential therapeutic for ER+ve breast cancers and possibly other MYB-dependent cancers. PMID:26812885

  4. CDK4 coexpression with Ras generates malignant human epidermal tumorigenesis.

    PubMed

    Lazarov, Mirella; Kubo, Yoshiaki; Cai, Ti; Dajee, Maya; Tarutani, Masahito; Lin, Qun; Fang, Min; Tao, Shiying; Green, Cheryl L; Khavari, Paul A

    2002-10-01

    Ras acts with other proteins to induce neoplasia. By itself, however, strong Ras signaling can suppress proliferation of normal cells. In primary epidermal cells, we found that oncogenic Ras transiently decreases cyclin-dependent kinase (CDK) 4 expression in association with cell cycle arrest in G1 phase. CDK4 co-expression circumvents Ras growth suppression and induces invasive human neoplasia resembling squamous cell carcinoma. Tumorigenesis is dependent on CDK4 kinase function, with cyclin D1 required but not sufficient for this process. In facilitating escape from G1 growth restraints, Ras and CDK4 alter the composition of cyclin D and cyclin E complexes and promote resistance to growth inhibition by INK4 cyclin-dependent kinase inhibitors. These data identify a new role for oncogenic Ras in CDK4 regulation and highlight the functional importance of CDK4 suppression in preventing uncontrolled growth.

  5. An essential pathway links FLT3-ITD, HCK and CDK6 in acute myeloid leukemia

    PubMed Central

    Lopez, Sophie; Voisset, Edwige; Tisserand, Julie C.; Mosca, Cyndie; Prebet, Thomas; Santamaria, David; Dubreuil, Patrice; Sepulveda, Paulo De

    2016-01-01

    CDK4/CDK6 and RB proteins drive the progression through the G1 phase of the cell cycle. In acute myeloid leukemia (AML), the activity of the CDK/Cyclin D complex is increased. The mechanism involved is unknown, as are the respective roles played by CDK4 or CDK6 in this process. Here, we report that AML cells carrying FLT3-ITD mutations are dependent on CDK6 for cell proliferation while CDK4 is not essential. We showed that FLT3-ITD signaling is responsible for CDK6 overexpression, through a pathway involving the SRC-family kinase HCK. Accordingly, FLT3-ITD failed to transform primary hematopoietic progenitor cells from Cdk6−/− mice. Our results demonstrate that CDK6 is the primary target of CDK4/CDK6 inhibitors in FLT3-ITD positive AML. Furthermore, we delineate an essential protein kinase pathway -FLT3/HCK/CDK6- in the context of AML with FLT3-ITD mutations. PMID:27323399

  6. Cdk4 deficiency inhibits skin tumor development but does not affect normal keratinocyte proliferation.

    PubMed

    Rodriguez-Puebla, Marcelo L; Miliani de Marval, Paula L; LaCava, Margaret; Moons, David S; Kiyokawa, Hiroaki; Conti, Claudio J

    2002-08-01

    Most human tumors have mutations that result in deregulation of the cdk4/cyclin-Ink4-Rb pathway. Overexpression of D-type cyclins or cdk4 and inactivation of Ink4 inhibitors are common in human tumors. Conversely, lack of cyclin D1 expression results in significant reduction in mouse skin and mammary tumor development. However, complete elimination of tumor development was not observed in these models, suggesting that other cyclin/cdk complexes play an important role in tumorigenesis. Here we described the effects of cdk4 deficiency on mouse skin proliferation and tumor development. Cdk4 deficiency resulted in a 98% reduction in the number of tumors generated through the two-stage carcinogenesis model. The absence of cdk4 did not affect normal keratinocyte proliferation and both wild-type and cdk4 knockout epidermis are equally affected after topical treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), resulting in epidermal hyperplasia. In similar fashion, cdk4 knockout keratinocytes proliferated well in an in vivo model of wound-induced proliferation. Biochemical studies in mouse epidermis showed that cdk6 activity increased twofold in cdk4-deficient mice compared to wild-type siblings. These results suggest that therapeutic approaches to inhibit cdk4 activity could provide a target to inhibit tumor development with minimal or no effect in normal tissue.

  7. Efficacy of CDK4 inhibition against sarcomas depends on their levels of CDK4 and p16ink4 mRNA

    PubMed Central

    Perez, Marco; Muñoz-Galván, Sandra; Jiménez-García, Manuel P.; Marín, Juan J.; Carnero, Amancio

    2015-01-01

    Sarcomas are malignant tumors accounting for a high percentage of cancer morbidity and mortality in children and young adults. Surgery and radiation therapy are the accepted treatments for most sarcomas; however, patients with metastatic disease are treated with systemic chemotherapy. Many tumors display marginal levels of chemoresponsiveness and new treatment approaches are needed. Deregulation of the G1 checkpoint is crucial for various oncogenic transformation processes, suggesting that many cancer cell types depend on CDK4/6 activity. Thus, CDK4/6 activity appears to represent a promising therapeutic target for cancer treatment. In the present work, we explore the efficacy of CDK4 inhibition using palbociclib (PD0332991), a highly selective inhibitor of CDK4/6, in a panel of sarcoma cell lines and sarcoma tumor xenografts (PDXs). Palbociclib induces senescence in these cell lines and the responsiveness of these cell lines correlated with their levels of CDK4 mRNA. Palbociclib is also active in vivo against sarcomas displaying high levels of CDK4 but not against sarcomas displaying low levels of CDK4 and high levels of p16ink4a. The analysis of tumors growing after palbociclib showed a clear decrease in the CDK4 levels, indicating that clonal selection occurred in these treated tumors. In summary, our data support the efficacy of CDK4 inhibitors against sarcomas displaying increased CDK4 levels, particularly fibrosarcomas and MPNST. Our results also suggest that high levels of p16ink4a may indicate poor efficacy of CDK4 inhibitors. PMID:26528855

  8. A selective chemical probe for exploring the role of CDK8 and CDK19 in human disease

    PubMed Central

    Esdar, Christina; Waalboer, Dennis; Adeniji-Popoola, Olajumoke; Ortiz-Ruiz, Maria-Jesus; Mallinger, Aurélie; Samant, Rahul S.; Czodrowski, Paul; Musil, Djordje; Schwarz, Daniel; Schneider, Klaus; Stubbs, Mark; Ewan, Ken; Fraser, Elizabeth; TePoele, Robert; Court, Will; Box, Gary; Valenti, Melanie; de Haven Brandon, Alexis; Gowan, Sharon; Rohdich, Felix; Raynaud, Florence; Schneider, Richard; Poeschke, Oliver; Blaukat, Andree; Workman, Paul; Schiemann, Kai; Eccles, Suzanne A.; Wienke, Dirk; Blagg, Julian

    2015-01-01

    There is unmet need for chemical tools to explore the role of the Mediator complex in human pathologies ranging from cancer to cardiovascular disease. Here we determine that CCT251545, a small molecule WNT-pathway inhibitor discovered through cell-based screening, is a potent and selective chemical probe for the human Mediator complex-associated protein kinases CDK8 and CDK19 with >100-fold selectivity over 291 other kinases. X-ray crystallography demonstrates a Type 1 binding mode involving insertion of the CDK8 C-terminus into the ligand binding site. In contrast to Type II inhibitors of CDK8/19, CCT251545 displays potent cell-based activity. We show that CCT251545 and close analogues alter WNT-pathway regulated gene expression and other on-target effects of modulating CDK8/19 including genes regulated by STAT1. Consistent with this we find that phosphorylation of STAT1SER727 is a biomarker of CDK8 kinase activity in vitro and in vivo. Finally, we demonstrate in vivo activity of CCT251545 in WNT-dependent tumors. PMID:26502155

  9. The pan-neural bHLH proteins DEADPAN and ASENSE regulate mitotic activity and cdk inhibitor dacapo expression in the Drosophila larval optic lobes.

    PubMed

    Wallace, K; Liu, T H; Vaessin, H

    2000-01-01

    Developmental regulators and cell cycle regulators have to interface in order to ensure appropriate cell proliferation during organogenesis. Our analysis of the roles of the pan-neural genes deadpan and asense defines critical roles for these genes in regulation of mitotic activities in the larval optic lobes. Loss of deadpan results in reduced cell proliferation, while ectopic deadpan expression causes over-proliferation. In contrast, loss of asense results in increased proliferation, while ectopic asense expression causes reduced proliferation. Consistent with these observations endogenous Deadpan is expressed in mitotic areas of the optic lobes, and endogenous Asense is expressed in cells that will become quiescent. Altered Deadpan or Asense expression results in altered expression of the cyclin dependent kinase inhibitor gene dacapo. Thus, regulation of mitotic activity during optic lobe development may, at least in part, involve deadpan and asense mediated regulation of the cyclin dependent kinase inhibitor gene dacapo. genesis 26:77-85, 2000.

  10. Hepatocyte growth factor (HGF) inhibits skeletal muscle cell differentiation: a role for the bHLH protein twist and the cdk inhibitor p27.

    PubMed

    Leshem, Y; Spicer, D B; Gal-Levi, R; Halevy, O

    2000-07-01

    Hepatocyte growth factor (HGF) plays a crucial role in regulating the differentiation of both fetal and adult skeletal myoblasts. This study aimed at defining the intracellular factors that mediate the effect of HGF on adult myoblast differentiation. HGF increased Twist expression while decreasing p27(kip1) protein levels and not affecting the induction of p21(Cip1/Waf1) in satellite cells. Like HGF, overexpression of Twist did not affect p21 expression while inhibiting muscle-specific proteins. Both ectopic Twist-antisense (Twist-AS) and p27 partially rescued the effects of HGF on bromodeoxyuridine (BrdU) incorporation and myosin heavy chain (MHC) expression in muscle satellite cells; the two plasmids together effected full rescue, suggesting that HGF independently regulates these two factors to mediate its effects. Ectopic p27 promoted differentiation in the presence of HGF by blocking the induction of Twist. Using Twist-AS to lower Twist levels restored the HGF-dependent reduction of p27 and MHC. In the presence of ectopic HGF, satellite cells formed thin mononuclear myotubes. Neither ectopic p27, Twist-AS, or their combination reversed this change in cell morphology, suggesting that HGF acts through additional mediators to inhibit downstream events during myogenesis. Taken together, the results suggest that the effects of HGF on muscle cell proliferation and differentiation are mediated through changes in the expression levels of the myogenic-inhibitory basic helix-loop-helix (bHLH) protein Twist and the cell-cycle inhibitor p27.

  11. Compensatory induction of MYC expression by sustained CDK9 inhibition via a BRD4-dependent mechanism

    PubMed Central

    Lu, Huasong; Xue, Yuhua; Yu, Guoying K; Arias, Carolina; Lin, Julie; Fong, Susan; Faure, Michel; Weisburd, Ben; Ji, Xiaodan; Mercier, Alexandre; Sutton, James; Luo, Kunxin; Gao, Zhenhai; Zhou, Qiang

    2015-01-01

    CDK9 is the kinase subunit of positive transcription elongation factor b (P-TEFb) that enables RNA polymerase (Pol) II's transition from promoter-proximal pausing to productive elongation. Although considerable interest exists in CDK9 as a therapeutic target, little progress has been made due to lack of highly selective inhibitors. Here, we describe the development of i-CDK9 as such an inhibitor that potently suppresses CDK9 phosphorylation of substrates and causes genome-wide Pol II pausing. While most genes experience reduced expression, MYC and other primary response genes increase expression upon sustained i-CDK9 treatment. Essential for this increase, the bromodomain protein BRD4 captures P-TEFb from 7SK snRNP to deliver to target genes and also enhances CDK9's activity and resistance to inhibition. Because the i-CDK9-induced MYC expression and binding to P-TEFb compensate for P-TEFb's loss of activity, only simultaneously inhibiting CDK9 and MYC/BRD4 can efficiently induce growth arrest and apoptosis of cancer cells, suggesting the potential of a combinatorial treatment strategy. DOI: http://dx.doi.org/10.7554/eLife.06535.001 PMID:26083714

  12. βTrCP controls the lysosome-mediated degradation of CDK1, whose accumulation correlates with tumor malignancy.

    PubMed

    Herrero-Ruiz, Joaquín; Mora-Santos, Mar; Giráldez, Servando; Sáez, Carmen; Japón, Miguel A; Tortolero, Maria; Romero, Francisco

    2014-09-15

    In mammals, cell cycle progression is controlled by cyclin-dependent kinases, among which CDK1 plays important roles in the regulation of the G2/M transition, G1 progression and G1/S transition. CDK1 is highly regulated by its association to cyclins, phosphorylation and dephosphorylation, changes in subcellular localization, and by direct binding of CDK inhibitor proteins. CDK1 steady-state protein levels are held constant throughout the cell cycle by a coordinated regulation of protein synthesis and degradation. We show that CDK1 is ubiquitinated by the E3 ubiquitin ligase SCFβTrCP and degraded by the lysosome. Furthermore, we found that DNA damage not only triggers the stabilization of inhibitory phosphorylation sites on CDK1 and repression of CDK1 gene expression, but also regulates βTrCP-induced CDK1 degradation in a cell type-dependent manner. Specifically, treatment with the chemotherapeutic agent doxorubicin in certain cell lines provokes CDK1 degradation and induces apoptosis, whereas in others it inhibits destruction of the protein. These observations raise the possibility that different tumor types, depending on their pathogenic spectrum mutations, may display different sensitivity to βTrCP-induced CDK1 degradation after DNA damage. Finally, we found that CDK1 accumulation in patients' tumors shows a negative correlation with βTrCP and a positive correlation with the degree of tumor malignancy.

  13. βTrCP controls the lysosome-mediated degradation of CDK1, whose accumulation correlates with tumor malignancy

    PubMed Central

    Herrero-Ruiz, Joaquín; Mora-Santos, Mar; Giráldez, Servando; Sáez, Carmen; Japón, Miguel Á.; Tortolero, Maria; Romero, Francisco

    2014-01-01

    In mammals, cell cycle progression is controlled by cyclin-dependent kinases, among which CDK1 plays important roles in the regulation of the G2/M transition, G1 progression and G1/S transition. CDK1 is highly regulated by its association to cyclins, phosphorylation and dephosphorylation, changes in subcellular localization, and by direct binding of CDK inhibitor proteins. CDK1 steady-state protein levels are held constant throughout the cell cycle by a coordinated regulation of protein synthesis and degradation. We show that CDK1 is ubiquitinated by the E3 ubiquitin ligase SCFβTrCP and degraded by the lysosome. Furthermore, we found that DNA damage not only triggers the stabilization of inhibitory phosphorylation sites on CDK1 and repression of CDK1 gene expression, but also regulates βTrCP-induced CDK1 degradation in a cell type-dependent manner. Specifically, treatment with the chemotherapeutic agent doxorubicin in certain cell lines provokes CDK1 degradation and induces apoptosis, whereas in others it inhibits destruction of the protein. These observations raise the possibility that different tumor types, depending on their pathogenic spectrum mutations, may display different sensitivity to βTrCP-induced CDK1 degradation after DNA damage. Finally, we found that CDK1 accumulation in patients’ tumors shows a negative correlation with βTrCP and a positive correlation with the degree of tumor malignancy. PMID:25149538

  14. Accumulation of cytoplasmic Cdk1 is associated with cancer growth and survival rate in epithelial ovarian cancer

    PubMed Central

    Shin, Ha-Yeon; Chung, Joon-Yong; Kang, Eun Suk; Lee, Eun-ju; Kim, Jae-Hoon

    2016-01-01

    Cyclin dependent kinase 1 (Cdk1) have previously reported correlation with cancer growth and a key regulator for cell cycle. Mostly, Cdk1′s function of nucleus for cell cycle is well known to be associated with cancer, but cytoplasmic Cdk1′s traits are not clearly identified, yet. We revealed that tissue microarray blocks of epithelial ovarian cancer (n = 249) showed increased level of cytoplasmic Cdk1 (p < 0.001), but not in nucleus (p = 0.192) of histologic cell type independently. On survival analysis, Cdk1 overexpression conferred a significantly worse prognosis in 5-year overall survival (Log-rank p = 0.028, Hazard ratio = 2.016, 95% CI = 1.097 to 4.635). Also, the expression of Cdk1 was increased in ovarian cancer cell lines and Gene Expression Omnibus datasets. When the expression and activity of Cdk1 were inhibited by si-Cdk1 or RO-3306 which is a potent Cdk1 inhibitor, the growth of ovarian cancer was diminished. Moreover, combined treatment with RO-3306 and cisplatin in ovarian cancer significantly elevated anti-cancer effects than single-agent treatment. In conclusion, cytoplasmic Cdk1 expression which was elevated in ovarian cancer predicts a poor overall survival. The inhibition of Cdk1 expression and activity reduced ovarian cancer growth. PMID:27385216

  15. miR-30e controls DNA damage-induced stress responses by modulating expression of the CDK inhibitor p21WAF1/CIP1 and caspase-3

    PubMed Central

    Sohn, Dennis; Peters, Dominik; Piekorz, Roland P.; Budach, Wilfried; Jänicke, Reiner U.

    2016-01-01

    MicroRNAs (miRNAs), a class of small non-coding RNAs that usually cause gene silencing by translational repression or degradation of mRNAs, are implicated in DNA damage-induced stress responses. To identify senescence-associated miRNAs, we performed microarray analyses using wild-type and p53-deficient HCT116 colon carcinoma cells that following gamma-irradiation (γIR) are driven into senescence and apoptosis, respectively. Several miRNAs including miR-30e were found upregulated in a p53-dependent manner specifically in senescent cells, but not in apoptotic cells. Overexpression of miR-30e in HCT116 cells not only inhibited γIR-, etoposide- or miR-34a-induced caspase-3-like DEVDase activities and cell death, but greatly accelerated and augmented their senescent phenotype. Consistently, procaspase-3 protein, but not mRNA decreased in the presence of miR-30e, whereas expression of the cyclin-dependent kinase inhibitor p21 increased both at the mRNA and protein level. Performing luciferase reporter gene assays, we identified the 3′-UTR of the caspase-3 mRNA as a direct miR-30e target. In contrast, although miR-30e was unable to bind to the p21 mRNA, it increased expression of a luciferase construct containing the p21 promoter, suggesting that the miR-30e-mediated upregulation of p21 occurs indirectly at the transcriptional level. Interestingly, despite suppressing procaspase-3 expression, miR-30e was unable to protect RKO colon carcinoma cells from DNA damage-induced death or to induce senescence, as miR-30e completely fails to upregulate p21 in these cells. These data suggest that miR-30e functions in a cell type-dependent manner as an important molecular switch for DNA damage-induced stress responses and may thus represent a target of therapeutic value. PMID:26895377

  16. Cyclin-Dependent Kinase Inhibitors as Anticancer Therapeutics.

    PubMed

    Law, Mary E; Corsino, Patrick E; Narayan, Satya; Law, Brian K

    2015-11-01

    Cyclin-dependent kinases (CDKs) have been considered promising drug targets for a number of years, but most CDK inhibitors have failed rigorous clinical testing. Recent studies demonstrating clear anticancer efficacy and reduced toxicity of CDK4/6 inhibitors such as palbociclib and multi-CDK inhibitors such as dinaciclib have rejuvenated the field. Favorable results with palbociclib and its recent U.S. Food and Drug Administration approval demonstrate that CDK inhibitors with narrow selectivity profiles can have clinical utility for therapy based on individual tumor genetics. A brief overview of results obtained with ATP-competitive inhibitors such as palbociclib and dinaciclib is presented, followed by a compilation of new avenues that have been pursued toward the development of novel, non-ATP-competitive CDK inhibitors. These creative ways to develop CDK inhibitors are presented along with crystal structures of these agents complexed with CDK2 to highlight differences in their binding sites and mechanisms of action. The recent successes of CDK inhibitors in the clinic, combined with the potential for structure-based routes to the development of non-ATP-competitive CDK inhibitors, and evidence that CDK inhibitors may have use in suppressing chromosomal instability and in synthetic lethal drug combinations inspire optimism that CDK inhibitors will become important weapons in the fight against cancer.

  17. CDK2 Inhibition Causes Anaphase Catastrophe in Lung Cancer through the Centrosomal Protein CP110

    PubMed Central

    Hu, Shanhu; Danilov, Alexey V.; Godek, Kristina; Orr, Bernardo; Tafe, Laura J.; Rodriguez-Canales, Jaime; Behrens, Carmen; Mino, Barbara; Moran, Cesar A.; Memoli, Vincent A.; Mustachio, Lisa Maria; Galimberti, Fabrizio; Ravi, Saranya; DeCastro, Andrew; Lu, Yun; Sekula, David; Andrew, Angeline S; Wistuba, Ignacio I.; Freemantle, Sarah; Compton, Duane A.; Dmitrovsky, Ethan

    2015-01-01

    Aneuploidy is frequently detected in human cancers and is implicated in carcinogenesis. Pharmacological targeting of aneuploidy is an attractive therapeutic strategy as this would preferentially eliminate malignant over normal cells. We previously discovered that CDK2 inhibition causes lung cancer cells with more than two centrosomes to undergo multipolar cell division leading to apoptosis, defined as anaphase catastrophe. Cells with activating KRAS mutations were especially sensitive to CDK2 inhibition. Mechanisms of CDK2-mediated anaphase catastrophe and how activated KRAS enhances this effect were investigated. Live-cell imaging provided direct evidence that following CDK2 inhibition, lung cancer cells develop multipolar anaphase and undergo multipolar cell division with the resulting progeny apoptotic. Small interfering RNA (siRNA)-mediated repression of the CDK2 target and centrosome protein CP110 induced anaphase catastrophe of lung cancer cells. In contrast, CP110 overexpression antagonized CDK2 inhibitor-mediated anaphase catastrophe. Furthermore, activated KRAS mutations sensitized lung cancer cells to CDK2 inhibition by deregulating CP110 expression. Thus, CP110 is a critical mediator of CDK2-inhibition-driven anaphase catastrophe. Independent examination of murine and human paired normal-malignant lung tissues revealed marked upregulation of CP110 in malignant versus normal lung. Human lung cancers with KRAS mutations had significantly lower CP110 expression as compared to KRAS wild-type cancers. Thus, a direct link was found between CP110 and CDK2 inhibitor antineoplastic response. CP110 plays a mechanistic role in response of lung cancer cells to CDK2 inhibition, especially in the presence of activated KRAS mutations. PMID:25808870

  18. Modulation of aberrant CDK5 signaling rescues impaired neurogenesis in models of Alzheimer's disease.

    PubMed

    Crews, L; Patrick, C; Adame, A; Rockenstein, E; Masliah, E

    2011-02-10

    Recent studies show that in Alzheimer's disease (AD), alterations in neurogenesis contribute to the neurodegenerative process. Neurodegeneration in AD has been associated with aberrant signaling through the cyclin-dependent kinase-5 (CDK5) pathway via its activators p35/p25; however, the role of CDK5 in the mechanisms of defective adult neurogenesis in AD is unknown. First, to study AD-like abnormal activation of CDK5 signaling in an in vitro model of neurogenesis, neuronal progenitor cells (NPCs) were infected with a viral vector expressing p35, and exposed to amyloid-β protein (Aβ(1-42)). These conditions resulted in impaired maturation and neurite outgrowth in vitro, and these effects were reversed by pharmacological or genetic inhibition of CDK5. Similarly, neurogenesis was impaired in a transgenic mouse model of AD that expresses high levels of amyloid precursor protein (APP), and this effect was reversed in transgenic mice crossed with a CDK5 heterozygous-deficient mouse line. A similar rescue effect was observed in APP transgenic mice treated with Roscovitine, a pharmacological inhibitor of CDK5. Taken together, these data suggest that the CDK5 signaling pathway has a critical role in maintaining the integrity of NPCs and neuronal maturation in the adult hippocampus. Moreover, potential therapeutic approaches could focus on modulating the aberrant activity of CDK5 to target the neurogenic and neurodegenerative alterations in AD.

  19. A Kinase-Independent Activity of Cdk9 Modulates Glucocorticoid Receptor-Mediated Gene Induction

    PubMed Central

    2015-01-01

    A gene induction competition assay has recently uncovered new inhibitory activities of two transcriptional cofactors, NELF-A and NELF-B, in glucocorticoid-regulated transactivation. NELF-A and -B are also components of the NELF complex, which participates in RNA polymerase II pausing shortly after the initiation of gene transcription. We therefore asked if cofactors (Cdk9 and ELL) best known to affect paused polymerase could reverse the effects of NELF-A and -B. Unexpectedly, Cdk9 and ELL augmented, rather than prevented, the effects of NELF-A and -B. Furthermore, Cdk9 actions are not blocked either by Ckd9 inhibitors (DRB or flavopiridol) or by two Cdk9 mutants defective in kinase activity. The mode and site of action of NELF-A and -B mutants with an altered NELF domain are similarly affected by wild-type and kinase-dead Cdk9. We conclude that Cdk9 is a new modulator of GR action, that Ckd9 and ELL have novel activities in GR-regulated gene expression, that NELF-A and -B can act separately from the NELF complex, and that Cdk9 possesses activities that are independent of Cdk9 kinase activity. Finally, the competition assay has succeeded in ordering the site of action of several cofactors of GR transactivation. Extension of this methodology should be helpful in determining the site and mode of action of numerous additional cofactors and in reducing unwanted side effects. PMID:24559102

  20. Cdk7 mediates RPB1-driven mRNA synthesis in Toxoplasma gondii

    PubMed Central

    Deshmukh, Abhijit S.; Mitra, Pallabi; Maruthi, Mulaka

    2016-01-01

    Cyclin-dependent kinase 7 in conjunction with CyclinH and Mat1 activates cell cycle CDKs and is a part of the general transcription factor TFIIH. Role of Cdk7 is well characterized in model eukaryotes however its relevance in protozoan parasites has not been investigated. This important regulator of key processes warrants closer examination particularly in this parasite given its unique cell cycle progression and flexible mode of replication. We report functional characterization of TgCdk7 and its partners TgCyclinH and TgMat1. Recombinant Cdk7 displays kinase activity upon binding its cyclin partner and this activity is further enhanced in presence of Mat1. The activated kinase phosphorylates C-terminal domain of TgRPB1 suggesting its role in parasite transcription. Therefore, the function of Cdk7 in CTD phosphorylation and RPB1 mediated transcription was investigated using Cdk7 inhibitor. Unphosphorylated CTD binds promoter DNA while phosphorylation by Cdk7 triggers its dissociation from DNA with implications for transcription initiation. Inhibition of Cdk7 in the parasite led to strong reduction in Serine 5 phosphorylation of TgRPB1-CTD at the promoters of constitutively expressed actin1 and sag1 genes with concomitant reduction of both nascent RNA synthesis and 5′-capped transcripts. Therefore, we provide compelling evidence for crucial role of TgCdk7 kinase activity in mRNA synthesis. PMID:27759017

  1. Crystal structure of a human cyclin-dependent kinase 6 complexwith a flavonol inhibitor, Fisetin

    SciTech Connect

    Lu, Heshu; Chang, Debbie J.; Baratte, Blandine; Meijer, Laurent; Schulze-Gahmen, Ursula

    2005-01-10

    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 anti-mitotic and/or anti-neurodegenerative 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. 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 affinity and specificity.

  2. Inhibition of CDK9 as a therapeutic strategy for inflammatory arthritis

    PubMed Central

    Hellvard, Annelie; Zeitlmann, Lutz; Heiser, Ulrich; Kehlen, Astrid; Niestroj, André; Demuth, Hans-Ulrich; Koziel, Joanna; Delaleu, Nicolas; Jan Potempa; Mydel, Piotr

    2016-01-01

    Rheumatoid arthritis is characterised by synovial inflammation and proliferation of fibroblast-like synoviocytes. The induction of apoptosis has long been proposed as a target for proliferative autoimmune diseases, and has further been shown to act as a successful treatment of experimental models of arthritis, such as collagen-induced arthritis. Here we examined the effects of specific oral small-molecule inhibitors of the transcription regulating cyclin-dependent kinase 9 on the development and progression of collagen-induced arthritis. DBA/1 mice were immunised with bovine collagen type II and treated orally with specific CDK9 inhibitors. The effects of CDK9 inhibition on RNA levels and protein expression, apoptosis induction, caspase activation and lymphocyte phenotype were further analysed. Mice showed a significant delay in disease onset and a reduction in disease severity following treatment with CDK9 inhibitors. Inhibiting CDK9 activity in peripheral blood mononuclear cells resulted in the loss of Mcl-1 expression at both the protein and RNA levels, along with a subsequent increase in apoptosis. CDK9 specific inhibitors may be a potential alternative treatment not only of cancer, but also for autoimmune- and inflammatory diseases. Taken together, these results show that transient inhibition of CDK9 induces apoptosis in leukocyte subsets and modulates the immune response. PMID:27511630

  3. CDK2 Regulates HIV-1 Transcription by Phosphorylation of CDK9 on Serine 90

    PubMed Central

    2012-01-01

    Background HIV-1 transcription is activated by the viral Tat protein that recruits host positive transcription elongation factor-b (P-TEFb) containing CDK9/cyclin T1 to the HIV-1 promoter. P-TEFb in the cells exists as a lower molecular weight CDK9/cyclin T1 dimer and a high molecular weight complex of 7SK RNA, CDK9/cyclin T1, HEXIM1 dimer and several additional proteins. Our previous studies implicated CDK2 in HIV-1 transcription regulation. We also found that inhibition of CDK2 by iron chelators leads to the inhibition of CDK9 activity, suggesting a functional link between CDK2 and CDK9. Here, we investigate whether CDK2 phosphorylates CDK9 and regulates its activity. Results The siRNA-mediated knockdown of CDK2 inhibited CDK9 kinase activity and reduced CDK9 phosphorylation. Stable shRNA-mediated CDK2 knockdown inhibited HIV-1 transcription, but also increased the overall level of 7SK RNA. CDK9 contains a motif (90SPYNR94) that is consensus CDK2 phosphorylation site. CDK9 was phosphorylated on Ser90 by CDK2 in vitro. In cultured cells, CDK9 phosphorylation was reduced when Ser90 was mutated to an Ala. Phosphorylation of CDK9 on Ser90 was also detected with phospho-specific antibodies and it was reduced after the knockdown of CDK2. CDK9 expression decreased in the large complex for the CDK9-S90A mutant and was correlated with a reduced activity and an inhibition of HIV-1 transcription. In contrast, the CDK9-S90D mutant showed a slight decrease in CDK9 expression in both the large and small complexes but induced Tat-dependent HIV-1 transcription. Molecular modeling showed that Ser 90 of CDK9 is located on a flexible loop exposed to solvent, suggesting its availability for phosphorylation. Conclusion Our data indicate that CDK2 phosphorylates CDK9 on Ser 90 and thereby contributes to HIV-1 transcription. The phosphorylation of Ser90 by CDK2 represents a novel mechanism of HIV-1 regulated transcription and provides a new strategy for activation of latent HIV-1

  4. Oncogenic Functions of cdK4 and cdK6

    DTIC Science & Technology

    1998-07-01

    frequently leads to dysregulated cdk4/cdk6 activity in human tumors, as do mutations in cdk4 that prevent its association with p16INK4a ( Motokura et al...novel cyclin D partner. Mol. Cell. Biol. 14: 2077-2086. Morgan, D. 1995. Principals of CDK regulation. Nature 474: 131-134. Motokura , T., T. Bloom

  5. Role of CDK5 as a Tumor Suppressor Gene in Non-Small Cell Lung Cancer

    DTIC Science & Technology

    2014-08-01

    since 1) EGFR is mutated or amplified frequently in human lung adenocarcinoma, 2) EGFR employs the RAS signal transduction pathway, as well as other...effector pathways, and 3) EGFR mutations correlate with sensitivity to EGFR inhibitors, raising the question of whether Cdk5 ablation can modify...and Egfr . To accomplish this, we have generated a novel inducible autochthonous lung cancer mouse model, CE- Cdk5f/f, in which mutant Egfr expression

  6. Rb and FZR1/Cdh1 determine CDK4/6-cyclin D requirement in C. elegans and human cancer cells.

    PubMed

    The, Inge; Ruijtenberg, Suzan; Bouchet, Benjamin P; Cristobal, Alba; Prinsen, Martine B W; van Mourik, Tim; Koreth, John; Xu, Huihong; Heck, Albert J R; Akhmanova, Anna; Cuppen, Edwin; Boxem, Mike; Muñoz, Javier; van den Heuvel, Sander

    2015-01-06

    Cyclin-dependent kinases 4 and 6 (CDK4/6) in complex with D-type cyclins promote cell cycle entry. Most human cancers contain overactive CDK4/6-cyclin D, and CDK4/6-specific inhibitors are promising anti-cancer therapeutics. Here, we investigate the critical functions of CDK4/6-cyclin D kinases, starting from an unbiased screen in the nematode Caenorhabditis elegans. We found that simultaneous mutation of lin-35, a retinoblastoma (Rb)-related gene, and fzr-1, an orthologue to the APC/C co-activator Cdh1, completely eliminates the essential requirement of CDK4/6-cyclin D (CDK-4/CYD-1) in C. elegans. CDK-4/CYD-1 phosphorylates specific residues in the LIN-35 Rb spacer domain and FZR-1 amino terminus, resembling inactivating phosphorylations of the human proteins. In human breast cancer cells, simultaneous knockdown of Rb and FZR1 synergistically bypasses cell division arrest induced by the CDK4/6-specific inhibitor PD-0332991. Our data identify FZR1 as a candidate CDK4/6-cyclin D substrate and point to an APC/C(FZR1) activity as an important determinant in response to CDK4/6-inhibitors.

  7. Rb and FZR1/Cdh1 determine CDK4/6-cyclin D requirement in C. elegans and human cancer cells

    PubMed Central

    The, Inge; Ruijtenberg, Suzan; Bouchet, Benjamin P.; Cristobal, Alba; Prinsen, Martine B. W.; van Mourik, Tim; Koreth, John; Xu, Huihong; Heck, Albert J. R.; Akhmanova, Anna; Cuppen, Edwin; Boxem, Mike; Muñoz, Javier; van den Heuvel, Sander

    2015-01-01

    Cyclin-dependent kinases 4 and 6 (CDK4/6) in complex with D-type cyclins promote cell cycle entry. Most human cancers contain overactive CDK4/6-cyclin D, and CDK4/6-specific inhibitors are promising anti-cancer therapeutics. Here, we investigate the critical functions of CDK4/6-cyclin D kinases, starting from an unbiased screen in the nematode Caenorhabditis elegans. We found that simultaneous mutation of lin-35, a retinoblastoma (Rb)-related gene, and fzr-1, an orthologue to the APC/C co-activator Cdh1, completely eliminates the essential requirement of CDK4/6-cyclin D (CDK-4/CYD-1) in C. elegans. CDK-4/CYD-1 phosphorylates specific residues in the LIN-35 Rb spacer domain and FZR-1 amino terminus, resembling inactivating phosphorylations of the human proteins. In human breast cancer cells, simultaneous knockdown of Rb and FZR1 synergistically bypasses cell division arrest induced by the CDK4/6-specific inhibitor PD-0332991. Our data identify FZR1 as a candidate CDK4/6-cyclin D substrate and point to an APC/CFZR1 activity as an important determinant in response to CDK4/6-inhibitors. PMID:25562820

  8. Role of CDK5/cyclin complexes in ischemia-induced death and survival of renal tubular cells.

    PubMed

    Guevara, Tatiana; Sancho, Mónica; Pérez-Payá, Enrique; Orzáez, Mar

    2014-01-01

    Ischemia reperfusion processes induce damage in renal tubules and compromise the viability of kidney transplants. Understanding the molecular events responsible for tubule damage and recovery would help to develop new strategies for organ preservation. CDK5 has been traditionally considered a neuronal kinase with dual roles in cell death and survival. Here, we demonstrate that CDK5 and their regulators p35/p25 and cyclin I are also expressed in renal tubular cells. We show that treatment with CDK inhibitors promotes the formation of pro-survival CDK5/cyclin I complexes and enhances cell survival upon an ischemia reperfusion pro-apoptotic insult. These findings support the benefit of treating with CDK inhibitors for renal preservation, assisting renal tubule protection.

  9. Ubiquitination of p27 is regulated by Cdk-dependent phosphorylation and trimeric complex formation

    PubMed Central

    Montagnoli, Alessia; Fiore, Francesca; Eytan, Esther; Carrano, Andrea C.; Draetta, Giulio F.; Hershko, Avram; Pagano, Michele

    1999-01-01

    The cellular abundance of the cyclin-dependent kinase (Cdk) inhibitor p27 is regulated by the ubiquitin–proteasome system. Activation of p27 degradation is seen in proliferating cells and in many types of aggressive human carcinomas. p27 can be phosphorylated on threonine 187 by Cdks, and cyclin E/Cdk2 overexpression can stimulate the degradation of wild-type p27, but not of a threonine 187-to-alanine p27 mutant [p27(T187A)]. However, whether threonine 187 phosphorylation stimulates p27 degradation through the ubiquitin–proteasome system or an alternative pathway is still not known. Here, we demonstrate that p27 ubiquitination (as assayed in vivo and in an in vitro reconstituted system) is cell-cycle regulated and that Cdk activity is required for the in vitro ubiquitination of p27. Furthermore, ubiquitination of wild-type p27, but not of p27(T187A), can occur in G1-enriched extracts only upon addition of cyclin E/Cdk2 or cyclin A/Cdk2. Using a phosphothreonine 187 site-specific antibody for p27, we show that threonine 187 phosphorylation of p27 is also cell-cycle dependent, being present in proliferating cells but undetectable in G1 cells. Finally, we show that in addition to threonine 187 phosphorylation, efficient p27 ubiquitination requires formation of a trimeric complex with the cyclin and Cdk subunits. In fact, cyclin B/Cdk1 which can phosphorylate p27 efficiently, but cannot form a stable complex with it, is unable to stimulate p27 ubiquitination by G1 extracts. Furthermore, another p27 mutant [p27(CK−)] that can be phosphorylated by cyclin E/Cdk2 but cannot bind this kinase complex, is refractory to ubiquitination. Thus throughout the cell cycle, both phosphorylation and trimeric complex formation act as signals for the ubiquitination of a Cdk inhibitor. PMID:10323868

  10. The structure of cyclin E1/CDK2: implications for CDK2 activation and CDK2-independent roles.

    PubMed

    Honda, Reiko; Lowe, Edward D; Dubinina, Elena; Skamnaki, Vicky; Cook, Atlanta; Brown, Nick R; Johnson, Louise N

    2005-02-09

    Cyclin E, an activator of phospho-CDK2 (pCDK2), is important for cell cycle progression in metazoans and is frequently overexpressed in cancer cells. It is essential for entry to the cell cycle from G0 quiescent phase, for the assembly of prereplication complexes and for endoreduplication in megakaryotes and giant trophoblast cells. We report the crystal structure of pCDK2 in complex with a truncated cyclin E1 (residues 81-363) at 2.25 A resolution. The N-terminal cyclin box fold of cyclin E1 is similar to that of cyclin A and promotes identical changes in pCDK2 that lead to kinase activation. The C-terminal cyclin box fold shows significant differences from cyclin A. It makes additional interactions with pCDK2, especially in the region of the activation segment, and contributes to CDK2-independent binding sites of cyclin E. Kinetic analysis with model peptide substrates show a 1.6-fold increase in kcat for pCDK2/cyclin E1 (81-363) over kcat of pCDK2/cyclin E (full length) and pCDK2/cyclin A. The structural and kinetic results indicate no inherent substrate discrimination between pCDK2/cyclin E and pCDK2/cyclin A with model substrates.

  11. Artemisinin blocks prostate cancer growth and cell cycle progression by disrupting Sp1 interactions with the cyclin-dependent kinase-4 (CDK4) promoter and inhibiting CDK4 gene expression.

    PubMed

    Willoughby, Jamin A; Sundar, Shyam N; Cheung, Mark; Tin, Antony S; Modiano, Jaime; Firestone, Gary L

    2009-01-23

    Artemisinin, a naturally occurring component of Artemisia annua, or sweet wormwood, is a potent anti-malaria compound that has recently been shown to have anti-proliferative effects on a number of human cancer cell types, although little is know about the molecular mechanisms of this response. We have observed that artemisinin treatment triggers a stringent G1 cell cycle arrest of LNCaP (lymph node carcinoma of the prostate) human prostate cancer cells that is accompanied by a rapid down-regulation of CDK2 and CDK4 protein and transcript levels. Transient transfection with promoter-linked luciferase reporter plasmids revealed that artemisinin strongly inhibits CDK2 and CDK4 promoter activity. Deletion analysis of the CDK4 promoter revealed a 231-bp artemisinin-responsive region between -1737 and -1506. Site-specific mutations revealed that the Sp1 site at -1531 was necessary for artemisinin responsiveness in the context of the CDK4 promoter. DNA binding assays as well as chromatin immunoprecipitation assays demonstrated that this Sp1-binding site in the CDK4 promoter forms a specific artemisinin-responsive DNA-protein complex that contains the Sp1 transcription factor. Artemisinin reduced phosphorylation of Sp1, and when dephosphorylation of Sp1 was inhibited by treatment of cells with the phosphatase inhibitor okadaic acid, the ability of artemisinin to down-regulate Sp1 interactions with the CDK4 promoter was ablated, rendering the CDK4 promoter unresponsive to artemisinin. Finally, overexpression of Sp1 mostly reversed the artemisinin down-regulation of CDK4 promoter activity and partially reversed the cell cycle arrest. Taken together, our results demonstrate that a key event in the artemisinin anti-proliferative effects in prostate cancer cells is the transcriptional down-regulation of CDK4 expression by disruption of Sp1 interactions with the CDK4 promoter.

  12. 1α,25 dihydroxi-vitamin D₃ modulates CDK4 and CDK6 expression and localization.

    PubMed

    Irazoqui, Ana P; Heim, Nadia B; Boland, Ricardo L; Buitrago, Claudia G

    2015-03-27

    We recently reported that the vitamin D receptor (VDR) and p38 MAPK participate in pro-differentiation events triggered by 1α,25(OH)₂-vitamin D₃ [1,25D] in skeletal muscle cells. Specifically, our studies demonstrated that 1,25D promotes G0/G1 arrest of cells inducing cyclin D3 and cyclin dependent kinases inhibitors (CKIs) p21(Waf1/Cip1) and p27(Kip1) expression in a VDR and p38 MAPK dependent manner. In this work we present data indicating that cyclin-dependent kinases (CDKs) 4 and 6 also play a role in the mechanism by which 1,25D stimulates myogenesis. To investigate VDR involvement in hormone regulation of CDKs 4 and 6, we significantly reduced its expression by the use of a shRNA against mouse VDR, generating the skeletal muscle cell line C2C12-VDR. Investigation of changes in cellular cycle regulating proteins by immunoblotting showed that the VDR is involved in the 1,25D -induced CDKs 4 and 6 protein levels at 6 h of hormone treatment. CDK4 levels remains high during S phase peak and G0/G1 arrest while CDK6 expression decreases at 12 h and increases again al 24 h. The up-regulation of CDKs 4 and 6 by 1,25D (6 h) was abolished in C2C12 cells pre-treated with the ERK1/2 inhibitor, UO126. Moreover, CDKs 4 and 6 expression induced by the hormone nor was detected when α and β isoforms of p38 MAPK were inhibited by compound SB203580. Confocal images show that there is not co-localization between VDR and CDKs at 6 h of hormone treatment, however CDK4 and VDR co-localizates in nucleus after 12 h of 1,25D exposure. Of relevance, at this time 1,25D promotes CDK6 localization in a peri-nuclear ring. Our data demonstrate that the VDR, ERK1/2 and p38 MAPK are involved in the control of CDKs 4 and 6 by 1,25D in skeletal muscle cells sustaining the operation of a VDR and MAPKs -dependent mechanism in hormone modulation of myogenesis.

  13. The emerging roles and therapeutic potential of cyclin-dependent kinase 11 (CDK11) in human cancer

    PubMed Central

    Zhou, Yubing; Shen, Jacson K.; Hornicek, Francis J.; Kan, Quancheng; Duan, Zhenfeng

    2016-01-01

    Overexpression and/or hyperactivation of cyclin-dependent kinases (CDKs) are common features of most cancer types. CDKs have been shown to play important roles in tumor cell proliferation and growth by controlling cell cycle, transcription, and RNA splicing. CDK4/6 inhibitor palbociclib has been recently approved by the FDA for the treatment of breast cancer. CDK11 is a serine/threonine protein kinase in the CDK family and recent studies have shown that CDK11 also plays critical roles in cancer cell growth and proliferation. A variety of genetic and epigenetic events may cause universal overexpression of CDK11 in human cancers. Inhibition of CDK11 has been shown to lead to cancer cell death and apoptosis. Significant evidence has suggested that CDK11 may be a novel and promising therapeutic target for the treatment of cancers. This review will focus on the emerging roles of CDK11 in human cancers, and provide a proof-of-principle for continued efforts toward targeting CDK11 for effective cancer treatment. PMID:27049727

  14. Engineering an analog-sensitive CDK12 cell line using CRISPR/Cas

    PubMed Central

    Bartkowiak, Bartlomiej; Yan, Christopher; Greenleaf, Arno L.

    2015-01-01

    The RNA Polymerase II C-terminal domain (CTD) kinase CDK12 has been implicated as a tumor suppressor and regulator of DNA damage response genes. Although much has been learned about CDK12 and its activity, due to the lack of a specific inhibitor and the complications posed by long term RNAi depletion, much is still unknown about the particulars of CDK12 function. Therefore gaining a better understanding of CDK12’s roles at the molecular level will be challenging without the development of additional tools. In order to address these issues we have used the CRISPR/Cas gene engineering system to create a mammalian cell line in which the only functional copy of CDK12 is selectively inhibitable by a cell-permeable adenine analog (analog-sensitive CDK12). Inhibition of CDK12 results in a perturbation of the phosphorylation patterns on the CTD and an arrest in cellular proliferation. This cell line should serve as a powerful tool for future studies.* PMID:26189575

  15. Cdk9 T-loop Phosphorylation is Regulated by the Calcium Signaling Pathway

    PubMed Central

    Ramakrishnan, Rajesh; Rice, Andrew P.

    2011-01-01

    Eukaryotic RNA polymerase II transcriptional elongation is a tightly regulated process and is dependent upon positive transcription elongation factor-b (P-TEFb). The core P-TEFb complex is composed of Cdk9 and Cyclin T and is essential for the expression of most protein coding genes. Cdk9 kinase function is dependent upon phosphorylation of Thr186 in its T-loop. In this study, we examined kinases and signaling pathways that influence Cdk9 T-loop phosphorylation. Using an RNAi screen in HeLa cells, we found that Cdk9 T-loop phosphorylation is regulated by Calcium/Calmodulin- dependent kinase 1D (CaMK1D). Using small molecules inhibitors in HeLa cells and primary CD4+ T lymphocytes, we found that the Ca2+ signaling pathway is required for Cdk9 T-loop phosphorylation. Inhibition of Ca2+ signaling led to dephosphorylation of Thr186 on Cdk9. In reporter plasmid assays, inhibition of the Ca2+ signaling pathway repressed the PCNA promoter and HIV-1 Tat transactivation of the HIV-1 LTR, but not HTLV-1 Tax transactivation of the HTLV-1 LTR, suggesting that perturbation of the Ca2+ pathway and reduction of Cdk9 T-loop phosphorylation inhibits transcription units that have a rigorous requirement for P-TEFb function. PMID:21448926

  16. Cdk9 T-loop phosphorylation is regulated by the calcium signaling pathway.

    PubMed

    Ramakrishnan, Rajesh; Rice, Andrew P

    2012-02-01

    Eukaryotic RNA polymerase II transcriptional elongation is a tightly regulated process and is dependent upon positive transcription elongation factor-b (P-TEFb). The core P-TEFb complex is composed of Cdk9 and Cyclin T and is essential for the expression of most protein coding genes. Cdk9 kinase function is dependent upon phosphorylation of Thr186 in its T-loop. In this study, we examined kinases and signaling pathways that influence Cdk9 T-loop phosphorylation. Using an RNAi screen in HeLa cells, we found that Cdk9 T-loop phosphorylation is regulated by Ca(2+)/calmodulin-dependent kinase 1D (CaMK1D). Using small molecules inhibitors in HeLa cells and primary CD4(+) T lymphocytes, we found that the Ca(2+) signaling pathway is required for Cdk9 T-loop phosphorylation. Inhibition of Ca(2+) signaling led to dephosphorylation of Thr186 on Cdk9. In reporter plasmid assays, inhibition of the Ca(2+) signaling pathway repressed the PCNA promoter and HIV-1 Tat transactivation of the HIV-1 LTR, but not HTLV-1 Tax transactivation of the HTLV-1 LTR, suggesting that perturbation of the Ca(2+) pathway and reduction of Cdk9 T-loop phosphorylation inhibits transcription units that have a rigorous requirement for P-TEFb function.

  17. Inhibiting CDK2: A Study with First Principles and Classical Methods

    NASA Astrophysics Data System (ADS)

    Heady, Lucy

    2005-03-01

    Cyclin-dependent kinases (CDKs) are a group of proteins responsible for controlling entry to different phases of the cell cycle, and are therefore promising targets for the treatment of cancerous tumours. Using DFT we have calculated the binding energy of a number of different inhibitors to the ATP binding pocket of CDK2. Data for these calculations were taken from X- ray crystal structures. The binding energies calculated predict the correct rank order of the inhibitors considered and correlate well with the available experimental values of binding affinity with the exception of the inhibitor SU9516. This suggests that the crystal structure is not showing all of the direct interactions. Dynamical effects in the binding pocket have been investigated in a series of classical molecular dynamics simulations. During the simulation of CDK2 bound to SU9516, a major structural change occurs bringing the inhibitor into close contact with a polar lysine residue. This missing interaction accounts for the discrepancy in the binding energy. It also explains the observed greater potency of SU9516 for CDK2 over CDK4, as in CDK4 this polar lysine is not present.

  18. CDK4/6 inhibition induces epithelial cell cycle arrest and ameliorates acute kidney injury

    PubMed Central

    DiRocco, Derek P.; Bisi, John; Roberts, Patrick; Strum, Jay; Wong, Kwok-Kin; Sharpless, Norman

    2013-01-01

    Acute kidney injury (AKI) is common and urgently requires new preventative therapies. Expression of a cyclin-dependent kinase (CDK) inhibitor transgene protects against AKI, suggesting that manipulating the tubular epithelial cell cycle may be a viable therapeutic strategy. Broad spectrum small molecule CDK inhibitors are protective in some kidney injury models, but these have toxicities and epithelial proliferation is eventually required for renal repair. Here, we tested a well-tolerated, novel and specific small molecule inhibitor of CDK4 and CDK6, PD 0332991, to investigate the effects of transient cell cycle inhibition on epithelial survival in vitro and kidney injury in vivo. We report that CDK4/6 inhibition induced G0/G1 cycle arrest in cultured human renal proximal tubule cells (hRPTC) at baseline and after injury. Induction of transient G0/G1 cycle arrest through CDK4/6 inhibition protected hRPTC from DNA damage and caspase 3/7 activation following exposure to the nephrotoxins cisplatin, etoposide, and antimycin A. In vivo, mice treated with PD 0332991 before ischemia-reperfusion injury (IRI) exhibited dramatically reduced epithelial progression through S phase 24 h after IRI. Despite reduced epithelial proliferation, PD 0332991 ameliorated kidney injury as reflected by improved serum creatinine and blood urea nitrogen levels 24 h after injury. Inflammatory markers and macrophage infiltration were significantly decreased in injured kidneys 3 days following IRI. These results indicate that induction of proximal tubule cell cycle arrest with specific CDK4/6 inhibitors, or “pharmacological quiescence,” represents a novel strategy to prevent AKI. PMID:24338822

  19. p16/CDKN2 and CDK4 gene mutations in sporadic melanoma development and progression.

    PubMed

    Piccinin, S; Doglioni, C; Maestro, R; Vukosavljevic, T; Gasparotto, D; D'Orazi, C; Boiocchi, M

    1997-02-20

    The p16/CDKN2(MTS1) gene encoding for the p16 inhibitor of cyclin D/CDK4 complexes is frequently mutated and deleted in a large fraction of melanoma cell lines, and p16 germline mutations have also been observed in familial melanomas. Moreover, a CDK4 gene mutation, responsible for a functional resistance of CDK4 kinase to p16 inhibitory activity, has been described to occur in some cases of familial melanoma. These data strongly support the idea that deregulation of the CDK4/cyclin D pathway, via CDKN2 or CDK4 mutations, is of biological significance in the development of melanoma. To shed light on the role of these alterations in the development and progression of sporadic melanoma, 12 primary melanomas and 9 corresponding metastases were analyzed for CDKN2 and CDK4 gene mutations. Of the 12 primary melanomas analyzed, 4 showed the presence of mutational inactivation of the p 16 protein and 2 carried silent mutations. No metastases showed the presence of CDKN2 mutations, indicating that mutations of this cyclin-dependent kinase inhibitor is not common in the progression of sporadic melanoma. On the other hand, the absence, in the metastases, of the CDKN2 mutation detected in the corresponding primary tumors suggests that 9p21 homozygous deletion may play a major role in the metastatic spreading of this type of tumor. None of the cases analyzed showed the presence of an Arg24Cys mutation, which functionally protects CDK4 from p16 inhibition. This indicates that CDK4 mutation plays a minor role in the development and progression of sporadic melanoma.

  20. Molecular Dynamics Simulations and Classical Multidimensional Scaling Unveil New Metastable States in the Conformational Landscape of CDK2

    PubMed Central

    Pisani, Pasquale; Rastelli, Giulio

    2016-01-01

    Protein kinases are key regulatory nodes in cellular networks and their function has been shown to be intimately coupled with their structural flexibility. However, understanding the key structural mechanisms of large conformational transitions remains a difficult task. CDK2 is a crucial regulator of cell cycle. Its activity is finely tuned by Cyclin E/A and the catalytic segment phosphorylation, whereas its deregulation occurs in many types of cancer. ATP competitive inhibitors have failed to be approved for clinical use due to toxicity issues raised by a lack of selectivity. However, in the last few years type III allosteric inhibitors have emerged as an alternative strategy to selectively modulate CDK2 activity. In this study we have investigated the conformational variability of CDK2. A low dimensional conformational landscape of CDK2 was modeled using classical multidimensional scaling on a set of 255 crystal structures. Microsecond-scale plain and accelerated MD simulations were used to populate this landscape by using an out-of-sample extension of multidimensional scaling. CDK2 was simulated in the apo-form and in complex with the allosteric inhibitor 8-anilino-1-napthalenesulfonic acid (ANS). The apo-CDK2 landscape analysis showed a conformational equilibrium between an Src-like inactive conformation and an active-like form. These two states are separated by different metastable states that share hybrid structural features with both forms of the kinase. In contrast, the CDK2/ANS complex landscape is compatible with a conformational selection picture where the binding of ANS in proximity of the αC helix causes a population shift toward the inactive conformation. Interestingly, the new metastable states could enlarge the pool of candidate structures for the development of selective allosteric CDK2 inhibitors. The method here presented should not be limited to the CDK2 case but could be used to systematically unmask similar mechanisms throughout the human

  1. Phosphorylation of the centrosomal protein, Cep169, by Cdk1 promotes its dissociation from centrosomes in mitosis.

    PubMed

    Mori, Yusuke; Inoue, Yoko; Taniyama, Yuki; Tanaka, Sayori; Terada, Yasuhiko

    2015-12-25

    Cep169 is a centrosomal protein conserved among vertebrates. In our previous reports, we showed that mammalian Cep169 interacts and collaborates with CDK5RAP2 to regulate microtubule (MT) dynamics and stabilization. Although Cep169 is required for MT regulation, its precise cellular function remains largely elusive. Here we show that Cep169 associates with centrosomes during interphase, but dissociates from these structures from the onset of mitosis, although CDK5RAP2 (Cep215) is continuously located at the centrosomes throughout cell cycle. Interestingly, treatment with purvalanol A, a Cdk1 inhibitor, nearly completely blocked the dissociation of Cep169 from centrosomes during mitosis. In addition, mass spectrometry analyses identified 7 phosphorylated residues of Cep169 corresponding to consensus phosphorylation sequence for Cdk1. These data suggest that the dissociation of Cep169 from centrosomes is controlled by Cdk1/Cyclin B during mitosis, and that Cep169 might regulate MT dynamics of mitotic spindle.

  2. 1α,25 dihydroxi-vitamin D{sub 3} modulates CDK4 and CDK6 expression and localization

    SciTech Connect

    Irazoqui, Ana P.; Heim, Nadia B.; Boland, Ricardo L.; Buitrago, Claudia G.

    2015-03-27

    We recently reported that the vitamin D receptor (VDR) and p38 MAPK participate in pro-differentiation events triggered by 1α,25(OH){sub 2}-vitamin D{sub 3} [1,25D] in skeletal muscle cells. Specifically, our studies demonstrated that 1,25D promotes G0/G1 arrest of cells inducing cyclin D3 and cyclin dependent kinases inhibitors (CKIs) p21{sup Waf1/Cip1} and p27{sup Kip1} expression in a VDR and p38 MAPK dependent manner. In this work we present data indicating that cyclin-dependent kinases (CDKs) 4 and 6 also play a role in the mechanism by which 1,25D stimulates myogenesis. To investigate VDR involvement in hormone regulation of CDKs 4 and 6, we significantly reduced its expression by the use of a shRNA against mouse VDR, generating the skeletal muscle cell line C2C12-VDR. Investigation of changes in cellular cycle regulating proteins by immunoblotting showed that the VDR is involved in the 1,25D –induced CDKs 4 and 6 protein levels at 6 h of hormone treatment. CDK4 levels remains high during S phase peak and G0/G1 arrest while CDK6 expression decreases at 12 h and increases again al 24 h. The up-regulation of CDKs 4 and 6 by 1,25D (6 h) was abolished in C2C12 cells pre-treated with the ERK1/2 inhibitor, UO126. Moreover, CDKs 4 and 6 expression induced by the hormone nor was detected when α and β isoforms of p38 MAPK were inhibited by compound SB203580. Confocal images show that there is not co-localization between VDR and CDKs at 6 h of hormone treatment, however CDK4 and VDR co-localizates in nucleus after 12 h of 1,25D exposure. Of relevance, at this time 1,25D promotes CDK6 localization in a peri-nuclear ring. Our data demonstrate that the VDR, ERK1/2 and p38 MAPK are involved in the control of CDKs 4 and 6 by 1,25D in skeletal muscle cells sustaining the operation of a VDR and MAPKs –dependent mechanism in hormone modulation of myogenesis. - Highlights: • 1,25D modulates CDKs 4 and 6 expression in skeletal muscle cells. • CDK4 co

  3. CDK5 and Its Activator P35 in Normal Pituitary and in Pituitary Adenomas: Relationship to VEGF Expression

    PubMed Central

    Xie, Weiyan; Wang, Hongyun; He, Yue; Li, Dan; Gong, Lei; Zhang, Yazhuo

    2014-01-01

    Pituitary tumors are monoclonal adenomas that account for about 10-15% of intracranial tumors. Cyclin-dependent kinase 5 (CDK5) regulates the activities of various proteins and cellular processes in the nervous system, but its potential roles in pituitary adenomas are poorly understood. The kinase activity of CDK5 requires association with an activating protein, p35 (also known as CDK5 activator 1, p35). Here, we show that functional CDK5, associated with p35, is present in normal human pituitary and in pituitary tumors. Furthermore, p35 mRNA and protein levels were higher in pituitary adenomas than in the normal glands, suggesting that CDK5 activity might be upregulated in pituitary tumors. Inhibition of CDK5 activity in rat pituitary cells, reduced the expression of vascular endothelial growth factor (VEGF), a protein that regulates vasculogenesis and angiogenesis. Our results suggest that increased CDK5-mediated VEGF expression might play a crucial role in the development of pituitary adenomas, and that roscovitine and other CDK5 inhibitors could be useful as anticancer agents. PMID:24550687

  4. High glucose increases Cdk5 activity in podocytes via transforming growth factor-β1 signaling pathway

    SciTech Connect

    Zhang, Yue; Li, Hongbo; Hao, Jun; Zhou, Yi; Liu, Wei

    2014-08-15

    Podocytes are highly specialized and terminally differentiated glomerular cells that play a vital role in the development and progression of diabetic nephropathy (DN). Cyclin-dependent kinase 5 (Cdk5), who is an atypical but essential member of the Cdk family of proline-directed serine/threonine kinases, has been shown as a key regulator of podocyte differentiation, proliferation and morphology. Our previous studies demonstrated that the expression of Cdk5 was significantly increased in podocytes of diabetic rats, and was closely related with podocyte injury of DN. However, the mechanisms of how expression and activity of Cdk5 are regulated under the high glucose environment have not yet been fully elucidated. In this study, we showed that high glucose up-regulated the expression of Cdk5 and its co-activator p35 with a concomitant increase in Cdk5 kinase activity in conditionally immortalized mouse podocytes in vitro. When exposed to 30 mM glucose, transforming growth factor-β1 (TGF-β1) was activated. Most importantly, we found that SB431542, the Tgfbr1 inhibitor, significantly decreased the expression of Cdk5 and p35 and Cdk5 kinase activity in high glucose-treated podocytes. Moreover, high glucose increased the expression of early growth response-1 (Egr-1) via TGF-β1-ERK1/2 pathway in podocytes and inhibition of Egr-1 by siRNA decreased p35 expression and Cdk5 kinase activity. Furthermore, inhibition of Cdk5 kinase activity effectively alleviated podocyte apoptosis induced by high glucose or TGF-β1. Thus, the TGF-β1-ERK1/2-Egr-1 signaling pathway may regulate the p35 expression and Cdk5 kinase activity in high glucose-treated podocytes, which contributes to podocyte injury of DN. - Highlights: • HG up-regulated the expression of Cdk5 and p35, and Cdk5 activity in podocytes. • HG activated TGF-β1 pathway and SB431542 inhibited Cdk5 expression and activity. • HG increased the expression of Egr-1 via TGF-β1-ERK1/2 pathway. • Inhibition of Egr-1

  5. Germ line transmission of the Cdk4(R24C) mutation facilitates tumorigenesis and escape from cellular senescence.

    PubMed

    Rane, Sushil G; Cosenza, Stephen C; Mettus, Richard V; Reddy, E Premkumar

    2002-01-01

    Mutations in CDK4 and its key kinase inhibitor p16(INK4a) have been implicated in the genesis and progression of familial human melanoma. The importance of the CDK4 locus in human cancer first became evident following the identification of a germ line CDK4-Arg24Cys (R24C) mutation, which abolishes the ability of CDK4 to bind to p16(INK4a). To determine the role of the Cdk4(R24C) germ line mutation in the genesis of other cancer types, we introduced the R24C mutation in the Cdk4 locus of mice by using Cre-loxP-mediated "knock-in" technology. Cdk4(R24C/R24C) mouse embryo fibroblasts (MEFs) displayed increased Cdk4 kinase activity resulting in hyperphosphorylation of all three members of the Rb family, pRb, p107, and p130. MEFs derived from Cdk4(R24C/R24C) mice displayed decreased doubling times, escape from replicative senescence, and escape sensitivity to contact-induced growth arrest. These MEFs also exhibited a high degree of susceptibility to oncogene-induced transformation, suggesting that the Cdk4(R24C) mutation can serve as a primary event in the progression towards a fully transformed phenotype. In agreement with the in vitro data, homozygous Cdk4(R24C/R24C) mice developed tumors of various etiology within 8 to 10 months of their life span. The majority of these tumors were found in the pancreas, pituitary, brain, mammary tissue, and skin. In addition, Cdk4(R24C/R24C) mice showed extraordinary susceptibility to carcinogens and developed papillomas within the first 8 to 10 weeks following cutaneous application of the carcinogens 9,10-di-methyl-1,2-benz[a]anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA). This report formally establishes that the activation of Cdk4 is sufficient to promote cancer in many tissues. The observation that a wide variety of tumors develop in mice harboring the Cdk4(R24C) mutation offers a genetic proof that Cdk4 activation may constitute a central event in the genesis of many types of cancers in addition to melanoma.

  6. Phosphorylation of CDK2 on threonine 160 influences silencing of sex chromosome during male meiosis.

    PubMed

    Wang, Lu; Liu, Wenjing; Zhao, Weidong; Song, Gendi; Wang, Guishuan; Wang, Xiaorong; Sun, Fei

    2014-06-01

    In mammalian meiosis, the X and Y chromosomes are largely unsynapsed and transcriptionally silenced during the pachytene stage of meiotic prophase (meiotic sex chromosome inactivation), forming a specialized nuclear territory called sex or XY body. An increasing number of proteins and noncoding RNAs were found to localize to the sex body and take part in influencing expression of sex chromosome genes. Cyclin-dependent kinase 2 (Cdk2 (-/-)) spermatocytes show incomplete sex chromosome pairing. Here, we further showed that phosphorylation of CDK2 isoform 1 (p-CDK2(39) [39 kDa]) on threonine 160 localizes to the sites of asynapsis and the sex body, interacting with phosphorylated gamma-H2AX. Meanwhile, p-CDK2(39) is frequently mislocalized throughout the sex body, and meiotic sex chromosome inactivation is disrupted in PWK×C57BL/6J hybrid mice. Furthermore, pachytene spermatocytes treated with mevastatin (an inhibitor of p-CDK2) showed overexpression of sex chromosome-linked genes. Our results highlight an important role for p-CDK2(39) in influencing silencing of the sex chromosomes during male meiosis by interacting with gamma-H2AX.

  7. Cdk1, but not Cdk2, is the sole Cdk that is essential and sufficient to drive resumption of meiosis in mouse oocytes.

    PubMed

    Adhikari, Deepak; Zheng, Wenjing; Shen, Yan; Gorre, Nagaraju; Ning, Yao; Halet, Guillaume; Kaldis, Philipp; Liu, Kui

    2012-06-01

    Mammalian oocytes are arrested at the prophase of meiosis I during fetal or postnatal development, and the meiosis is resumed by the preovulatory surge of luteinizing hormone. The in vivo functional roles of cyclin-dependent kinases (Cdks) during the resumption of meiosis in mammalian oocytes are largely unknown. Previous studies have shown that deletions of Cdk3, Cdk4 or Cdk6 in mice result in viable animals with normal oocyte maturation, indicating that these Cdks are not essential for the meiotic maturation of oocytes. In addition, conventional knockout of Cdk1 and Cdk2 leads to embryonic lethality and postnatal follicular depletion, respectively, making it impossible to study the functions of Cdk1 and Cdk2 in oocyte meiosis. In this study, we generated conditional knockout mice with oocyte-specific deletions of Cdk1 and Cdk2. We showed that the lack of Cdk1, but not of Cdk2, leads to female infertility due to a failure of the resumption of meiosis in the oocyte. Re-introduction of Cdk1 mRNA into Cdk1-null oocytes largely resumed meiosis. Thus, Cdk1 is the sole Cdk that is essential and sufficient to drive resumption of meiosis in mouse oocytes. We also found that Cdk1 maintains the phosphorylation status of protein phosphatase 1 and lamin A/C in oocytes in order for meiosis resumption to occur.

  8. Discovery of Potent, Selective, and Orally Bioavailable Small-Molecule Modulators of the Mediator Complex-Associated Kinases CDK8 and CDK19

    PubMed Central

    2016-01-01

    The Mediator complex-associated cyclin-dependent kinase CDK8 has been implicated in human disease, particularly in colorectal cancer where it has been reported as a putative oncogene. Here we report the discovery of 109 (CCT251921), a potent, selective, and orally bioavailable inhibitor of CDK8 with equipotent affinity for CDK19. We describe a structure-based design approach leading to the discovery of a 3,4,5-trisubstituted-2-aminopyridine series and present the application of physicochemical property analyses to successfully reduce in vivo metabolic clearance, minimize transporter-mediated biliary elimination while maintaining acceptable aqueous solubility. Compound 109 affords the optimal compromise of in vitro biochemical, pharmacokinetic, and physicochemical properties and is suitable for progression to animal models of cancer. PMID:26796641

  9. The regulation and functions of cdk7.

    PubMed

    Shuttleworth, J

    1995-01-01

    cdk7 started its life rather anonymously as a kinase called MO15, identified during a search for cDNA's which encode protein kinases related to cdc2. For several years its function remained obscure, but during the last 18 months MO15 has revealed itself as the catalytic subunit of cdk activating kinase, associating with at least two other subunits including a new cyclin, cyclin H. MO15(cdk7) has therefore been established paradoxically as both a new member and a regulator of the cyclin dependent kinase family. New evidence now suggests that cdk7 is also involved in the processes of transcription initiation and DNA repair, associating with the general transcription factor TFIIH. The engima of cdk7 is likely to remain for a while yet, and perhaps even more surprises are in store.

  10. Cyclin Dependent Kinase 9 Inhibitors for Cancer Therapy.

    PubMed

    Sonawane, Yogesh A; Taylor, Margaret A; Napoleon, John Victor; Rana, Sandeep; Contreras, Jacob I; Natarajan, Amarnath

    2016-10-13

    Cyclin dependent kinase (CDK) inhibitors have been the topic of intense research for nearly 2 decades due to their widely varied and critical functions within the cell. Recently CDK9 has emerged as a druggable target for the development of cancer therapeutics. CDK9 plays a crucial role in transcription regulation; specifically, CDK9 mediated transcriptional regulation of short-lived antiapoptotic proteins is critical for the survival of transformed cells. Focused chemical libraries based on a plethora of scaffolds have resulted in mixed success with regard to the development of selective CDK9 inhibitors. Here we review the regulation of CDK9, its cellular functions, and common core structures used to target CDK9, along with their selectivity profile and efficacy in vitro and in vivo.

  11. CDK5 downregulation enhances synaptic plasticity.

    PubMed

    Posada-Duque, Rafael Andrés; Ramirez, Omar; Härtel, Steffen; Inestrosa, Nibaldo C; Bodaleo, Felipe; González-Billault, Christian; Kirkwood, Alfredo; Cardona-Gómez, Gloria Patricia

    2017-01-01

    CDK5 is a serine/threonine kinase that is involved in the normal function of the adult brain and plays a role in neurotransmission and synaptic plasticity. However, its over-regulation has been associated with Tau hyperphosphorylation and cognitive deficits. Our previous studies have demonstrated that CDK5 targeting using shRNA-miR provides neuroprotection and prevents cognitive deficits. Dendritic spine morphogenesis and forms of long-term synaptic plasticity-such as long-term potentiation (LTP)-have been proposed as essential processes of neuroplasticity. However, whether CDK5 participates in these processes remains controversial and depends on the experimental model. Using wild-type mice that received injections of CDK5 shRNA-miR in CA1 showed an increased LTP and recovered the PPF in deficient LTP of APPswe/PS1Δ9 transgenic mice. On mature hippocampal neurons CDK5, shRNA-miR for 12 days induced increased dendritic protrusion morphogenesis, which was dependent on Rac activity. In addition, silencing of CDK5 increased BDNF expression, temporarily increased phosphorylation of CaMKII, ERK, and CREB; and facilitated calcium signaling in neurites. Together, our data suggest that CDK5 downregulation induces synaptic plasticity in mature neurons involving Ca(2+) signaling and BDNF/CREB activation.

  12. Cdk5 promotes DNA replication stress checkpoint activation through RPA-32 phosphorylation, and impacts on metastasis free survival in breast cancer patients

    PubMed Central

    Chiker, Sara; Pennaneach, Vincent; Loew, Damarys; Dingli, Florent; Biard, Denis; Cordelières, Fabrice P; Gemble, Simon; Vacher, Sophie; Bieche, Ivan; Hall, Janet; Fernet, Marie

    2015-01-01

    Cyclin dependent kinase 5 (Cdk5) is a determinant of PARP inhibitor and ionizing radiation (IR) sensitivity. Here we show that Cdk5-depleted (Cdk5-shRNA) HeLa cells show higher sensitivity to S-phase irradiation, chronic hydroxyurea exposure, and 5-fluorouracil and 6-thioguanine treatment, with hydroxyurea and IR sensitivity also seen in Cdk5-depleted U2OS cells. As Cdk5 is not directly implicated in DNA strand break repair we investigated in detail its proposed role in the intra-S checkpoint activation. While Cdk5-shRNA HeLa cells showed altered basal S-phase dynamics with slower replication velocity and fewer active origins per DNA megabase, checkpoint activation was impaired after a hydroxyurea block. Cdk5 depletion was associated with reduced priming phosphorylations of RPA32 serines 29 and 33 and SMC1-Serine 966 phosphorylation, lower levels of RPA serine 4 and 8 phosphorylation and DNA damage measured using the alkaline Comet assay, gamma-H2AX signal intensity, RPA and Rad51 foci, and sister chromatid exchanges resulting in impaired intra-S checkpoint activation and subsequently higher numbers of chromatin bridges. In vitro kinase assays coupled with mass spectrometry demonstrated that Cdk5 can carry out the RPA32 priming phosphorylations on serines 23, 29, and 33 necessary for this checkpoint activation. In addition we found an association between lower Cdk5 levels and longer metastasis free survival in breast cancer patients and survival in Cdk5-depleted breast tumor cells after treatment with IR and a PARP inhibitor. Taken together, these results show that Cdk5 is necessary for basal replication and replication stress checkpoint activation and highlight clinical opportunities to enhance tumor cell killing. PMID:26237679

  13. CDC6 controls dynamics of the first embryonic M-phase entry and progression via CDK1 inhibition.

    PubMed

    El Dika, Mohammed; Laskowska-Kaszub, Katarzyna; Koryto, Magdalena; Dudka, Damian; Prigent, Claude; Tassan, Jean-Pierre; Kloc, Malgorzata; Polanski, Zbigniew; Borsuk, Ewa; Kubiak, Jacek Z

    2014-12-01

    CDC6 is essential for S-phase to initiate DNA replication. It also regulates M-phase exit by inhibiting the activity of the major M-phase protein kinase CDK1. Here we show that addition of recombinant CDC6 to Xenopus embryo cycling extract delays the M-phase entry and inhibits CDK1 during the whole M-phase. Down regulation of endogenous CDC6 accelerates the M-phase entry, abolishes the initial slow and progressive phase of histone H1 kinase activation and increases the level of CDK1 activity during the M-phase. All these effects are fully rescued by the addition of recombinant CDC6 to the extracts. Diminution of CDC6 level in mouse zygotes by two different methods results in accelerated entry into the first cell division showing physiological relevance of CDC6 in intact cells. Thus, CDC6 behaves as CDK1 inhibitor regulating not only the M-phase exit, but also the M-phase entry and progression via limiting the level of CDK1 activity. We propose a novel mechanism of M-phase entry controlled by CDC6 and counterbalancing cyclin B-mediated CDK1 activation. Thus, CDK1 activation proceeds with concomitant inhibition by CDC6, which tunes the timing of the M-phase entry during the embryonic cell cycle.

  14. Cdk2 plays a critical role in hepatocyte cell cycle progression and survival in the setting of cyclin D1 expression in vivo.

    PubMed

    Hanse, Eric A; Nelsen, Christopher J; Goggin, Melissa M; Anttila, Chelsea K; Mullany, Lisa K; Berthet, Cyril; Kaldis, Philipp; Crary, Gretchen S; Kuriyama, Ryoko; Albrecht, Jeffrey H

    2009-09-01

    Cdk2 was once believed to play an essential role in cell cycle progression, but cdk2(-/-) mice have minimal phenotypic abnormalities. In this study, we examined the role of cdk2 in hepatocyte proliferation, centrosome duplication and survival. Cdk2(-/-) hepatocytes underwent mitosis and had normal centrosome content after mitogen stimulation. Unlike wild-type cells, cdk2(-/-) liver cells failed to undergo centrosome overduplication in response to ectopic cyclin D1 expression. After mitogen stimulation in culture or partial hepatectomy in vivo, cdk2(-/-) hepatocytes demonstrated diminished proliferation. Cyclin D1 is a key mediator of cell cycle progression in hepatocytes, and transient expression of this protein is sufficient to promote robust proliferation of these cells in vivo. In cdk2(-/-) mice and animals treated with the cdk2 inhibitor seliciclib, cyclin D1 failed to induce hepatocyte cell cycle progression. Surprisingly, cdk2 ablation or inhibition led to massive hepatocyte and animal death following cyclin D1 transfection. In a transgenic model of chronic hepatic cyclin D1 expression, seliciclib induced hepatocyte injury and animal death, suggesting that cdk2 is required for survival of cyclin D1-expressing cells even in the absence of substantial proliferation. In conclusion, our studies demonstrate that cdk2 plays a role in liver regeneration. Furthermore, it is essential for centrosome overduplication, proliferation and survival of hepatocytes that aberrantly express cyclin D1 in vivo. These studies suggest that cdk2 may warrant further investigation as a target for therapy of liver tumors with constitutive cyclin D1 expression.

  15. The lethal response to Cdk1 inhibition depends on sister chromatid alignment errors generated by KIF4 and isoform 1 of PRC1

    PubMed Central

    Voets, Erik; Marsman, Judith; Demmers, Jeroen; Beijersbergen, Roderick; Wolthuis, Rob

    2015-01-01

    Cyclin-dependent kinase 1 (Cdk1) is absolutely essential for cell division. Complete ablation of Cdk1 precludes the entry of G2 phase cells into mitosis, and is early embryonic lethal in mice. Dampening Cdk1 activation, by reducing gene expression or upon treatment with cell-permeable Cdk1 inhibitors, is also detrimental for proliferating cells, but has been associated with defects in mitotic progression, and the formation of aneuploid daughter cells. Here, we used a large-scale RNAi screen to identify the human genes that critically determine the cellular toxicity of Cdk1 inhibition. We show that Cdk1 inhibition leads to fatal sister chromatid alignment errors and mitotic arrest in the spindle checkpoint. These problems start early in mitosis and are alleviated by depletion of isoform 1 of PRC1 (PRC1-1), by gene ablation of its binding partner KIF4, or by abrogation of KIF4 motor activity. Our results show that, normally, Cdk1 activity must rise above the level required for mitotic entry. This prevents KIF4-dependent PRC1-1 translocation to astral microtubule tips and safeguards proper chromosome congression. We conclude that cell death in response to Cdk1 inhibitors directly relates to chromosome alignment defects generated by insufficient repression of PRC1-1 and KIF4 during prometaphase. PMID:26423135

  16. A novel approach to the discovery of small molecule ligands of CDK2

    PubMed Central

    Martin, Mathew P.; Alam, Riazul; Betzi, Stephane; Ingles, Donna J.; Zhu, Jin-Yi

    2012-01-01

    In an attempt to identify novel small molecule ligands of CDK2 with potential as allosteric inhibitors, we devised a robust and cost-effective fluorescence-based high-throughput screening assay. The assay is based on the specific interaction of CDK2 with the extrinsic fluorophore 8-anilino-1-naphthalene sulfonate (ANS), which binds to a large allosteric pocket adjacent to the ATP site. Hit compounds which displace ANS directly or indirectly from CDK2 are readily classified as ATP site binders or allosteric ligands through the use of staurosporine, which blocks the ATP site without displacing ANS. Pilot screening of 1,453 compounds led to the discovery of 12 compounds with displacement activities (EC50 values) ranging from 6 to 44 μM, all of which were classified as ATP site-directed ligands. Four new Type I inhibitor scaffolds were confirmed by X-ray crystallography. While this small compound library contained only ATP-site directed ligands, the application of this assay to large compound libraries has the potential to reveal previously unrecognized chemical scaffolds suitable for structure-based design of CDK2 inhibitors with new mechanisms of action. PMID:22893598

  17. Relative Resistance of Cdk5-phosphorylated CRMP2 to Dephosphorylation*S⃞

    PubMed Central

    Cole, Adam R.; Soutar, Marc P. M.; Rembutsu, Makoto; van Aalten, Lidy; Hastie, C. James; Mclauchlan, Hilary; Peggie, Mark; Balastik, Martin; Lu, Kun Ping; Sutherland, Calum

    2008-01-01

    Collapsin response mediator protein 2 (CRMP2) binds to microtubules and regulates axon outgrowth in neurons. This action is regulated by sequential phosphorylation by the kinases cyclin-dependent kinase 5 (Cdk5) and glycogen synthase kinase 3 (GSK3) at sites that are hyperphosphorylated in Alzheimer disease. The increased phosphorylation in Alzheimer disease could be due to increases in Cdk5 and/or GSK3 activity or, alternatively, through decreased activity of a CRMP phosphatase. Here we establish that dephosphorylation of CRMP2 at the residues targeted by GSK3 (Ser-518/Thr-514/Thr-509) is carried out by a protein phosphatase 1 family member in vitro, in neuroblastoma cells, and primary cortical neurons. Inhibition of GSK3 activity using insulin-like growth factor-1 or the highly selective inhibitor CT99021 causes rapid dephosphorylation of CRMP2 at these sites. In contrast, pharmacological inhibition of Cdk5 using purvalanol results in only a gradual and incomplete dephosphorylation of CRMP2 at the site targeted by Cdk5 (Ser-522), suggesting a distinct phosphatase targets this residue. A direct comparison of dephosphorylation at the Cdk5 versus GSK3 sites in vitro shows that the Cdk5 site is comparatively resistant to phosphatase treatment. The presence of the peptidyl-prolyl isomerase enzyme, Pin1, does not affect dephosphorylation of Ser-522 in vitro, in cells, or in Pin1 transgenic mice. Instead, the relatively high resistance of this site to phosphatase treatment is at least in part due to the presence of basic residues located nearby. Similar sequences in Tau are also highly resistant to phosphatase treatment. We propose that relative resistance to phosphatases might be a common feature of Cdk5 substrates and could contribute to the hyperphosphorylation of CRMP2 and Tau observed in Alzheimer disease. PMID:18460467

  18. Immunohistochemical detection of CDK4 and p16INK4 proteins in cutaneous malignant melanoma.

    PubMed

    Wang, Y L; Uhara, H; Yamazaki, Y; Nikaido, T; Saida, T

    1996-02-01

    p16INK4 gene, which encodes a specific inhibitor of cyclin-dependent kinase 4 (CDK4), has been recently reported as an important tumour suppressor gene. It is mapped to chromosome 9p21, which is frequently deleted or mutated in many tumour cell lines including malignant melanoma. Since the CDK4/cyclin D complex propels a cell to go through the G1 check point of the cell cycle, a critical phase of cell division, alteration of the p16INK4 gene could lead a cell to uncontrolled proliferation and malignant transformation. To clarify any role for p16INK4 and CDK4 proteins in the development of human malignant melanoma, we have examined, immunohistochemically, the expression of these two proteins in melanocytic neoplasms including 19 primary lesions of non-familial melanoma. Intense nuclear and/or cytoplasmic expression of the CDK4 protein was observed in 11 of 19 cases (58%) of melanoma. In contrast, virtually no nuclear or cytoplasmic staining for CDK4 protein was detected in 28 benign melanocytic naevi, including six Spitz naevi. Expression of p16INK4 protein was observed in three of 19 melanomas (16%) and in 17 of 28 benign naevi (61%). Inverse expression of CDK4 and p16INK4, at individual cell level, was detected in one case of melanoma. The present study suggests that CDK4 overexpression is characteristic for malignant melanoma, and probably reflects its autonomous accelerated cell proliferation. The expression rate of p16INK4 protein in malignant melanoma was lower than that in benign naevi, although the significance of p16INK4 deletion in melanoma development has not been definitely confirmed.

  19. miR-455 inhibits breast cancer cell proliferation through targeting CDK14.

    PubMed

    Wang, Bing; Zou, Aimei; Ma, Liqiang; Chen, Xiong; Wang, Lie; Zeng, Ximing; Tan, Ting

    2017-03-11

    Breast cancer is the most frequently occurring cancer in women worldwide, microRNAs (miRNAs) play critical role in the initiation and progression of breast cancer. Here, we studied the effect of miR-455 on cell proliferation of breast cancer, and found that miR-455 was downregulated in breast cancer tissues and cells. Its overexpression inhibited cell proliferation, whereas its knockdown promoted cell proliferation of breast cancer. We found a Cdc2-related protein kinase CDK14 was the target of miR-455, when the 3(')UTR of CDK14 was cloned into luciferase reporter vector and transfected into cells, miR-455 mimic could inhibit the luciferase activity in a dose-dependent manner, miR-455 inhibitor increased the luciferase activity, but the mutant miR-455 mimic couldn't change the luciferase activity, suggesting miR-455 directly bound to the 3(')UTR of CDK14. Meanwhile, we also found miR-455 inhibited Cyclin D1 expression and promoted p21 expression, confirming miR-455 inhibited cell proliferation. Double knockdown of miR-455 and CDK14 inhibited the proliferation of breast cancer cell, confirming miR-455 inhibiting cell proliferation by targeting CDK14. Moreover, miR-455 levels were negatively correlated with CDK14 levels in breast cancer tissues. Our finding revealed miR-455 inhibits breast cancer cell proliferation through targeting CDK14, it might be a target for breast cancer therapy.

  20. The CDK-APC/C Oscillator Predominantly Entrains Periodic Cell-Cycle Transcription

    PubMed Central

    Rahi, Sahand Jamal; Pecani, Kresti; Ondracka, Andrej; Oikonomou, Catherine; Cross, Frederick R.

    2016-01-01

    Throughout cell cycle progression, the expression of multiple transcripts oscillate, and whether these are under the centralized control of the CDK-APC/C proteins or can be driven by a de-centralized transcription factor (TF) cascade is a fundamental question for understanding cell cycle regulation. In budding yeast, we find that the transcription of nearly all genes, as assessed by RNA-seq or fluorescence microscopy in single cells, is dictated by CDK-APC/C. Three exceptional genes are transcribed in a pulsatile pattern in a variety of CDK-APC/C arrests. Pursuing one of these transcripts, the SIC1 inhibitor of B-type cyclins, we use a combination of mathematical modeling and experimentation to provide evidence that, counter-intuitively, Sic1 provides a failsafe mechanism promoting nuclear division when levels of mitotic cyclins are low. PMID:27058667

  1. Chloroquine alleviates etoposide-induced centrosome amplification by inhibiting CDK2 in adrenocortical tumor cells

    PubMed Central

    Chen, T-Y; Syu, J-S; Lin, T-C; Cheng, H-l; Lu, F-l; Wang, C-Y

    2015-01-01

    The antitumor drug etoposide (ETO) is widely used in treating several cancers, including adrenocortical tumor (ACT). However, when used at sublethal doses, tumor cells still survive and are more susceptible to the recurring tumor due to centrosome amplification. Here, we checked the effect of sublethal dose of ETO in ACT cells. Sublethal dose of ETO treatment did not induce cell death but arrested the ACT cells in G2/M phase. This resulted in centrosome amplification and aberrant mitotic spindle formation leading to genomic instability and cellular senescence. Under such conditions, Chk2, cyclin A/CDK2 and ERK1/2 were aberrantly activated. Pharmacological inactivation of Chk2, CDK2 or ERK1/2 or depletion of CDK2 or Chk2 inhibited the centrosome amplification in ETO-treated ACT cells. In addition, autophagy was activated by ETO and was required for ACT cell survival. Chloroquine, the autophagy inhibitor, reduced ACT cell growth and inhibited ETO-induced centrosome amplification. Chloroquine alleviated CDK2 and ERK, but not Chk2, activation and thus inhibited centrosome amplification in either ETO- or hydroxyurea-treated ACT cells. In addition, chloroquine also inhibited centrosome amplification in osteosarcoma U2OS cell lines when treated with ETO or hydroxyurea. In summary, we have demonstrated that chloroquine inhibited ACT cell growth and alleviated DNA damage-induced centrosome amplification by inhibiting CDK2 and ERK activity, thus preventing genomic instability and recurrence of ACT. PMID:26690546

  2. An Erk/Cdk5 axis controls the diabetogenic actions of PPARγ

    PubMed Central

    Banks, Alexander S.; McAllister, Fiona E.; Camporez, João Paulo G.; Zushin, Peter-James H.; Jurczak, Michael J.; Laznik-Bogoslavski, Dina; Shulman, Gerald I.; Gygi, Steven P.; Spiegelman, Bruce M.

    2014-01-01

    Obesity-linked insulin resistance is a major precursor to the development of type 2 diabetes. Previous work has shown that phosphorylation of PPARγ at serine 273 by Cdk5 stimulates diabetogenic gene expression in adipose tissues1. Inhibition of this modification is a key therapeutic mechanism for anti-diabetic PPARγ ligand drugs, such as the thiazolidinediones and PPARγ partial/non-agonists2. To better understand the importance of this obesity-linked PPARγ phosphorylation, we created mice that ablated Cdk5 specifically in adipose tissues. Surprisingly, these mice have both a paradoxical increase in PPARγ phosphorylation at S273 and worsened insulin resistance. Unbiased proteomic studies show that ERK kinases are activated in these KO animals. We show here that ERK directly phosphorylates S273 of PPARγ in a robust manner and that Cdk5 suppresses ERKs through direct action on a novel site in MEK, the ERK kinase. Importantly, pharmacological MEK and ERK inhibition markedly improves insulin resistance in both obese wild type and ob/ob mice, and also completely reverses the deleterious effects of the Cdk5 ablation. These data show that an ERK/Cdk5 axis controls PPARγ function and suggest that MEK/ERK inhibitors may hold promise for the treatment of type 2 diabetes. PMID:25409143

  3. Fluorescent peptide biosensor for monitoring CDK4/cyclin D kinase activity in melanoma cell extracts, mouse xenografts and skin biopsies.

    PubMed

    Prével, Camille; Pellerano, Morgan; González-Vera, Juan A; Henri, Pauline; Meunier, Laurent; Vollaire, Julien; Josserand, Véronique; Morris, May C

    2016-11-15

    Melanoma constitutes the most aggressive form of skin cancer, which further metastasizes into a deadly form of cancer. The p16(INK4a)-Cyclin D-CDK4/6-pRb pathway is dysregulated in 90% of melanomas. CDK4/Cyclin D kinase hyperactivation, associated with mutation of CDK4, amplification of Cyclin D or loss of p16(INK4a) leads to increased risk of developing melanoma. This kinase therefore constitutes a key biomarker in melanoma and an emerging pharmacological target, however there are no tools enabling direct detection or quantification of its activity. Here we report on the design and application of a fluorescent peptide biosensor to quantify CDK4 activity in melanoma cell extracts, skin biopsies and melanoma xenografts. This biosensor provides sensitive means of comparing CDK4 activity between different melanoma cell lines and further responds to CDK4 downregulation by siRNA or small-molecule inhibitors. By affording means of monitoring CDK4 hyperactivity consequent to cancer-associated molecular alterations in upstream signaling pathways that converge upon this kinase, this biosensor offers an alternative to immunological identification of melanoma-specific biomarkers, thereby constituting an attractive tool for diagnostic purposes, providing complementary functional information to histological analysis, of particular utility for detection of melanoma onset in precancerous lesions. This is indeed the first fluorescent peptide biosensor which has been successfully implemented to monitor kinase activity in skin samples and melanoma tumour xenografts. Moreover by enabling to monitor response to CDK4 inhibitors, this biosensor constitutes an attractive companion assay to identify compounds of therapeutic relevance for melanoma.

  4. Cdk2 deficiency decreases ras/CDK4-dependent malignant progression, but not myc-induced tumorigenesis.

    PubMed

    Macias, Everardo; Kim, Yongbaek; Miliani de Marval, Paula L; Klein-Szanto, Andres; Rodriguez-Puebla, Marcelo L

    2007-10-15

    We have previously shown that forced expression of CDK4 in mouse skin (K5CDK4 mice) results in increased susceptibility to squamous cell carcinoma (SCC) development in a chemical carcinogenesis protocol. This protocol induces skin papilloma development, causing a selection of cells bearing activating Ha-ras mutations. We have also shown that myc-induced epidermal proliferation and oral tumorigenesis (K5Myc mice) depends on CDK4 expression. Biochemical analysis of K5CDK4 and K5Myc epidermis as well as skin tumors showed that keratinocyte proliferation is mediated by CDK4 sequestration of p27Kip1 and p21Cip1, and activation of CDK2. Here, we studied the role of CDK2 in epithelial tumorigenesis. In normal skin, loss of CDK2 rescues CDK4-induced, but not myc-induced epidermal hyperproliferation. Ablation of CDK2 in K5CDK4 mice results in decreased incidences and multiplicity of skin tumors as well as malignant progression to SCC. Histopathologic analysis showed that K5CDK4 tumors are drastically more aggressive than K5CDK4/CDK2-/- tumors. On the other hand, we show that CDK2 is dispensable for myc-induced tumorigenesis. In contrast to our previous report of K5Myc/CDK4-/-, K5Myc/CDK2-/- mice developed oral tumors with the same frequency as K5Myc mice. Overall, we have established that ras-induced tumors are more susceptible to CDK2 ablation than myc-induced tumors, suggesting that the efficacy of targeting CDK2 in tumor development and malignant progression is dependent on the oncogenic pathway involved.

  5. Suppression of Vimentin Phosphorylation by the Avian Reovirus p17 through Inhibition of CDK1 and Plk1 Impacting the G2/M Phase of the Cell Cycle

    PubMed Central

    Chiu, Hung-Chuan; Huang, Wei-Ru; Liao, Tsai-Ling; Wu, Hung-Yi; Munir, Muhammad; Shih, Wing-Ling; Liu, Hung-Jen

    2016-01-01

    The p17 protein of avian reovirus (ARV) causes cell cycle retardation in a variety of cell lines; however, the underlying mechanism(s) by which p17 regulates the cell cycle remains largely unknown. We demonstrate for the first time that p17 interacts with CDK1 and vimentin as revealed by reciprocal co-immunoprecipitation and GST pull-down assays. Both in vitro and in vivo studies indicated that direct interaction of p17 and CDK1/vimentin was mapped within the amino terminus (aa 1–60) of p17 and central region (aa 27–118) of CDK1/vimentin. Furthermore, p17 was found to occupy the Plk1-binding site within the vimentin, thereby blocking Plk1 recruitment to CDK1-induced vimentin phosphorylation at Ser 56. Interaction of p17 to CDK1 or vimentin interferes with CDK1-catalyzed phosphorylation of vimentin at Ser 56 and subsequently vimentin phosphorylation at Ser 82 by Plk1. Furthermore, we have identified upstream signaling pathways and cellular factor(s) targeted by p17 and found that p17 regulates inhibitory phosphorylation of CDK1 and blocks vimentin phosphorylation at Ser 56 and Ser 82. The p17-mediated inactivation of CDK1 is dependent on several mechanisms, which include direct interaction with CDK1, p17-mediated suppression of Plk1 by activating the Tpr/p53 and ATM/Chk1/PP2A pathways, and p17-mediated cdc25C degradation via an ubiquitin- proteasome pathway. Additionally, depletion of p53 with a shRNA as well as inhibition of ATM and vimentin by inhibitors diminished virus yield while Tpr and CDK1 knockdown increased virus yield. Taken together, results demonstrate that p17 suppresses both CDK1 and Plk1functions, disrupts vimentin phosphorylation, causes G2/M cell cycle arrest and thus benefits virus replication. PMID:27603133

  6. Brain-derived neurotrophic factor-dependent cdk1 inhibition prevents G2/M progression in differentiating tetraploid neurons.

    PubMed

    Ovejero-Benito, María C; Frade, José M

    2013-01-01

    Neurodegeneration is often associated with DNA synthesis in neurons, the latter usually remaining for a long time as tetraploid cells before dying by apoptosis. The molecular mechanism preventing G2/M transition in these neurons remains unknown, but it may be reminiscent of the mechanism that maintains tetraploid retinal ganglion cells (RGCs) in a G2-like state during normal development, thus preventing their death. Here we show that this latter process, known to depend on brain-derived neurotrophic factor (BDNF), requires the inhibition of cdk1 by TrkB. We demonstrate that a subpopulation of chick RGCs previously shown to become tetraploid co-expresses TrkB and cdk1 in vivo. By using an in vitro system that recapitulates differentiation and cell cycle re-entry of chick retinal neurons we show that BDNF, employed at concentrations specific for the TrkB receptor, reduces the expression of cdk1 in TrkB-positive, differentiating neurons. In this system, BDNF also inhibits the activity of both endogenous cdk1 and exogenously-expressed cdk1/cyclin B1 complex. This inhibition correlates with the phosphorylation of cdk1 at Tyr15, an effect that can be prevented with K252a, a tyrosine kinase inhibitor commonly used to prevent the activity of neurotrophins through their Trk receptors. The effect of BDNF on cdk1 activity is Tyr15-specific since BDNF cannot prevent the activity of a constitutively active form of cdk1 (Tyr15Phe) when expressed in differentiating retinal neurons. We also show that BDNF-dependent phosphorylation of cdk1 at Tyr15 could not be blocked with MK-1775, a Wee1-selective inhibitor, indicating that Tyr15 phosphorylation in cdk1 does not seem to occur through the canonical mechanism observed in proliferating cells. We conclude that the inhibition of both expression and activity of cdk1 through a BDNF-dependent mechanism contributes to the maintenance of tetraploid RGCs in a G2-like state.

  7. Phosphorylation of cyclin-dependent kinase 5 (Cdk5) at Tyr-15 is inhibited by Cdk5 activators and does not contribute to the activation of Cdk5.

    PubMed

    Kobayashi, Hiroyuki; Saito, Taro; Sato, Ko; Furusawa, Kotaro; Hosokawa, Tomohisa; Tsutsumi, Koji; Asada, Akiko; Kamada, Shinji; Ohshima, Toshio; Hisanaga, Shin-ichi

    2014-07-11

    Cdk5 is a member of the cyclin-dependent kinase (Cdk) family. In contrast to other Cdks that promote cell proliferation, Cdk5 plays a role in regulating various neuronal functions, including neuronal migration, synaptic activity, and neuron death. Cdks responsible for cell proliferation need phosphorylation in the activation loop for activation in addition to binding a regulatory subunit cyclin. Cdk5, however, is activated only by binding to its activator, p35 or p39. Furthermore, in contrast to Cdk1 and Cdk2, which are inhibited by phosphorylation at Tyr-15, the kinase activity of Cdk5 is reported to be stimulated when phosphorylated at Tyr-15 by Src family kinases or receptor-type tyrosine kinases. We investigated the activation mechanism of Cdk5 by phosphorylation at Tyr-15. Unexpectedly, however, it was found that Tyr-15 phosphorylation occurred only on monomeric Cdk5, and the coexpression of activators, p35/p25, p39, or Cyclin I, inhibited the phosphorylation. In neuron cultures, too, the activation of Fyn tyrosine kinase did not increase Tyr-15 phosphorylation of Cdk5. Further, phospho-Cdk5 at Tyr-15 was not detected in the p35-bound Cdk5. In contrast, expression of active Fyn increased p35 in neurons. These results indicate that phosphorylation at Tyr-15 is not an activation mechanism of Cdk5 but, rather, indicate that tyrosine kinases could activate Cdk5 by increasing the protein amount of p35. These results call for reinvestigation of how Cdk5 is regulated downstream of Src family kinases or receptor tyrosine kinases in neurons, which is an important signaling cascade in a variety of neuronal activities.

  8. Androgen receptor is a potential novel prognostic marker and oncogenic target in osteosarcoma with dependence on CDK11

    PubMed Central

    Liao, Yunfei; Sassi, Slim; Halvorsen, Stefan; Feng, Yong; Shen, Jacson; Gao, Yan; Cote, Gregory; Choy, Edwin; Harmon, David; Mankin, Henry; Hornicek, Francis; Duan, Zhenfeng

    2017-01-01

    Osteosarcoma is the most common bone cancer in children and adolescents. Previously, we have found that cyclin-dependent kinase 11 (CDK11) signaling was essential for osteosarcoma cell growth and survival. Subsequently, CDK11 siRNA gene targeting, expression profiling, and network reconstruction of differentially expressed genes were performed between CDK11 knock down and wild type osteosarcoma cells. Reconstructed network of the differentially expressed genes pointed to the AR as key to CDK11 signaling in osteosarcoma. CDK11 increased transcriptional activation of AR gene in osteosarcoma cell lines. AR protein was highly expressed in various osteosarcoma cell lines and patient tumor tissues. Tissue microarray analysis showed that the disease-free survival rate for patients with high-expression of AR was significantly shorter than for patients with low-expression of AR. In addition, AR gene expression knockdown via siRNA greatly inhibited cell growth and viability. Similar results were found in osteosarcoma cells treated with AR inhibitor. These findings suggest that CDK11 is involved in the regulation of AR pathway and AR can be a potential novel prognostic marker and therapeutic target for osteosarcoma treatment. PMID:28262798

  9. CDK4 regulates cancer stemness and is a novel therapeutic target for triple-negative breast cancer

    PubMed Central

    Dai, Meiou; Zhang, Chenjing; Ali, Ayad; Hong, Xinyuan; Tian, Jun; Lo, Chieh; Fils-Aimé, Nadège; Burgos, Sergio A.; Ali, Suhad; Lebrun, Jean-Jacques

    2016-01-01

    Triple negative breast cancers exhibit very aggressive features and poor patient outcomes. These tumors are enriched in cancer stem cells and exhibit resistance to most treatments and chemotherapy. In this study, we found the cyclin-dependent kinase (CDK4) to act as a cancer stem cell regulator and novel prognostic marker in triple negative breast cancers. We found CDK4 to be highly expressed in these tumors and its expression to correlate with poor overall and relapse free survival outcomes, high tumor grade and poor prognostic features of triple negative breast cancer patients. Moreover, we found that blocking CDK4 expression or kinase activity, using a pharmacological inhibitor prevented breast cancer stem cell self-renewal. Interestingly, suppression of CDK4 expression or kinase activity reversed the basal-B TNBC mesenchymal phenotype to an epithelial- and luminal-like phenotype which correlates with better clinical prognosis. Finally, blocking CDK4 activity efficiently eliminated both normal and chemotherapy-resistant cancer cells in triple negative breast cancers, highlighting CDK4 as a promising novel therapeutic target for these aggressive breast tumors. PMID:27759034

  10. Tamoxifen inhibits CDK5 kinase activity by interacting with p35/p25 and modulates the pattern of tau phosphorylation.

    PubMed

    Corbel, Caroline; Zhang, Bing; Le Parc, Annabelle; Baratte, Blandine; Colas, Pierre; Couturier, Cyril; Kosik, Kenneth S; Landrieu, Isabelle; Le Tilly, Véronique; Bach, Stéphane

    2015-04-23

    Cyclin-dependent kinase 5 (CDK5) is a multifunctional enzyme that plays numerous roles, notably in brain development. CDK5 is activated through its association with the activators, p35 and p39, rather than by cyclins. Proteolytic procession of the N-terminal part of its activators has been linked to Alzheimer's disease and various other neuropathies. The interaction with the proteolytic product p25 prolongs CDK5 activation and modifies the substrate specificity. In order to discover small-molecule inhibitors of the interaction between CDK5 and p25, we have used a bioluminescence resonance energy transfer (BRET)-based screening assay. Among the 1,760 compounds screened, the generic drug tamoxifen has been identified. The inhibition of the CDK5 activity by tamoxifen was notably validated by monitoring the phosphorylation state of tau protein. The study of the molecular mechanism of inhibition indicates that tamoxifen interacts with p25 to block the CDK5/p25 interaction and pave the way for new treatments of tauopathies.

  11. Cyclic AMP induces IPC leukemia cell apoptosis via CRE-and CDK-dependent Bim transcription.

    PubMed

    Huseby, S; Gausdal, G; Keen, T J; Kjærland, E; Krakstad, C; Myhren, L; Brønstad, K; Kunick, C; Schwede, F; Genieser, H-G; Kleppe, R; Døskeland, S O

    2011-12-08

    The IPC-81 cell line is derived from the transplantable BNML model of acute myelogenic leukemia (AML), known to be a reliable predictor of the clinical efficiency of antileukemic agents, like the first-line AML anthracycline drug daunorubicin (DNR). We show here that cAMP acted synergistically with DNR to induce IPC cell death. The DNR-induced death differed from that induced by cAMP by (1) not involving Bim induction, (2) being abrogated by GSK3β inhibitors, (3) by being promoted by the HSP90/p23 antagonist geldanamycin and truncated p23 and (4) by being insensitive to the CRE binding protein (CREB) antagonist ICER and to cyclin-dependent protein kinase (CDK) inhibitors. In contrast, the apoptosis induced by cAMP correlated tightly with Bim protein expression. It was abrogated by Bim (BCL2L11) downregulation, whether achieved by the CREB antagonist ICER, by CDK inhibitors, by Bim-directed RNAi, or by protein synthesis inhibitor. The forced expression of BimL killed IPC-81(WT) cells rapidly, Bcl2-overexpressing cells being partially resistant. The pivotal role of CREB and CDK activity for Bim transcription is unprecedented. It is also noteworthy that newly developed cAMP analogs specifically activating PKA isozyme I (PKA-I) were able to induce IPC cell apoptosis. Our findings support the notion that AML cells may possess targetable death pathways not exploited by common anti-cancer agents.

  12. Cdk5: An Emerging Kinase in Pain Signaling

    PubMed Central

    Pareek, Tej Kumar; Zipp, Lisa; Letterio, John J

    2015-01-01

    Pain is an important survival mechanism for an organism. It can turn into severe mental and physical disorder however, if the molecular and/or cellular pathways involved in pain signaling are altered. Chronic pain is characterized by an altered pain perception that includes allodynia (a response to a normally non-noxious stimulus) and hyperalgesia (an exaggerated response to a normally noxious stimulus). Past few years of pain research has been mainly focused on precise understanding of the molecular and cellular nociceptive signatures altered during chronic pain, so that more effective pain relievers can be developed. The importance of protein kinases in normal cellular homeostasis and disease pathogenesis has evolved rapidly in the past few decades. The recent advancement defining the role of multiple protein kinases in regulating neuronal plasticity and pain sensitization has gained enough attention of pharmaceutical industry to develop specific and selective kinase inhibitors as analgesics. Cyclin-dependent kinase 5 (Cdk5) is one such emerging kinase in pain biology. We will discuss here the recent advancement and therapeutic potential of Cdk5 in pain signaling.

  13. Expression of CDK7, Cyclin H, and MAT1 Is Elevated in Breast Cancer and Is Prognostic in Estrogen Receptor–Positive Breast Cancer

    PubMed Central

    Patel, Hetal; Abduljabbar, Rezvan; Lai, Chun-Fui; Periyasamy, Manikandan; Harrod, Alison; Gemma, Carolina; Steel, Jennifer H.; Patel, Naina; Busonero, Claudia; Jerjees, Dena; Remenyi, Judit; Smith, Sally; Gomm, Jennifer J.; Magnani, Luca; Győrffy, Balázs; Jones, Louise J.; Fuller-Pace, Frances; Shousha, Sami; Buluwela, Laki; Rakha, Emad A.; Ellis, Ian O.; Coombes, R. Charles; Ali, Simak

    2017-01-01

    Purpose CDK-activating kinase (CAK) is required for the regulation of the cell cycle and is a trimeric complex consisting of cyclin-dependent kinase 7 (CDK7), Cyclin H, and the accessory protein, MAT1. CDK7 also plays a critical role in regulating transcription, primarily by phosphorylating RNA polymerase II, as well as transcription factors such as estrogen receptor-α (ER). Deregulation of cell cycle and transcriptional control are general features of tumor cells, highlighting the potential for the use of CDK7 inhibitors as novel cancer therapeutics. Experimental Design mRNA and protein expression of CDK7 and its essential cofactors cyclin H and MAT1 were evaluated in breast cancer samples to determine if their levels are altered in cancer. Immunohistochemical staining of >900 breast cancers was used to determine the association with clinicopathologic features and patient outcome. Results We show that expressions of CDK7, cyclin H, and MAT1 are all closely linked at the mRNA and protein level, and their expression is elevated in breast cancer compared with the normal breast tissue. Intriguingly, CDK7 expression was inversely proportional to tumor grade and size, and outcome analysis showed an association between CAK levels and better outcome. Moreover, CDK7 expression was positively associated with ER expression and in particular with phosphorylation of ER at serine 118, a site important for ER transcriptional activity. Conclusions Expressions of components of the CAK complex, CDK7, MAT1, and Cyclin H are elevated in breast cancer and correlate with ER. Like ER, CDK7 expression is inversely proportional to poor prognostic factors and survival. PMID:27301701

  14. The CDK inhibitor AT7519M in patients with relapsed or refractory chronic lymphocytic leukemia (CLL) and mantle cell lymphoma. A Phase II study of the Canadian Cancer Trials Group.

    PubMed

    Seftel, Matthew D; Kuruvilla, John; Kouroukis, Tom; Banerji, Versha; Fraser, Graeme; Crump, Michael; Kumar, Rajat; Chalchal, Haji I; Salim, Muhammad; Laister, Rob C; Crocker, Susan; Gibson, Spencer B; Toguchi, Marcia; Lyons, John F; Xu, Hao; Powers, Jean; Sederias, Joana; Seymour, Lesley; Hay, Annette E

    2017-06-01

    AT7519M is a small molecule inhibitor of cyclin-dependent kinases 1, 2, 4, 5, and 9 with in vitro activity against lymphoid malignancies. In two concurrent Phase II trials, we evaluated AT7519M in relapsed or refractory chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) using the recommended Phase II dosing of 27 mg/m(2) twice weekly for 2 of every 3 weeks. Primary objective was objective response rate (ORR). Nineteen patients were accrued (7 CLL, 12 MCL). Four CLL patients achieved stable disease (SD). Two MCL patients achieved partial response (PR), and 6 had SD. One additional MCL patient with SD subsequently achieved PR 9 months after completion of AT7519M. Tumor lysis syndrome was not reported. In conclusion, AT7519M was safely administered to patients with relapsed/refractory CLL and MCL. In CLL, some patients had tumor reductions, but the ORR was low. In MCL, activity was noted with ORR of 27%.

  15. Synaptic NMDA receptor stimulation activates PP1 by inhibiting its phosphorylation by Cdk5

    PubMed Central

    Hou, Hailong; Sun, Lu; Siddoway, Benjamin A.; Petralia, Ronald S.; Yang, Hongtian; Gu, Hua; Nairn, Angus C.

    2013-01-01

    The serine/threonine protein phosphatase protein phosphatase 1 (PP1) is known to play an important role in learning and memory by mediating local and downstream aspects of synaptic signaling, but how PP1 activity is controlled in different forms of synaptic plasticity remains unknown. We find that synaptic N-methyl-d-aspartate (NMDA) receptor stimulation in neurons leads to activation of PP1 through a mechanism involving inhibitory phosphorylation at Thr320 by Cdk5. Synaptic stimulation led to proteasome-dependent degradation of the Cdk5 regulator p35, inactivation of Cdk5, and increased auto-dephosphorylation of Thr320 of PP1. We also found that neither inhibitor-1 nor calcineurin were involved in the control of PP1 activity in response to synaptic NMDA receptor stimulation. Rather, the PP1 regulatory protein, inhibitor-2, formed a complex with PP1 that was controlled by synaptic stimulation. Finally, we found that inhibitor-2 was critical for the induction of long-term depression in primary neurons. Our work fills a major gap regarding the regulation of PP1 in synaptic plasticity. PMID:24189275

  16. Mutation testing in melanoma families: INK4A, CDK4 and INK4D

    PubMed Central

    Newton Bishop, J A; Harland, M; Bennett, D C; Bataille, V; Goldstein, A M; Tucker, M A; Ponder, B A J; Cuzick, J; Selby, P; Bishop, D T

    1999-01-01

    The INK4A gene which codes for the cyclin-dependent kinase (CDK) inhibitor INK4A or p16 underlies susceptibility to melanoma in some families. Germline mutations in the gene that codes for the target protein of p16, CDK4, underlie susceptibility in very rare families. We report mutation screening of the INK4A and CDK4 genes in 42 UK families. A total of nine families were identified with INK4A mutations and none with CDK4 exon 2 mutations. These mutations were in 8/22 (35%) families with three or more cases of melanoma and 1/20 (5%) families with only two cases. In one of these nine families a novel single base pair substitution was identified, Gly67Arg. In an attempt to identify another melanoma susceptibility gene, a member of the INK4 family, the p19 INK4D gene has been studied. The p19 gene was sequenced in DNA from the 42 UK families and six additional US families. No mutations were identified. © 1999 Cancer Research Campaign PMID:10390011

  17. Targeting the AKT/GSK3{beta}/Cyclin D1/Cdk4 Survival Signaling Pathway for Eradication of Tumor Radioresistance Acquired by Fractionated Radiotherapy

    SciTech Connect

    Shimura, Tsutomu; Kakuda, Satoshi; Ochiai, Yasushi; Kuwahara, Yoshikazu; Takai, Yoshihiro; Fukumoto, Manabu

    2011-06-01

    Purpose: Radioresistance is a major cause of treatment failure of radiotherapy (RT) in human cancer. We have recently revealed that acquired radioresistance of tumor cells induced by fractionated radiation is attributable to cyclin D1 overexpression as a consequence of the downregulation of GSK3{beta}-dependent cyclin D1 proteolysis mediated by a constitutively activated serine-threonine kinase, AKT. This prompted us to hypothesize that targeting the AKT/GSK3{beta}/cyclin D1 pathway may improve fractionated RT by suppressing acquired radioresistance of tumor cells. Methods and Materials: Two human tumor cell lines with acquired radioresistance were exposed to X-rays after incubation with either an AKT inhibitor, AKT/PKB signaling inhibitor-2 (API-2), or a Cdk4 inhibitor (Cdk4-I). Cells were then subjected to immunoblotting, clonogenic survival assay, cell growth analysis, and cell death analysis with TUNEL and annexin V staining. In vivo radiosensitivity was assessed by growth of human tumors xenografted into nude mice. Results: Treatment with API-2 resulted in downregulation of cyclin D1 expression in cells with acquired radioresistance. Cellular radioresistance disappeared completely both in vitro and in vivo with accompanying apoptosis when treated with API-2. Furthermore, inhibition of cyclin D1/Cdk4 by Cdk4-I was sufficient for abolishing radioresistance. Treatment with either API-2 or Cdk4-I was also effective in suppressing resistance to cis-platinum (II)-diamine-dichloride in the cells with acquired radioresistance. Interestingly, the radiosensitizing effect of API-2 was canceled by overexpression of cyclin D1 whereas Cdk4-I was still able to sensitize cells with cyclin D1 overexpression. Conclusion: Cyclin D1/Cdk4 is a critical target of the AKT survival signaling pathway responsible for tumor radioresistance. Targeting the AKT/GSK3{beta}/cyclin D1/Cdk4 pathway would provide a novel approach to improve fractionated RT and would have an impact on tumor

  18. Functional characterization of CDK5 and CDK5R1 mutations identified in patients with non-syndromic intellectual disability.

    PubMed

    Moncini, Silvia; Castronovo, Paola; Murgia, Alessandra; Russo, Silvia; Bedeschi, Maria Francesca; Lunghi, Marta; Selicorni, Angelo; Bonati, Maria Teresa; Riva, Paola; Venturin, Marco

    2016-04-01

    Cyclin-dependent kinase 5 (CDK5) and cyclin-dependent kinase 5, regulatory subunit 1 (CDK5R1), encoding CDK5 activator p35, have a fundamental role in central nervous system (CNS) development and function, and are involved in the pathogenesis of several neurodegenerative disorders, thus constituting strong candidate genes for the onset of intellectual disability (ID). We carried out a mutation screening of CDK5 and CDK5R1 coding regions and CDK5R1 3'-UTR on a cohort of 360 patients with non-syndromic ID (NS-ID) using denaturing high performance liquid chromatography (DHPLC) and direct sequencing. We found one novel silent mutation in CDK5 and one novel silent mutation in CDK5R1 coding regions, three novel intronic variations in CDK5, not causing any splicing defect, and four novel heterozygous variations in CDK5R1 3'-UTR. None of these variations was present in 450 healthy controls and single-nucleotide polymorphism (SNP) databases. The functional study of CDK5R1 p.A108V mutation evidenced an impaired p35 cleavage by the calcium-dependent protease calpain. Moreover, luciferase constructs containing the CDK5R1 3'-UTR mutations showed altered gene expression levels. Eight known polymorphisms were also identified displaying different frequencies in NS-ID patients compared with the controls. In particular, the minor allele of CDK5R1 3'-UTR rs735555 polymorphism was associated with increased risk for NS-ID. In conclusion, our data suggest that mutations and polymorphisms in CDK5 and CDK5R1 genes may contribute to the onset of the NS-ID phenotype.

  19. Mammalian cells cycle without the D-type cyclin-dependent kinases Cdk4 and Cdk6.

    PubMed

    Malumbres, Marcos; Sotillo, Rocío; Santamaría, David; Galán, Javier; Cerezo, Ana; Ortega, Sagrario; Dubus, Pierre; Barbacid, Mariano

    2004-08-20

    Cdk4 and Cdk6 are thought to be essential for initiation of the cell cycle in response to mitogenic stimuli. Previous studies have shown that Cdk4 is dispensable for proliferation in most cell types, an observation attributed to a putative compensatory role by Cdk6. Cdk6-null mice are viable and develop normally although hematopoiesis is slightly impaired. Embryos defective for Cdk4 and Cdk6 die during the late stages of embryonic development due to severe anemia. However, these embryos display normal organogenesis and most cell types proliferate normally. In vitro, embryonic fibroblasts lacking Cdk4 and Cdk6 proliferate and become immortal upon serial passage. Moreover, quiescent Cdk4/Cdk6-null cells respond to serum stimulation and enter S phase with normal kinetics although with lower efficiency. These results indicate that D-type cyclin-dependent kinases are not essential for cell cycle entry and suggest the existence of alternative mechanisms to initiate cell proliferation upon mitogenic stimulation.

  20. MDM2 turnover and expression of ATRX determine the choice between quiescence and senescence in response to CDK4 inhibition

    PubMed Central

    Dickson, Mark A.; Klein, Mary E.; O'Connor, Rachael; Wilder, Fatima O.; Socci, Nicholas D.; Tap, William D.; Schwartz, Gary K.; Singer, Samuel; Crago, Aimee M.; Koff, Andrew

    2015-01-01

    CDK4 inhibitors (CDK4i) earned Breakthrough Therapy Designation from the FDA last year and are entering phase III clinical trials in several cancers. However, not all tumors respond favorably to these drugs. CDK4 activity is critical for progression through G1 phase and into the mitotic cell cycle. Inhibiting this kinase induces Rb-positive cells to exit the cell cycle into either a quiescent or senescent state. In this report, using well-differentiated and dedifferentiated liposarcoma (WD/DDLS) cell lines, we show that the proteolytic turnover of MDM2 is required for CDK4i-induced senescence. Failure to reduce MDM2 does not prevent CDK4i-induced withdrawal from the cell cycle but the cells remain in a reversible quiescent state. Reducing MDM2 in these cells drives them into the more stable senescent state. CDK4i-induced senescence associated with loss of MDM2 is also observed in some breast cancer, lung cancer and glioma cell lines indicating that this is not limited to WD/DDLS cells in which MDM2 is overexpressed or in cells that contain wild type p53. MDM2 turnover depends on its E3 ligase activity and expression of ATRX. Interestingly, in seven patients the changes in MDM2 expression were correlated with outcome. These insights identify MDM2 and ATRX as new regulators controlling geroconversion, the process by which quiescent cells become senescent, and this insight may be exploited to improve the activity of CDK4i in cancer therapy. PMID:25803170

  1. Palbociclib treatment of FLT3-ITD+ AML cells uncovers a kinase-dependent transcriptional regulation of FLT3 and PIM1 by CDK6

    PubMed Central

    Uras, Iris Z.; Walter, Gina J.; Scheicher, Ruth; Bellutti, Florian; Prchal-Murphy, Michaela; Tigan, Anca S.; Valent, Peter; Heidel, Florian H.; Kubicek, Stefan; Scholl, Claudia; Fröhling, Stefan

    2016-01-01

    Up to 30% of patients with acute myeloid leukemia have constitutively activating internal tandem duplications (ITDs) of the FLT3 receptor tyrosine kinase. Such mutations are associated with a poor prognosis and a high propensity to relapse after remission. FLT3 inhibitors are being developed as targeted therapy for FLT3-ITD+ acute myeloid leukemia; however, their use is complicated by rapid development of resistance, which illustrates the need for additional therapeutic targets. We show that the US Food and Drug Administration–approved CDK4/6 kinase inhibitor palbociclib induces apoptosis of FLT3-ITD leukemic cells. The effect is specific for FLT3-mutant cells and is ascribed to the transcriptional activity of CDK6: CDK6 but not its functional homolog CDK4 is found at the promoters of the FLT3 and PIM1 genes, another important leukemogenic driver. There CDK6 regulates transcription in a kinase-dependent manner. Of potential clinical relevance, combined treatment with palbociclib and FLT3 inhibitors results in synergistic cytotoxicity. Simultaneously targeting two critical signaling nodes in leukemogenesis could represent a therapeutic breakthrough, leading to complete remission and overcoming resistance to FLT3 inhibitors. PMID:27099147

  2. Functional, chemical genomic, and super-enhancer screening identify sensitivity to cyclin D1/CDK4 pathway inhibition in Ewing sarcoma

    PubMed Central

    Crompton, Brian; Cowley, Glenn; Vazquez, Francisca; Weir, Barbara A.; Tsherniak, Aviad; Parasuraman, Sudha; Kim, Sunkyu; Alexe, Gabriela; Stegmaier, Kimberly

    2015-01-01

    Ewing sarcoma is an aggressive bone and soft tissue tumor in children and adolescents, with treatment remaining a clinical challenge. This disease is mediated by somatic chromosomal translocations of the EWS gene and a gene encoding an ETS transcription factor, most commonly, FLI1. While direct targeting of aberrant transcription factors remains a pharmacological challenge, identification of dependencies incurred by EWS/FLI1 expression would offer a new therapeutic avenue. We used a combination of super-enhancer profiling, near-whole genome shRNA-based and small-molecule screening to identify cyclin D1 and CDK4 as Ewing sarcoma-selective dependencies. We revealed that super-enhancers mark Ewing sarcoma specific expression signatures and EWS/FLI1 target genes in human Ewing sarcoma cell lines. Particularly, a super-enhancer regulates cyclin D1 and promotes its expression in Ewing sarcoma. We demonstrated that Ewing sarcoma cells require CDK4 and cyclin D1 for survival and anchorage-independent growth. Additionally, pharmacologic inhibition of CDK4 with selective CDK4/6 inhibitors led to cytostasis and cell death of Ewing sarcoma cell lines in vitro and growth delay in an in vivo Ewing sarcoma xenograft model. These results demonstrated a dependency in Ewing sarcoma on CDK4 and cyclin D1 and support exploration of CDK4/6 inhibitors as a therapeutic approach for patients with this disease. PMID:26337082

  3. Characterization of Human Cyclin-Dependent Kinase 12 (CDK12) and CDK13 Complexes in C-Terminal Domain Phosphorylation, Gene Transcription, and RNA Processing

    PubMed Central

    Liang, Kaiwei; Gao, Xin; Gilmore, Joshua M.; Florens, Laurence; Washburn, Michael P.; Smith, Edwin

    2015-01-01

    Cyclin-dependent kinase 9 (CDK9) and CDK12 have each been demonstrated to phosphorylate the RNA polymerase II C-terminal domain (CTD) at serine 2 of the heptad repeat, both in vitro and in vivo. CDK9, as part of P-TEFb and the super elongation complex (SEC), is by far the best characterized of CDK9, CDK12, and CDK13. We employed both in vitro and in vivo assays to further investigate the molecular properties of CDK12 and its paralog CDK13. We isolated Flag-tagged CDK12 and CDK13 and found that they associate with numerous RNA processing factors. Although knockdown of CDK12, CDK13, or their cyclin partner CCNK did not affect the bulk CTD phosphorylation levels in HCT116 cells, transcriptome sequencing (RNA-seq) analysis revealed that CDK12 and CDK13 losses in HCT116 cells preferentially affect expression of DNA damage response and snoRNA genes, respectively. CDK12 and CDK13 depletion also leads to a loss of expression of RNA processing factors and to defects in RNA processing. These findings suggest that in addition to implementing CTD phosphorylation, CDK12 and CDK13 may affect RNA processing through direct physical interactions with RNA processing factors and by regulating their expression. PMID:25561469

  4. Redundant Regulation of Cdk1 Tyrosine Dephosphorylation in Saccharomyces cerevisiae.

    PubMed

    Kennedy, Erin K; Dysart, Michael; Lianga, Noel; Williams, Elizabeth C; Pilon, Sophie; Doré, Carole; Deneault, Jean-Sebastien; Rudner, Adam D

    2016-03-01

    Cdk1 activity drives both mitotic entry and the metaphase-to-anaphase transition in all eukaryotes. The kinase Wee1 and the phosphatase Cdc25 regulate the mitotic activity of Cdk1 by the reversible phosphorylation of a conserved tyrosine residue. Mutation of cdc25 in Schizosaccharomyces pombe blocks Cdk1 dephosphorylation and causes cell cycle arrest. In contrast, deletion of MIH1, the cdc25 homolog in Saccharomyces cerevisiae, is viable. Although Cdk1-Y19 phosphorylation is elevated during mitosis in mih1∆ cells, Cdk1 is dephosphorylated as cells progress into G1, suggesting that additional phosphatases regulate Cdk1 dephosphorylation. Here we show that the phosphatase Ptp1 also regulates Cdk1 dephosphorylation in vivo and can directly dephosphorylate Cdk1 in vitro. Using a novel in vivo phosphatase assay, we also show that PP2A bound to Rts1, the budding yeast B56-regulatory subunit, regulates dephosphorylation of Cdk1 independently of a function regulating Swe1, Mih1, or Ptp1, suggesting that PP2A(Rts1) either directly dephosphorylates Cdk1-Y19 or regulates an unidentified phosphatase.

  5. Redundant Regulation of Cdk1 Tyrosine Dephosphorylation in Saccharomyces cerevisiae

    PubMed Central

    Kennedy, Erin K.; Dysart, Michael; Lianga, Noel; Williams, Elizabeth C.; Pilon, Sophie; Doré, Carole; Deneault, Jean-Sebastien; Rudner, Adam D.

    2016-01-01

    Cdk1 activity drives both mitotic entry and the metaphase-to-anaphase transition in all eukaryotes. The kinase Wee1 and the phosphatase Cdc25 regulate the mitotic activity of Cdk1 by the reversible phosphorylation of a conserved tyrosine residue. Mutation of cdc25 in Schizosaccharomyces pombe blocks Cdk1 dephosphorylation and causes cell cycle arrest. In contrast, deletion of MIH1, the cdc25 homolog in Saccharomyces cerevisiae, is viable. Although Cdk1-Y19 phosphorylation is elevated during mitosis in mih1∆ cells, Cdk1 is dephosphorylated as cells progress into G1, suggesting that additional phosphatases regulate Cdk1 dephosphorylation. Here we show that the phosphatase Ptp1 also regulates Cdk1 dephosphorylation in vivo and can directly dephosphorylate Cdk1 in vitro. Using a novel in vivo phosphatase assay, we also show that PP2A bound to Rts1, the budding yeast B56-regulatory subunit, regulates dephosphorylation of Cdk1 independently of a function regulating Swe1, Mih1, or Ptp1, suggesting that PP2ARts1 either directly dephosphorylates Cdk1-Y19 or regulates an unidentified phosphatase. PMID:26715668

  6. Histone H1 Phosphorylation by Cdk2 Selectively Modulates Mouse Mammary Tumor Virus Transcription through Chromatin Remodeling

    PubMed Central

    Bhattacharjee, Rabindra N.; Banks, Geoffrey C.; Trotter, Kevin W.; Lee, Huay-Leng; Archer, Trevor K.

    2001-01-01

    Transcriptional activation of the mouse mammary tumor virus (MMTV) promoter by ligand-bound glucocorticoid receptor (GR) is transient. Previously, we demonstrated that prolonged hormone exposure results in displacement of the transcription factor nuclear factor 1 (NF1) and the basal transcription complex from the promoter, the dephosphorylation of histone H1, and the establishment of a repressive chromatin structure. We have explored the mechanistic link between histone H1 dephosphorylation and silencing of the MMTV promoter by describing the putative kinase responsible for H1 phosphorylation. Both in vitro kinase assays and in vivo protein expression studies suggest that in hormone-treated cells the ability of cdk2 to phosphorylate histone H1 is decreased and the cdk2 inhibitory p21 protein level is increased. To address the role of cdk2 and histone H1 dephosphorylation in the silencing of the MMTV promoter, we used potent cdk2 inhibitors, Roscovitine and CVT-313, to generate an MMTV promoter which is associated predominantly with the dephosphorylated form of histone H1. Both Roscovitine and CVT-313 block phosphorylation of histone H1 and, under these conditions, the GR is unable to remodel chromatin, recruit transcription factors to the promoter, or stimulate MMTV mRNA accumulation. These results suggest a model where cdk2-directed histone H1 phosphorylation is a necessary condition to permit GR-mediated chromatin remodeling and activation of the MMTV promoter in vivo. PMID:11463824

  7. Functional specialization of chordate CDK1 paralogs during oogenic meiosis

    PubMed Central

    Øvrebø, Jan Inge; Campsteijn, Coen; Kourtesis, Ioannis; Hausen, Harald; Raasholm, Martina; Thompson, Eric M

    2015-01-01

    Cyclin-dependent kinases (CDKs) are central regulators of eukaryotic cell cycle progression. In contrast to interphase CDKs, the mitotic phase CDK1 is the only CDK capable of driving the entire cell cycle and it can do so from yeast to mammals. Interestingly, plants and the marine chordate, Oikopleura dioica, possess paralogs of the highly conserved CDK1 regulator. However, whereas in plants the 2 CDK1 paralogs replace interphase CDK functions, O. dioica has a full complement of interphase CDKs in addition to its 5 odCDK1 paralogs. Here we show specific sub-functionalization of odCDK1 paralogs during oogenesis. Differential spatiotemporal dynamics of the odCDK1a, d and e paralogs and the meiotic polo-like kinase 1 (Plk1) and aurora kinase determine the subset of meiotic nuclei in prophase I arrest that will seed growing oocytes and complete meiosis. Whereas we find odCDK1e to be non-essential, knockdown of the odCDK1a paralog resulted in the spawning of non-viable oocytes of reduced size. Knockdown of odCDK1d also resulted in the spawning of non-viable oocytes. In this case, the oocytes were of normal size, but were unable to extrude polar bodies upon exposure to sperm, because they were unable to resume meiosis from prophase I arrest, a classical function of the sole CDK1 during meiosis in other organisms. Thus, we reveal specific sub-functionalization of CDK1 paralogs, during the meiotic oogenic program. PMID:25714331

  8. Mechanism of p27 Unfolding for CDK2 Reactivation

    PubMed Central

    Rath, Soumya Lipsa; Senapati, Sanjib

    2016-01-01

    Cell-cycle regulatory protein, CDK2 is active when bound to its complementary partner protein, CyclinA or E. Recent discovery of the Kip/Cip family of proteins has indicated that the activity of CDK2 is also regulated by a member protein, p27. Although, the mechanism of CDK2 inhibition by p27 binding is known from crystal structure, little is known about the mechanism of CDK2 reactivation. Here, we execute classical and accelerated molecular dynamics simulations of unphosphorylated- and phosphorylated-p27 bound CDK2/CyclinA to unravel the CDK2 reactivation mechanism at molecular-to-atomic detail. Results suggest that the phosphorylation of p27 Y88 residue (pY88-p27) first disrupts the p27/CDK2 hybrid β-sheet and subsequently ejects the p27 310 helix from CDK2 catalytic cleft. The unbinding of p27 from CDK2/CyclinA complex, thus, follows a two-step unfolding mechanism, where the 310 helix ejection constitutes the rate-limiting step. Interestingly, the unfolding of p27 leaves CDK2/CyclinA in an active state, where the prerequisite CDK2-CyclinA interfacial contacts were regained and ATP achieved its native position for smooth transfer of phosphate. Our findings match very well with NMR chemical shift data that indicated the flip-out of p27 310 helix from CDK2 pocket and kinetic experiments that exhibited significant kinase activity of CDK2 when saturated with pY88-p27. PMID:27211815

  9. Mechanism of p27 Unfolding for CDK2 Reactivation.

    PubMed

    Rath, Soumya Lipsa; Senapati, Sanjib

    2016-05-23

    Cell-cycle regulatory protein, CDK2 is active when bound to its complementary partner protein, CyclinA or E. Recent discovery of the Kip/Cip family of proteins has indicated that the activity of CDK2 is also regulated by a member protein, p27. Although, the mechanism of CDK2 inhibition by p27 binding is known from crystal structure, little is known about the mechanism of CDK2 reactivation. Here, we execute classical and accelerated molecular dynamics simulations of unphosphorylated- and phosphorylated-p27 bound CDK2/CyclinA to unravel the CDK2 reactivation mechanism at molecular-to-atomic detail. Results suggest that the phosphorylation of p27 Y88 residue (pY88-p27) first disrupts the p27/CDK2 hybrid β-sheet and subsequently ejects the p27 310 helix from CDK2 catalytic cleft. The unbinding of p27 from CDK2/CyclinA complex, thus, follows a two-step unfolding mechanism, where the 310 helix ejection constitutes the rate-limiting step. Interestingly, the unfolding of p27 leaves CDK2/CyclinA in an active state, where the prerequisite CDK2-CyclinA interfacial contacts were regained and ATP achieved its native position for smooth transfer of phosphate. Our findings match very well with NMR chemical shift data that indicated the flip-out of p27 310 helix from CDK2 pocket and kinetic experiments that exhibited significant kinase activity of CDK2 when saturated with pY88-p27.

  10. Ischemic Stroke Injury Is Mediated by Aberrant Cdk5

    PubMed Central

    Meyer, Douglas A.; Torres-Altoro, Melissa I.; Tan, Zhenjun; Tozzi, Alessandro; Di Filippo, Massimiliano; DiNapoli, Vincent; Plattner, Florian; Kansy, Janice W.; Benkovic, Stanley A.; Huber, Jason D.; Miller, Diane B.; Greengard, Paul; Calabresi, Paolo; Rosen, Charles L.

    2014-01-01

    Ischemic stroke is one of the leading causes of morbidity and mortality. Treatment options are limited and only a minority of patients receive acute interventions. Understanding the mechanisms that mediate neuronal injury and death may identify targets for neuroprotective treatments. Here we show that the aberrant activity of the protein kinase Cdk5 is a principal cause of neuronal death in rodents during stroke. Ischemia induced either by embolic middle cerebral artery occlusion (MCAO) in vivo or by oxygen and glucose deprivation in brain slices caused calpain-dependent conversion of the Cdk5-activating cofactor p35 to p25. Inhibition of aberrant Cdk5 during ischemia protected dopamine neurotransmission, maintained field potentials, and blocked excitotoxicity. Furthermore, pharmacological inhibition or conditional knock-out (CKO) of Cdk5 prevented neuronal death in response to ischemia. Moreover, Cdk5 CKO dramatically reduced infarctions following MCAO. Thus, targeting aberrant Cdk5 activity may serve as an effective treatment for stroke. PMID:24920629

  11. Role of the Cdk Inhibitor Sic 1 in Start

    DTIC Science & Technology

    1998-08-01

    Cold Spring Harbor Laboratory Cold Spring Harbor , New York 11724 REPORT DATE: August 1998 TYPE OF REPORT: Annual PREPARED FOR...AND ADDRESS(ES) Cold Spring Harbor Laboratory Cold Spring Harbor , New York 11724 8. PERFORMING ORGANIZATION REPORT NUMBER 9. SPONSORING f...Meeting at Cold Spring Harbor Laboratory. Futcher, B., Yang, Q.-H., Sherlock, G., Marshak, D. and Schneider, B. SIC1 and other

  12. Biochemical Characterization of Complexes with p21, a CDK Inhibitor

    DTIC Science & Technology

    1998-08-01

    additional experiments to further characterize p28 and p40 , two potentially novel proteins that co-fractionated with p21 on glycerol gradients, sizing...well as with amino- and carboxy-terminal fragments of p21. Neither p28 nor p40 was captured in preliminary binding experiments, suggesting that these...an additional step of 1.0 HMGNB (25 mM Z 75 HEPES [pH 7.6], 1 M NaCI, 10% glycerol, 0.1% Nonidet P-40 [NP-40], 5 mM U P-mercaptoethanol, and 0.2 mM

  13. Role of CDK4 in Breast Development and Cancer

    DTIC Science & Technology

    2008-04-01

    induced DNA damage checkpoint responses. 15. SUBJECT TERMS CDK4 , Breast Development, Oncogenes, Cell Cycle, Breast Cancer 16. SECURITY...3A of appended publication) show that 97% of the Cdk4 (+/+):MMTV-neu mice develop breast cancer between 28 to 75 weeks of age. The rest of the mice...were found to develop salivary gland tumors. In sharp contrast, only 14% of the Cdk4 (neo/neo):MMTV-neu mice develop signs of breast cancer and this

  14. Myt1 inhibition of Cyclin A/Cdk1 is essential for fusome integrity and premeiotic centriole engagement in Drosophila spermatocytes

    PubMed Central

    Varadarajan, Ramya; Ayeni, Joseph; Jin, Zhigang; Homola, Ellen; Campbell, Shelagh D.

    2016-01-01

    Regulation of cell cycle arrest in premeiotic G2 phase coordinates germ cell maturation and meiotic cell division with hormonal and developmental signals by mechanisms that control Cyclin B synthesis and inhibitory phosphorylation of the M-phase kinase, Cdk1. In this study, we investigated how inhibitory phosphorylation of Cdk1 by Myt1 kinase regulates premeiotic G2 phase of Drosophila male meiosis. Immature spermatocytes lacking Myt1 activity exhibit two distinct defects: disrupted intercellular bridges (fusomes) and premature centriole disengagement. As a result, the myt1 mutant spermatocytes enter meiosis with multipolar spindles. These myt1 defects can be suppressed by depletion of Cyclin A activity or ectopic expression of Wee1 (a partially redundant Cdk1 inhibitory kinase) and phenocopied by expression of a Cdk1F mutant defective for inhibitory phosphorylation. We therefore conclude that Myt1 inhibition of Cyclin A/Cdk1 is essential for normal fusome behavior and centriole engagement during premeiotic G2 arrest of Drosophila male meiosis. The novel meiotic functions we discovered for Myt1 kinase are spatially and temporally distinct from previously described functions of Myt1 as an inhibitor of Cyclin B/Cdk1 to regulate G2/MI timing. PMID:27170181

  15. The Smad3/Smad4/CDK9 complex promotes renal fibrosis in mice with unilateral ureteral obstruction.

    PubMed

    Qu, Xinli; Jiang, Mengjie; Sun, Yu Bo Yang; Jiang, Xiaoyun; Fu, Ping; Ren, Yi; Wang, Die; Dai, Lie; Caruana, Georgina; Bertram, John F; Nikolic-Paterson, David J; Li, Jinhua

    2015-12-01

    Transforming growth factor-β1 (TGF-β1)/Smad signaling has a central role in the pathogenesis of renal fibrosis. Smad3 and Smad4 are pro-fibrotic, while Smad2 is anti-fibrotic. However, these Smads form heterogeneous complexes, the functions of which are poorly understood. Here we studied Smad complex function in renal fibrosis using the mouse model of unilateral ureteric obstruction. Mice heterozygous for Smad3/4 (Smad3/4(+/-)) exhibited substantial protection from renal fibrosis through day 7 of obstruction, whereas Smad2/3(+/-) and Smad2/4(+/-) mice showed only modest protection. Formation of Smad3/Smad4/CDK9 complexes was an early event following obstruction in wild-type mice, which involved nuclear phosphorylation of the linker regions of Smad3. Significantly, Smad3 or Smad4 deficiency decreased the formation of Smad4/CDK9 or Smad3/CDK9 complex, Smad3 linker phosphorylation, and fibrosis but at different degrees. In vitro, TGF-β1 stimulation of collagen I promoter activity involved formation of Smad3/Smad4/CDK9 complexes, and overexpression of each component gave additive increases in collagen promoter activity. Co-administration of a CDK9 inhibitor and Smad3-specific inhibition achieved better protection from TGF-β1-induced fibrotic response in vitro and renal interstitial fibrosis in vivo. Thus formation of Smad3/Smad4/CDK9 complex drives renal fibrosis during ureteral obstruction. Formation of this complex represents a novel target for antifibrotic therapies.

  16. CDK2 differentially controls normal cell senescence and cancer cell proliferation upon exposure to reactive oxygen species

    SciTech Connect

    Hwang, Chae Young; Lee, Seung-Min; Park, Sung Sup; Kwon, Ki-Sun

    2012-08-17

    Highlights: Black-Right-Pointing-Pointer H{sub 2}O{sub 2} differently adjusted senescence and proliferation in normal and cancer cells. Black-Right-Pointing-Pointer H{sub 2}O{sub 2} exposure transiently decreased PCNA levels in normal cells. Black-Right-Pointing-Pointer H{sub 2}O{sub 2} exposure transiently increased CDK2 activity in cancer cells. Black-Right-Pointing-Pointer p21{sup Cip1} is likely dispensable when H{sub 2}O{sub 2} induces senescence in normal cells. Black-Right-Pointing-Pointer Suggestively, CDK2 and PCNA play critical roles in H{sub 2}O{sub 2}-induced cell fate decision. -- Abstract: Reactive oxygen species modulate cell fate in a context-dependent manner. Sublethal doses of H{sub 2}O{sub 2} decreased the level of proliferating cell nuclear antigen (PCNA) in normal cells (including primary human dermal fibroblasts and IMR-90 cells) without affecting cyclin-dependent kinase 2 (CDK2) activity, leading to cell cycle arrest and subsequent senescence. In contrast, exposure of cancer cells (such as HeLa and MCF7 cells) to H{sub 2}O{sub 2} increased CDK2 activity with no accompanying change in the PCNA level, leading to cell proliferation. A CDK2 inhibitor, CVT-313, prevented H{sub 2}O{sub 2}-induced cancer cell proliferation. These results support the notion that the cyclin/CDK2/p21{sup Cip1}/PCNA complex plays an important role as a regulator of cell fate decisions.

  17. CDK2 activation in mouse epidermis induces keratinocyte proliferation but does not affect skin tumor development.

    PubMed

    Macias, Everardo; Miliani de Marval, Paula L; De Siervi, Adriana; Conti, Claudio J; Senderowicz, Adrian M; Rodriguez-Puebla, Marcelo L

    2008-08-01

    It has been widely assumed that elevated CDK2 kinase activity plays a contributory role in tumorigenesis. We have previously shown that mice overexpressing CDK4 under control of the keratin 5 promoter (K5CDK4 mice) develop epidermal hyperplasia and increased susceptibility to squamous cell carcinomas. In this model, CDK4 overexpression results in increased CDK2 activity associated with the noncatalytic function of CDK4, sequestration of p21(Cip1) and p27(Kip1). Furthermore, we have shown that ablation of Cdk2 reduces Ras-Cdk4 tumorigenesis, suggesting that increased CDK2 activity plays an important role in Ras-mediated tumorigenesis. To investigate this hypothesis, we generated two transgenic mouse models of elevated CDK2 kinase activity, K5Cdk2 and K5Cdk4(D158N) mice. The D158N mutation blocks CDK4 kinase activity without interfering with its binding capability. CDK2 activation via overexpression of CDK4(D158N), but not of CDK2, resulted in epidermal hyperplasia. We observed elevated levels of p21(Cip1) in K5Cdk2, but not in K5Cdk4(D158N), epidermis, suggesting that CDK2 overexpression elicits a p21(Cip1) response to maintain keratinocyte homeostasis. Surprisingly, we found that neither CDK2 overexpression nor the indirect activation of CDK2 enhanced skin tumor development. Thus, although the indirect activation of CDK2 is sufficient to induce keratinocyte hyperproliferation, activation of CDK2 alone does not induce malignant progression in Ras-mediated tumorigenesis.

  18. The Establishment of a Hyperactive Structure Allows the Tumour Suppressor Protein p53 to Function through P-TEFb during Limited CDK9 Kinase Inhibition

    PubMed Central

    Albert, Thomas K.; Antrecht, Claudia; Kremmer, Elisabeth; Meisterernst, Michael

    2016-01-01

    CDK9 is the catalytic subunit of positive elongation factor b (P-TEFb) that controls the transition of RNA polymerase II (RNAPII) into elongation. CDK9 inhibitors block mRNA synthesis and trigger activation of the stress-sensitive p53 protein. This in turn induces transcription of CDKN1A (p21) and other cell cycle control genes. It is presently unclear if and how p53 circumvents a general P-TEFb-requirement when it activates its target genes. Our investigations using a panel of specific inhibitors reason for a critical role of CDK9 also in the case of direct inhibition of the kinase. At the prototypic p21 gene, the activator p53 initially accumulates at the pre-bound upstream enhancer followed—with significant delay—by de novo binding to a secondary enhancer site within the first intron of p21. This is accompanied by recruitment of the RNAPII initiation machinery to both elements. ChIP and functional analyses reason for a prominent role of CDK9 itself and elongation factor complexes PAF1c and SEC involved in pause and elongation control. It appears that the strong activation potential of p53 facilitates gene activation in the situation of global repression of RNAPII transcription. The data further underline the fundamental importance of CDK9 for class II gene transcription. PMID:26745862

  19. Downregulation of microRNA-637 Increases Risk of Hypoxia-Induced Pulmonary Hypertension by Modulating Expression of Cyclin Dependent Kinase 6 (CDK6) in Pulmonary Smooth Muscle Cells

    PubMed Central

    Sang, Hai-yan; Jin, Ying-li; Zhang, Wen-qi; Chen, Li-bo

    2016-01-01

    Background The objective of this study was to investigate the molecular mechanism by which miR-637 interferes with the expression of CDK6, which contributes to the development of pulmonary hypertension (PH) with chronic obstructive pulmonary disease (COPD). Material/Methods We used an online miRNA database to identify CDK6 as a virtual target of miR-637, and validated the hypothesis using luciferase assay. Furthermore, we transfected SMCs with miR-637 mimics and inhibitor, and expression of CDK6 was determined using Western blot and real-time PCR. Results In this study, we identified CDK6 as a target of miR-637 in smooth muscle cells (SMCs), and determined the expression of miR-637 in SMCs from PH patients with COPD and normal controls. We also identified the exact miR-637 binding site in the 3′UTR of CDK6 by using a luciferase reporter system. The mRNA and protein expression levels of CDK6 in SMCs from PH patients with COPD were clearly upregulated compared with the normal controls. Cells exposed to hypoxia also showed notably increased CKD6 mRNA and protein expression levels, and when treated with miR-637 or CDK6 siRNA, this increase in CKD6 expression was clearly attenuated. Additionally, cell viability and cell cycle analysis showed that hypoxia markedly increased viability of SMCs by causing an accumulation in S phase, which was relieved by the introduction of miR-637 or CDK6 siRNA. Conclusions Our study proved that the CDK6 gene is a target of miR-637, and demonstrated the regulatory association between miR-637 and CDK6, suggesting a possible therapeutic target for PH, especially in patients with COPD. PMID:27794186

  20. Kinome-wide RNA interference screen reveals a role for PDK1 in acquired resistance to CDK4/6 inhibition in ER-positive breast cancer.

    PubMed

    Jansen, Valerie M; Bhola, Neil E; Bauer, Joshua A; Formisano, Luigi; Lee, Kyung-Min; Hutchinson, Katherine E; Witkiewicz, Agnieszka K; Moore, Preston D; Estrada, Monica Valeria; Sanchez, Violeta; Ericsson, Paula G; Sanders, Melinda; Pohlmann, Paula R; Pishvaian, Michael J; Riddle, David A; Wei, Wenyi; Dugger, Teresa C; Knudsen, Erik; Arteaga, Carlos L

    2017-03-01

    To discover mechanisms of resistance to CDK4/6 inhibitors, we used a kinome-wide siRNA screen to identify kinases that, when downregulated, promote sensitivity to ribociclib. We identified 3-phosphoinositide dependent protein kinase 1 (PDK1) as the top siRNA that sensitized ER+ MCF-7 cells to ribociclib. Pharmacological inhibition of PDK1 with GSK2334470 in combination with ribociclib or palbociclib, synergistically inhibited proliferation and increased apoptosis in a panel of ER+ breast cancer cell lines. Ribociclib-resistant MCF-7, T47D and HCC1428 cells, selected after chronic drug exposure, displayed increased levels of PDK1, P-RSK2, P-AKT and P-S6 compared to parental drug-sensitive cells. Cell cycle analysis revealed that CDK4/6 inhibition failed to induce G1 arrest, a reduction in S phase, and senescence in ribociclib-resistant cells, suggesting an upregulation of S-phase cyclins/CDKs. The resistant cells exhibited significantly higher levels of P-CDK2, cyclin A, cyclin D1, cyclin E and S477/T479 P-AKT, a CDK2-dependent phosphorylation site within AKT required for full kinase activity and limited to the S-phase of the cell cycle. Treatment with GSK2334470 or the CDK2 inhibitor dinaciclib re-sensitized ribociclib-resistant cells to CDK4/6 inhibitors; however, ribociclib/GSK2334470 inhibited the ribociclib-resistant cells more potently than ribociclib/dinaciclib. Ribociclib/GSK2334470 but not ribociclib/dinaciclib completely abrogated P-Rb, P-S6, P-RSK2, P-CDK2, cyclin A, cyclin D1 and cyclin E expression. Further, ribociclib in combination with GSK2334470 or the PI3Kα inhibitor alpelisib induced regression of MCF-7 xenografts. Finally, primary ER+ tumors displayed increased PDK1, P-S6 and cyclin D1 levels after short treatment with palbociclib. These data support a role for PI3K/PDK1 in mediating acquired resistance to CDK4/6 inhibitors.

  1. RBPJ maintains brain tumor–initiating cells through CDK9-mediated transcriptional elongation

    PubMed Central

    Xie, Qi; Wu, Qiulian; Kim, Leo; Miller, Tyler E.; Liau, Brian B.; Mack, Stephen C.; Yang, Kailin; Factor, Daniel C.; Fang, Xiaoguang; Huang, Zhi; Zhou, Wenchao; Alazem, Kareem; Wang, Xiuxing; Bernstein, Bradley E.; Bao, Shideng; Rich, Jeremy N.

    2016-01-01

    Glioblastomas co-opt stem cell regulatory pathways to maintain brain tumor–initiating cells (BTICs), also known as cancer stem cells. NOTCH signaling has been a molecular target in BTICs, but NOTCH antagonists have demonstrated limited efficacy in clinical trials. Recombining binding protein suppressor of hairless (RBPJ) is considered a central transcriptional mediator of NOTCH activity. Here, we report that pharmacologic NOTCH inhibitors were less effective than targeting RBPJ in suppressing tumor growth. While NOTCH inhibitors decreased canonical NOTCH gene expression, RBPJ regulated a distinct profile of genes critical to BTIC stemness and cell cycle progression. RBPJ was preferentially expressed by BTICs and required for BTIC self-renewal and tumor growth. MYC, a key BTIC regulator, bound the RBPJ promoter and treatment with a bromodomain and extraterminal domain (BET) family bromodomain inhibitor decreased MYC and RBPJ expression. Proteomic studies demonstrated that RBPJ binds CDK9, a component of positive transcription elongation factor b (P-TEFb), to target gene promoters, enhancing transcriptional elongation. Collectively, RBPJ links MYC and transcriptional control through CDK9, providing potential nodes of fragility for therapeutic intervention, potentially distinct from NOTCH. PMID:27322055

  2. RBPJ maintains brain tumor-initiating cells through CDK9-mediated transcriptional elongation.

    PubMed

    Xie, Qi; Wu, Qiulian; Kim, Leo; Miller, Tyler E; Liau, Brian B; Mack, Stephen C; Yang, Kailin; Factor, Daniel C; Fang, Xiaoguang; Huang, Zhi; Zhou, Wenchao; Alazem, Kareem; Wang, Xiuxing; Bernstein, Bradley E; Bao, Shideng; Rich, Jeremy N

    2016-07-01

    Glioblastomas co-opt stem cell regulatory pathways to maintain brain tumor-initiating cells (BTICs), also known as cancer stem cells. NOTCH signaling has been a molecular target in BTICs, but NOTCH antagonists have demonstrated limited efficacy in clinical trials. Recombining binding protein suppressor of hairless (RBPJ) is considered a central transcriptional mediator of NOTCH activity. Here, we report that pharmacologic NOTCH inhibitors were less effective than targeting RBPJ in suppressing tumor growth. While NOTCH inhibitors decreased canonical NOTCH gene expression, RBPJ regulated a distinct profile of genes critical to BTIC stemness and cell cycle progression. RBPJ was preferentially expressed by BTICs and required for BTIC self-renewal and tumor growth. MYC, a key BTIC regulator, bound the RBPJ promoter and treatment with a bromodomain and extraterminal domain (BET) family bromodomain inhibitor decreased MYC and RBPJ expression. Proteomic studies demonstrated that RBPJ binds CDK9, a component of positive transcription elongation factor b (P-TEFb), to target gene promoters, enhancing transcriptional elongation. Collectively, RBPJ links MYC and transcriptional control through CDK9, providing potential nodes of fragility for therapeutic intervention, potentially distinct from NOTCH.

  3. Inhibition of CDK4/6 protects against radiation-induced intestinal injury in mice

    PubMed Central

    Wei, Liang; Leibowitz, Brian J.; Wang, Xinwei; Epperly, Michael; Greenberger, Joel; Zhang, Lin

    2016-01-01

    Radiotherapy causes dose-limiting toxicity and long-term complications in rapidly renewing tissues, including the gastrointestinal tract. Currently, there is no FDA-approved agent for the prevention or treatment of radiation-induced intestinal injury. In this study, we have shown that PD 0332991 (PD), an FDA-approved selective inhibitor of cyclin-dependent kinase 4/6 (CDK4/6), prevents radiation-induced lethal intestinal injury in mice. Treating mice with PD or a structurally distinct CDK4/6 inhibitor prior to radiation blocked proliferation and crypt apoptosis and improved crypt regeneration. PD treatment also enhanced LGR5+ stem cell survival and regeneration after radiation. PD was an on-target inhibitor of RB phosphorylation and blocked G1/S transition in the intestinal crypts. PD treatment strongly but reversibly inhibited radiation-induced p53 activation, which blocked p53-upregulated modulator of apoptosis–dependent (PUMA-dependent) apoptosis without affecting p21-dependent suppression of DNA damage accumulation, with a repair bias toward nonhomologous end joining. Further, deletion of PUMA synergized with PD treatment for even greater intestinal radioprotection. Our results demonstrate that the cell cycle critically regulates the DNA damage response and survival of intestinal stem cells and support the concept that pharmacological quiescence is a potentially highly effective and selective strategy for intestinal radioprotection. PMID:27701148

  4. Overexpression of a novel activator of PAK4, the CDK5 kinase-associated protein CDK5RAP3, promotes hepatocellular carcinoma metastasis.

    PubMed

    Mak, Grace Wing-Yan; Chan, Mandy Man-Lok; Leong, Veronica Yee-Law; Lee, Joyce Man-Fong; Yau, Tai-On; Ng, Irene Oi-Lin; Ching, Yick-Pang

    2011-04-15

    The CDK5 kinase regulatory subunit-associated protein 3 (CDK5RAP3 or C53/LZAP) regulates apoptosis induced by genotoxic stress. Although CDK5RAP3 has been implicated in cancer progression, its exact role in carcinogenesis is not well established. In this article, we report that CDK5RAP3 has an important prometastatic function in hepatocarcinogenesis. An examination of human hepatocellular carcinoma (HCC) samples revealed at least twofold overexpression of CDK5RAP3 transcripts in 58% (39/67) of HCC specimens when compared with corresponding nontumorous livers. CDK5RAP3 overexpression was associated with more aggressive biological behavior. In HCC cell lines, stable overexpression of CDK5RAP3 promoted, and small interfering RNA-mediated knockdown inhibited, tumorigenic activity and metastatic potential. We found that overexpression of CDK5RAP3 and p21-activated protein kinase 4 (PAK4) correlated in human HCCs, and that CDK5RAP3 was a novel binding partner of PAK4, and this binding enhanced PAK4 activity. siRNA-mediated knockdown of PAK4 in CDK5RAP3-expressing HCC cells reversed the enhanced cell invasiveness mediated by CDK5RAP3 overexpression, implying that PAK4 is essential for CDK5RAP3 function. Taken together, our findings reveal that CDK5RAP3 is widely overexpressed in HCC and that overexpression of CDK5RAP3 promotes HCC metastasis through PAK4 activation.

  5. Knockdown of Expression of Cdk5 or p35 (a Cdk5 Activator) Results in Podocyte Apoptosis

    PubMed Central

    Zheng, Ya-Li; Zhang, Xia; Fu, Hai-Xia; Guo, Mei; Shukla, Varsha; Amin, Niranjana D.; E, Jing; Bao, Li; Luo, Hong-Yan; Li, Bo; Lu, Xiao-Hua; Gao, Yong-Cai

    2016-01-01

    Podocytes are terminally differentiated glomerular epithelial cells. Podocyte loss has been found in many renal diseases. Cdk5 is a cyclin-dependent protein kinase which is predominantly regulated by p35. To study the role of Cdk5/p35 in podocyte survival, we first applied western blotting (WB) analysis to confirm the time-course expression of Cdk5 and p35 during kidney development and in cultured immortalized mouse podocytes. We also demonstrated that p35 plays an important role in promoting podocyte differentiation by overexpression of p35 in podocytes. To deregulate the expression of Cdk5 or p35 in mouse podocytes, we used RNAi and analyzed cell function and apoptosis assaying for podocyte specific marker Wilms Tumor 1 (WT1) and cleaved caspase 3, respectively. We also counted viable cells using cell counting kit-8. We found that depletion of Cdk5 causes decreased expression of WT1 and apoptosis. It is noteworthy, however, that downregulation of p35 reduced Cdk5 activity, but had no effect on cleaved caspase 3 expression. It did, however, reduce expression of WT1, a transcription factor, and produced podocyte dysmorphism. On the other hand increased apoptosis could be detected in p35-deregulated podocytes using the TUNEL analysis and immunofluorescent staining with cleaved caspase3 antibody. Viability of podocytes was decreased in both Cdk5 and p35 knockdown cells. Knocking down Cdk5 or p35 gene by RNAi does not affect the cycline I expression, another Cdk5 activator in podocyes. We conclude that Cdk5 and p35 play a crucial role in maintaining podocyte differentiation and survival, and suggest these proteins as targets for therapeutic intervention in podocyte-damaged kidney diseases. PMID:27479491

  6. Genetic and pharmacological inhibition of CDK9 drives neutrophil apoptosis to resolve inflammation in zebrafish in vivo

    PubMed Central

    Hoodless, Laura J.; Lucas, Christopher D.; Duffin, Rodger; Denvir, Martin A.; Haslett, Christopher; Tucker, Carl S.; Rossi, Adriano G.

    2016-01-01

    Neutrophilic inflammation is tightly regulated and subsequently resolves to limit tissue damage and promote repair. When the timely resolution of inflammation is dysregulated, tissue damage and disease results. One key control mechanism is neutrophil apoptosis, followed by apoptotic cell clearance by phagocytes such as macrophages. Cyclin-dependent kinase (CDK) inhibitor drugs induce neutrophil apoptosis in vitro and promote resolution of inflammation in rodent models. Here we present the first in vivo evidence, using pharmacological and genetic approaches, that CDK9 is involved in the resolution of neutrophil-dependent inflammation. Using live cell imaging in zebrafish with labelled neutrophils and macrophages, we show that pharmacological inhibition, morpholino-mediated knockdown and CRISPR/cas9-mediated knockout of CDK9 enhances inflammation resolution by reducing neutrophil numbers via induction of apoptosis after tailfin injury. Importantly, knockdown of the negative regulator La-related protein 7 (LaRP7) increased neutrophilic inflammation. Our data show that CDK9 is a possible target for controlling resolution of inflammation. PMID:27833165

  7. CDK4 is an essential insulin effector in adipocytes

    PubMed Central

    Lagarrigue, Sylviane; Lopez-Mejia, Isabel C.; Denechaud, Pierre-Damien; Escoté, Xavier; Castillo-Armengol, Judit; Jimenez, Veronica; Chavey, Carine; Giralt, Albert; Lai, Qiuwen; Zhang, Lianjun; Martinez-Carreres, Laia; Delacuisine, Brigitte; Annicotte, Jean-Sébastien; Blanchet, Emilie; Huré, Sébastien; Abella, Anna; Tinahones, Francisco J.; Vendrell, Joan; Dubus, Pierre; Bosch, Fatima; Kahn, C. Ronald; Fajas, Lluis

    2015-01-01

    Insulin resistance is a fundamental pathogenic factor that characterizes various metabolic disorders, including obesity and type 2 diabetes. Adipose tissue contributes to the development of obesity-related insulin resistance through increased release of fatty acids, altered adipokine secretion, and/or macrophage infiltration and cytokine release. Here, we aimed to analyze the participation of the cyclin-dependent kinase 4 (CDK4) in adipose tissue biology. We determined that white adipose tissue (WAT) from CDK4-deficient mice exhibits impaired lipogenesis and increased lipolysis. Conversely, lipolysis was decreased and lipogenesis was increased in mice expressing a mutant hyperactive form of CDK4 (CDK4R24C). A global kinome analysis of CDK4-deficient mice following insulin stimulation revealed that insulin signaling is impaired in these animals. We determined that insulin activates the CCND3-CDK4 complex, which in turn phosphorylates insulin receptor substrate 2 (IRS2) at serine 388, thereby creating a positive feedback loop that maintains adipocyte insulin signaling. Furthermore, we found that CCND3 expression and IRS2 serine 388 phosphorylation are increased in human obese subjects. Together, our results demonstrate that CDK4 is a major regulator of insulin signaling in WAT. PMID:26657864

  8. Human CDK18 promotes replication stress signaling and genome stability

    PubMed Central

    Barone, Giancarlo; Staples, Christopher J.; Ganesh, Anil; Patterson, Karl W.; Bryne, Dominic P.; Myers, Katie N.; Patil, Abhijit A.; Eyers, Claire E.; Maslen, Sarah; Skehel, J. Mark; Eyers, Patrick A.; Collis, Spencer J.

    2016-01-01

    Cyclin-dependent kinases (CDKs) coordinate cell cycle checkpoints with DNA repair mechanisms that together maintain genome stability. However, the myriad mechanisms that can give rise to genome instability are still to be fully elucidated. Here, we identify CDK18 (PCTAIRE 3) as a novel regulator of genome stability, and show that depletion of CDK18 causes an increase in endogenous DNA damage and chromosomal abnormalities. CDK18-depleted cells accumulate in early S-phase, exhibiting retarded replication fork kinetics and reduced ATR kinase signaling in response to replication stress. Mechanistically, CDK18 interacts with RAD9, RAD17 and TOPBP1, and CDK18-deficiency results in a decrease in both RAD17 and RAD9 chromatin retention in response to replication stress. Importantly, we demonstrate that these phenotypes are rescued by exogenous CDK18 in a kinase-dependent manner. Collectively, these data reveal a rate-limiting role for CDK18 in replication stress signalling and establish it as a novel regulator of genome integrity. PMID:27382066

  9. Human CDK18 promotes replication stress signaling and genome stability.

    PubMed

    Barone, Giancarlo; Staples, Christopher J; Ganesh, Anil; Patterson, Karl W; Bryne, Dominic P; Myers, Katie N; Patil, Abhijit A; Eyers, Claire E; Maslen, Sarah; Skehel, J Mark; Eyers, Patrick A; Collis, Spencer J

    2016-10-14

    Cyclin-dependent kinases (CDKs) coordinate cell cycle checkpoints with DNA repair mechanisms that together maintain genome stability. However, the myriad mechanisms that can give rise to genome instability are still to be fully elucidated. Here, we identify CDK18 (PCTAIRE 3) as a novel regulator of genome stability, and show that depletion of CDK18 causes an increase in endogenous DNA damage and chromosomal abnormalities. CDK18-depleted cells accumulate in early S-phase, exhibiting retarded replication fork kinetics and reduced ATR kinase signaling in response to replication stress. Mechanistically, CDK18 interacts with RAD9, RAD17 and TOPBP1, and CDK18-deficiency results in a decrease in both RAD17 and RAD9 chromatin retention in response to replication stress. Importantly, we demonstrate that these phenotypes are rescued by exogenous CDK18 in a kinase-dependent manner. Collectively, these data reveal a rate-limiting role for CDK18 in replication stress signalling and establish it as a novel regulator of genome integrity.

  10. Rarity of CDK4 germline mutations in familial melanoma.

    PubMed

    Goldstein, A M; Chidambaram, A; Halpern, A; Holly, E A; Guerry IV, D; Sagebiel, R; Elder, D E; Tucker, M A

    2002-02-01

    To date, two genes have been implicated in melanoma pathogenesis. The first, CDKN2A, is a tumour suppressor gene with germline mutations detected in 20% of melanoma-prone families. The second, CDK4, is an oncogene with co-segregating germline mutations detected in only three kindreds worldwide. We examined 16 American melanoma-prone families for mutations in all coding exons of CDK4 and screened additional members of two previously reported families with the Arg24Cys germline CDK4 mutation to evaluate the penetrance of the mutation. No new CDK4 mutations were identified. In the two Arg24Cys families, the penetrance was estimated to be 63%. Overall, 12 out of 12 invasive melanoma patients, none out of one in situ melanoma patient, five out of 13 dysplastic naevi patients, two out of 15 unaffected family members, and none out of 10 spouses carried the Arg24Cys mutation. Dysplastic naevi did not strongly co-segregate with the Arg24Cys mutation. Thus the phenotype observed in melanoma-prone CDK4 families appears to be more complex than just the CDK4 mutation. Both genetic and environmental factors are likely to contribute to the occurrence of melanoma and dysplastic naevi in these families. In summary, although CDK4 is a melanoma susceptibility gene, it plays a minor role in hereditary melanoma.

  11. Development of highly potent and selective diaminothiazole inhibitors of cyclin-dependent kinases

    PubMed Central

    Schonbrunn, Ernst; Betzi, Stephane; Alam, Riazul; Martin, Mathew P.; Becker, Andreas; Han, Huijong; Francis, Rawle; Chakrasali, Ramappa; Jakkaraj, Sudhakar; Kazi, Aslamuzzaman; Sebti, Said M.; Cubitt, Christopher L.; Gebhard, Anthony W.; Hazlehurst, Lori A.; Tash, Joseph S.; Georg, Gunda I.

    2013-01-01

    Cyclin-dependent kinases (CDKs) are serine/threonine protein kinases that act as key regulatory elements in cell cycle progression. We describe the development of highly potent diaminothiazole inhibitors of CDK2 (IC50 = 0.0009 – 0.0015 µM) from a single hit compound with weak inhibitory activity (IC50 = 15 µM), discovered by high-throughput screening. Structure-based design was performed using 35 co-crystal structures of CDK2 liganded with distinct analogues of the parent compound. The profiling of compound 51 against a panel of 339 kinases revealed high selectivity for CDKs, with preference for CDK2 and CDK5 over CDK9, CDK1, CDK4 and CDK6. Compound 51 inhibited the proliferation of 13 out of 15 cancer cell lines with IC50 values between 0.27 and 6.9 µM, which correlated with the complete suppression of retinoblastoma phosphorylation and the onset of apoptosis. Combined, the results demonstrate the potential of this new inhibitors series for further development into CDK-specific chemical probes or therapeutics. PMID:23600925

  12. Cdk5 and the mystery of synaptic vesicle endocytosis.

    PubMed

    Nguyen, Chan; Bibb, James A

    2003-11-24

    Regulation of endocytosis by protein phosphorylation and dephosphorylation is critical to synaptic vesicle recycling. Two groups have now identified the neuronal kinase Cdk5 (cyclin-dependent kinase 5) as an important regulator of this process. Robinson and coworkers recently demonstrated that Cdk5 is necessary for synaptic vesicle endocytosis (SVE) (Tan et al., 2003), whereas a new report in this issue claims that Cdk5 negatively regulates SVE (Tomizawa et al., 2003). Careful examination of the data reveals a model that helps resolve the apparently contradictory nature of these reports.

  13. Discovery of 4,6-disubstituted pyrimidines as potent inhibitors of the heat shock factor 1 (HSF1) stress pathway and CDK9† †The authors declare the following competing financial interest(s): C. Rye, N. Chessum, L. Zani, M. Cheeseman, F. Raynaud, A. Hayes, A. Henley, E. de Billy, C. Lynch, S. Sharp, R. te Poele, L. O'Fee, P. Workman, and K. Jones are or have been employees of The Institute of Cancer Research which has a commercial interest in the development of HSF1 inhibitors. Authors who are or have been employed by The Institute of Cancer Research are subject to a “Rewards to Discoverers Scheme” which may reward contributors to a program that is subsequently licensed. ‡ ‡Electronic supplementary information (ESI) available: Experimental and assay procedures, crystallographic data, compound characterisation data. See DOI: 10.1039/c6md00159a Click here for additional data file.

    PubMed Central

    Rye, Carl S.; Chessum, Nicola E. A.; Lamont, Scott; Pike, Kurt G.; Faulder, Paul; Demeritt, Julie; Kemmitt, Paul; Tucker, Julie; Zani, Lorenzo; Cheeseman, Matthew D.; Isaac, Rosie; Goodwin, Louise; Boros, Joanna; Raynaud, Florence; Hayes, Angela; Henley, Alan T.; de Billy, Emmanuel; Lynch, Christopher J.; Sharp, Swee Y.; te Poele, Robert; Fee, Lisa O’; Foote, Kevin M.; Green, Stephen

    2016-01-01

    Heat shock factor 1 (HSF1) is a transcription factor that plays key roles in cancer, including providing a mechanism for cell survival under proteotoxic stress. Therefore, inhibition of the HSF1-stress pathway represents an exciting new opportunity in cancer treatment. We employed an unbiased phenotypic screen to discover inhibitors of the HSF1-stress pathway. Using this approach we identified an initial hit (1) based on a 4,6-pyrimidine scaffold (2.00 μM). Optimisation of cellular SAR led to an inhibitor with improved potency (25, 15 nM) in the HSF1 phenotypic assay. The 4,6-pyrimidine 25 was also shown to have high potency against the CDK9 enzyme (3 nM). PMID:27746890

  14. Signal transduction pathways that contribute to CDK1/cyclin B activation during the first mitotic division in sea urchin embryos.

    PubMed

    Salaün, Patrick; Le Breton, Magali; Morales, Julia; Bellé, Robert; Boulben, Sandrine; Mulner-Lorillon, Odile; Cormier, Patrick

    2004-06-10

    In sea urchins, fertilization triggers a rapid rise in protein synthesis necessary for activation of CDK1/cyclin B, the universal cell cycle regulator. It has been shown that FRAP/mTOR is required for eIF4E release from the translational repressor 4E-BP, a process that occurs upstream of de novo cyclin B synthesis. Here, we investigate whether PI 3-kinase acts independently or upstream from FRAP/mTOR in the signal transduction pathway that links fertilization to the activation of the CDK1/cyclin B complex in sea urchin egg. We found that wortmannin, a potent inhibitor of PI 3-kinase, partially inhibited the global increase in protein synthesis triggered by fertilization. Furthermore, wortmannin treatment induced partial inhibition of cyclin B translation triggered by fertilization, in correlation with an intermediate effect of the drug on 4E-BP degradation and on the dissociation of the 4E-BP/eIF4E complex induced by fertilization. Our results presented here suggest that PI 3-kinase activity is required for completion of mitotic divisions of the sea urchin embryo. Incubation of eggs with wortmannin or microinjection of wortmannin or LY 294002 affects drastically mitotic divisions induced by fertilization. In addition, we found that wortmannin treatment inhibits dephosphorylation of the tyrosine inhibitory site of CDK1. Taken together, these data suggest that PI 3-kinase acts upstream of at least two independent targets that function in the CDK1/cyclin B activation triggered by fertilization of sea urchin oocytes. We discuss the significance of these results concerning the cascade of reactions that impinge upon the activation of the CDK1/cyclin B complex that follows sea urchin oocyte fertilization.

  15. HBx-dependent cell cycle deregulation involves interaction with cyclin E/A-cdk2 complex and destabilization of p27Kip1.

    PubMed

    Mukherji, Atish; Janbandhu, Vaibhao C; Kumar, Vijay

    2007-01-01

    The HBx (X protein of hepatitis B virus) is a promiscuous transactivator implicated to play a key role in hepatocellular carcinoma. However, HBx-regulated molecular events leading to deregulation of cell cycle or establishment of a permissive environment for hepatocarcinogenesis are not fully understood. Our cell culture-based studies suggested that HBx had a profound effect on cell cycle progression even in the absence of serum. HBx presence led to an early and sustained level of cyclin-cdk2 complex during the cell cycle combined with increased protein kinase activity of cdk2 heralding an early proliferative signal. The increased cdk2 activity also led to an early proteasomal degradation of p27(Kip1) that could be reversed by HBx-specific RNA interference and blocked by a chemical inhibitor of cdk2 or the T187A mutant of p27. Further, our co-immunoprecipitation and in vitro binding studies with recombinant proteins suggested a direct interaction between HBx and the cyclin E/A-cdk2 complex. Interference with different signalling cascades known to be activated by HBx suggested a constitutive requirement of Src kinases for the association of HBx with these complexes. Notably, the HBx mutant that did not interact with cyclin E/A failed to destabilize p27(Kip1) or deregulate the cell cycle. Thus HBx appears to deregulate the cell cycle by interacting with the key cell cycle regulators independent of its well-established role in transactivation.

  16. CDK9-dependent transcriptional elongation in the innate interferon-stimulated gene response to respiratory syncytial virus infection in airway epithelial cells.

    PubMed

    Tian, Bing; Zhao, Yingxin; Kalita, Mridul; Edeh, Chukwudi B; Paessler, Slobodan; Casola, Antonella; Teng, Michael N; Garofalo, Roberto P; Brasier, Allan R

    2013-06-01

    Respiratory syncytial virus (RSV) is a negative-sense single-stranded RNA virus responsible for lower respiratory tract infections. During infection, the presence of double-stranded RNA (dsRNA) activates the interferon (IFN) regulatory factor 3 (IRF3) transcription factor, an event triggering expression of immediate early, IFN-stimulated genes (ISGs). We examine the role of transcriptional elongation in control of IRF3-dependent ISG expression. RSV infection induces ISG54, ISG56, and CIG5 gene expression in an IRF3-dependent manner demonstrated by IRF3 small interfering RNA (siRNA) silencing in both A549 epithelial cells and IRF3(-/-) MEFs. ISG expression was mediated by the recruitment of IRF3, CDK9, polymerase II (Pol II), and phospho-Ser(2) carboxy-terminal domain (CTD) Pol II to the IFN-stimulated response element (ISRE) binding sites of the IRF3-dependent ISG promoters in native chromatin. We find that RSV infection enhances the activated fraction of cyclin-dependent kinase 9 (CDK9) by promoting its association with bromodomain 4 (BRD4) and disrupting its association with the inhibitory 7SK small nuclear RNA. The requirement of CDK9 activity for ISG expression was shown by siRNA-mediated silencing of CDK9 and by a selective CDK9 inhibitor in A549 cells. In contrast, RSV-induced beta interferon (IFN-β) expression is not influenced by CDK9 inhibition. Using transcript-selective quantitative real-time reverse transcription-PCR (Q-RT-PCR) assays for the ISG54 gene, we observed that RSV induces transition from short to fully spliced mRNA transcripts and that this transition is blocked by CDK9 inhibition in both A549 and primary human small airway epithelial cells. These data indicate that transcription elongation plays a major role in RSV-induced ISG expression and is mediated by IRF3-dependent recruitment of activated CDK9. CDK9 activity may be a target for immunomodulation in RSV-induced lung disease.

  17. Cdk5 regulates PSD-95 ubiquitination in neurons

    PubMed Central

    Bianchetta, Michael J.; Lam, TuKiet T.; Jones, Stephen N.; Morabito, Maria A.

    2011-01-01

    The kinase Cdk5 and its activator p35 have been implicated in drug addiction, neurodegenerative diseases such as Alzheimer’s, learning and memory, and synapse maturation and plasticity. However the molecular mechanisms by which Cdk5 regulates synaptic plasticity are still unclear. PSD-95 is a major postsynaptic scaffolding protein of glutamatergic synapses that regulates synaptic strength and plasticity. PSD-95 is ubiquitinated by the Ubiquitin E3 Ligase Mdm2, and rapid and transient PSD-95 ubiquitination has been implicated in NMDA receptor-induced AMPA receptor endocytosis. Here we demonstrate that genetic or pharmacological reduction of Cdk5 activity increases the interaction of Mdm2 with PSD-95 and enhances PSD-95 ubiquitination without affecting PSD-95 protein levels in vivo in mice, suggesting a non-proteolytic function of ubiquitinated PSD-95 at synapses. We show that PSD-95 ubiquitination correlates with increased interaction with β-adaptin, a subunit of the clathrin adaptor protein complex AP-2. This interaction is increased by genetic reduction of Cdk5 activity or NMDA receptor stimulation and is dependent on Mdm2. Together these results support a function for Cdk5 in regulating PSD-95 ubiqutination and its interaction with AP-2 and suggest a mechanism by which PSD-95 may regulate NMDA receptor-induced AMPA receptor endocytosis. PMID:21849563

  18. Dissection of Cdk1–cyclin complexes in vivo

    PubMed Central

    Ear, Po Hien; Booth, Michael J.; Abd-Rabbo, Diala; Kowarzyk Moreno, Jacqueline; Hall, Conrad; Chen, Daici; Vogel, Jackie; Michnick, Stephen W.

    2013-01-01

    Cyclin-dependent kinases (Cdks) are regulatory enzymes with temporal and spatial selectivity for their protein substrates that are governed by cell cycle-regulated cyclin subunits. Specific cyclin–Cdk complexes bind to and phosphorylate target proteins, coupling their activity to cell cycle states. The identification of specific cyclin–Cdk substrates is challenging and so far, has largely been achieved through indirect correlation or use of in vitro techniques. Here, we use a protein-fragment complementation assay based on the optimized yeast cytosine deaminase to systematically identify candidate substrates of budding yeast Saccharomyces cerevisiae Cdk1 and show dependency on one or more regulatory cyclins. We identified known and candidate cyclin dependencies for many predicted protein kinase Cdk1 targets and showed elusory Clb3–Cdk1-specific phosphorylation of γ-tubulin, thus establishing the timing of this event in controlling assembly of the mitotic spindle. Our strategy can be generally applied to identify substrates and accessory subunits of multisubunit protein complexes. PMID:24019491

  19. Non-CDK-bound p27 (p27{sup NCDK}) is a marker for cell stress and is regulated through the Akt/PKB and AMPK-kinase pathways

    SciTech Connect

    Bjoerklund, Mia A.; Vaahtomeri, Kari; Peltonen, Karita; Viollet, Benoit; Maekelae, Tomi P.; Band, Arja M.; Laiho, Marikki

    2010-03-10

    p27Kip1 (p27) tumour suppressor protein is regulated by multiple mechanisms including its turnover, localization and complex formation with its key targets, cyclin-dependent kinases (CDK) and cyclins. We have earlier shown that p27 exists in cells in a form that lacks cyclin/CDK interactions (hence non-CDK, p27{sup NCDK}) but the nature of p27{sup NCDK} has remained unresolved. Here we demonstrate that the epitope recognized by the p27{sup NCDK}-specific antibody resides in the p27 CDK-interaction domain and that p27{sup NCDK} is regulated by the balance of CDK inhibitors and cyclin-CDK complexes. We find that signalling by cellular growth promoting pathways, like phosphoinositol 3-kinase (PI3K) and specifically Akt/PKB kinase, inversely correlates with p27{sup NCDK} levels whereas total p27 levels are unaffected. p27{sup NCDK}, but not total p27, is increased by cellular perturbations such as hyperosmotic and metabolic stress and activation of AMP-activated protein kinase (AMPK). By using AMPK catalytic subunit proficient and deficient cells we further demonstrate that the AMPK pathway governs p27{sup NCDK} responses to metabolic stress and PI3K inhibition. These results indicate that p27{sup NCDK} is a sensitive marker for both cell stress and proliferation over and above p27 and is regulated by Akt/PKB and AMPK pathways.

  20. Phosphorylation of XIAP by CDK1–cyclin-B1 controls mitotic cell death

    PubMed Central

    Hou, Ying; Allan, Lindsey A.

    2017-01-01

    ABSTRACT Regulation of cell death is crucial for the response of cancer cells to drug treatments that cause arrest in mitosis, and is likely to be important for protection against chromosome instability in normal cells. Prolonged mitotic arrest can result in cell death by activation of caspases and the induction of apoptosis. Here, we show that X-linked inhibitor of apoptosis (XIAP) plays a key role in the control of mitotic cell death. Ablation of XIAP expression sensitises cells to prolonged mitotic arrest caused by a microtubule poison. XIAP is stable during mitotic arrest, but its function is controlled through phosphorylation by the mitotic kinase CDK1–cyclin-B1 at S40. Mutation of S40 to a phosphomimetic residue (S40D) inhibits binding to activated effector caspases and abolishes the anti-apoptotic function of XIAP, whereas a non-phosphorylatable mutant (S40A) blocks apoptosis. By performing live-cell imaging, we show that phosphorylation of XIAP reduces the threshold for the onset of cell death in mitosis. This work illustrates that mitotic cell death is a form of apoptosis linked to the progression of mitosis through control by CDK1–cyclin-B1. PMID:27927753

  1. CDK1 substitutes for mTOR kinase to activate mitotic cap-dependent protein translation

    PubMed Central

    Shuda, Masahiro; Velásquez, Celestino; Cheng, Erdong; Cordek, Daniel G.; Kwun, Hyun Jin; Chang, Yuan; Moore, Patrick S.

    2015-01-01

    Mitosis is commonly thought to be associated with reduced cap-dependent protein translation. Here we show an alternative control mechanism for maintaining cap-dependent translation during mitosis revealed by a viral oncoprotein, Merkel cell polyomavirus small T (MCV sT). We find MCV sT to be a promiscuous E3 ligase inhibitor targeting the anaphase-promoting complex, which increases cell mitogenesis. MCV sT binds through its Large T stabilization domain region to cell division cycle protein 20 (Cdc20) and, possibly, cdc20 homolog 1 (Cdh1) E3 ligase adapters. This activates cyclin-dependent kinase 1/cyclin B1 (CDK1/CYCB1) to directly hyperphosphorylate eukaryotic initiation factor 4E (eIF4E)-binding protein (4E-BP1) at authentic sites, generating a mitosis-specific, mechanistic target of rapamycin (mTOR) inhibitor-resistant δ phospho-isoform not present in G1-arrested cells. Recombinant 4E-BP1 inhibits capped mRNA reticulocyte translation, which is partially reversed by CDK1/CYCB1 phosphorylation of 4E-BP1. eIF4G binding to the eIF4E–m7GTP cap complex is resistant to mTOR inhibition during mitosis but sensitive during interphase. Flow cytometry, with and without sT, reveals an orthogonal pH3S10+ mitotic cell population having higher inactive p4E-BP1T37/T46+ saturation levels than pH3S10– interphase cells. Using a Click-iT flow cytometric assay to directly measure mitotic protein synthesis, we find that most new protein synthesis during mitosis is cap-dependent, a result confirmed using the eIF4E/4G inhibitor drug 4E1RCat. For most cell lines tested, cap-dependent translation levels were generally similar between mitotic and interphase cells, and the majority of new mitotic protein synthesis was cap-dependent. These findings suggest that mitotic cap-dependent translation is generally sustained during mitosis by CDK1 phosphorylation of 4E-BP1 even under conditions of reduced mTOR signaling. PMID:25883264

  2. CDK1 substitutes for mTOR kinase to activate mitotic cap-dependent protein translation.

    PubMed

    Shuda, Masahiro; Velásquez, Celestino; Cheng, Erdong; Cordek, Daniel G; Kwun, Hyun Jin; Chang, Yuan; Moore, Patrick S

    2015-05-12

    Mitosis is commonly thought to be associated with reduced cap-dependent protein translation. Here we show an alternative control mechanism for maintaining cap-dependent translation during mitosis revealed by a viral oncoprotein, Merkel cell polyomavirus small T (MCV sT). We find MCV sT to be a promiscuous E3 ligase inhibitor targeting the anaphase-promoting complex, which increases cell mitogenesis. MCV sT binds through its Large T stabilization domain region to cell division cycle protein 20 (Cdc20) and, possibly, cdc20 homolog 1 (Cdh1) E3 ligase adapters. This activates cyclin-dependent kinase 1/cyclin B1 (CDK1/CYCB1) to directly hyperphosphorylate eukaryotic initiation factor 4E (eIF4E)-binding protein (4E-BP1) at authentic sites, generating a mitosis-specific, mechanistic target of rapamycin (mTOR) inhibitor-resistant δ phospho-isoform not present in G1-arrested cells. Recombinant 4E-BP1 inhibits capped mRNA reticulocyte translation, which is partially reversed by CDK1/CYCB1 phosphorylation of 4E-BP1. eIF4G binding to the eIF4E-m(7)GTP cap complex is resistant to mTOR inhibition during mitosis but sensitive during interphase. Flow cytometry, with and without sT, reveals an orthogonal pH3(S10+) mitotic cell population having higher inactive p4E-BP1(T37/T46+) saturation levels than pH3(S10-) interphase cells. Using a Click-iT flow cytometric assay to directly measure mitotic protein synthesis, we find that most new protein synthesis during mitosis is cap-dependent, a result confirmed using the eIF4E/4G inhibitor drug 4E1RCat. For most cell lines tested, cap-dependent translation levels were generally similar between mitotic and interphase cells, and the majority of new mitotic protein synthesis was cap-dependent. These findings suggest that mitotic cap-dependent translation is generally sustained during mitosis by CDK1 phosphorylation of 4E-BP1 even under conditions of reduced mTOR signaling.

  3. Heterochromatin remodeling by CDK12 contributes to learning in Drosophila

    PubMed Central

    Pan, Lixia; Xie, Wenbing; Li, Kai-Le; Yang, Zhihao; Xu, Jiang; Zhang, Wenhao; Liu, Lu-Ping; Ren, Xingjie; He, Zhimin; Wu, Junyu; Sun, Jin; Wei, Hui-Min; Wang, Daliang; Xie, Wei; Li, Wei; Ni, Jian-Quan; Sun, Fang-Lin

    2015-01-01

    Dynamic regulation of chromatin structure is required to modulate the transcription of genes in eukaryotes. However, the factors that contribute to the plasticity of heterochromatin structure are elusive. Here, we report that cyclin-dependent kinase 12 (CDK12), a transcription elongation-associated RNA polymerase II (RNAPII) kinase, antagonizes heterochromatin enrichment in Drosophila chromosomes. Notably, loss of CDK12 induces the ectopic accumulation of heterochromatin protein 1 (HP1) on euchromatic arms, with a prominent enrichment on the X chromosome. Furthermore, ChIP and sequencing analysis reveals that the heterochromatin enrichment on the X chromosome mainly occurs within long genes involved in neuronal functions. Consequently, heterochromatin enrichment reduces the transcription of neuronal genes in the adult brain and results in a defect in Drosophila courtship learning. Taken together, these results define a previously unidentified role of CDK12 in controlling the epigenetic transition between euchromatin and heterochromatin and suggest a chromatin regulatory mechanism in neuronal behaviors. PMID:26508632

  4. Heterochromatin remodeling by CDK12 contributes to learning in Drosophila.

    PubMed

    Pan, Lixia; Xie, Wenbing; Li, Kai-Le; Yang, Zhihao; Xu, Jiang; Zhang, Wenhao; Liu, Lu-Ping; Ren, Xingjie; He, Zhimin; Wu, Junyu; Sun, Jin; Wei, Hui-Min; Wang, Daliang; Xie, Wei; Li, Wei; Ni, Jian-Quan; Sun, Fang-Lin

    2015-11-10

    Dynamic regulation of chromatin structure is required to modulate the transcription of genes in eukaryotes. However, the factors that contribute to the plasticity of heterochromatin structure are elusive. Here, we report that cyclin-dependent kinase 12 (CDK12), a transcription elongation-associated RNA polymerase II (RNAPII) kinase, antagonizes heterochromatin enrichment in Drosophila chromosomes. Notably, loss of CDK12 induces the ectopic accumulation of heterochromatin protein 1 (HP1) on euchromatic arms, with a prominent enrichment on the X chromosome. Furthermore, ChIP and sequencing analysis reveals that the heterochromatin enrichment on the X chromosome mainly occurs within long genes involved in neuronal functions. Consequently, heterochromatin enrichment reduces the transcription of neuronal genes in the adult brain and results in a defect in Drosophila courtship learning. Taken together, these results define a previously unidentified role of CDK12 in controlling the epigenetic transition between euchromatin and heterochromatin and suggest a chromatin regulatory mechanism in neuronal behaviors.

  5. Emerging Drug Profile: Cyclin-Dependent Kinase Inhibitors

    PubMed Central

    Blachly, James S.; Byrd, John C.

    2013-01-01

    As the rational application of targeted therapies in cancer supplants traditional cytotoxic chemotherapy, there is an ever-greater need for a thorough understanding of the complex machinery of the cell and an application of this knowledge to the development of novel therapeutics and combinations of agents. Here, we review the current state of knowledge of the class of targeted agents known as cyclin-dependent kinase (CDK) inhibitors, with a focus on chronic lymphocytic leukemia (CLL). Flavopiridol (alvocidib) is the best studied of the CDK inhibitors, producing a dramatic cytotoxic effect in vitro and in vivo, with the principal limiting factor of acute tumor lysis. Unfortunately, flavopiridol has a narrow therapeutic window and is relatively non-selective with several off-target (i.e. non-CDK) effects, which prompted development of the second-generation CDK inhibitor dinaciclib. Dinaciclib appears to be both more potent and selective than flavopiridol, with at least an order of magnitude greater therapeutic index, and is currently in phase III clinical trials. In additional to flavopiridol and dinaciclib, we also review the current state of other members of this class, and provide commentary as to the future direction of combination therapy including CDK inhibitors. PMID:23488658

  6. Pharmacological cyclin dependent kinase inhibitors: Implications for colorectal cancer.

    PubMed

    Balakrishnan, Archana; Vyas, Arpita; Deshpande, Kaivalya; Vyas, Dinesh

    2016-02-21

    Colorectal cancer accounts for a significant proportion of cancer deaths worldwide. The need to develop more chemotherapeutic agents to combat this disease is critical. Cyclin dependent kinases (CDKs), along with its binding partner cyclins, serve to control the growth of cells through the cell cycle. A new class of drugs, termed CDK inhibitors, has been studied in preclinical and now clinical trials. These inhibitors are believed to act as an anti-cancer drug by blocking CDKs to block the uncontrolled cellular proliferation that is hallmark of cancers like colorectal cancer. CDK article provides overview of the emerging drug class of CDK inhibitors and provides a list of ones that are currently in clinical trials.

  7. Processing of cdk5 activator p35 to its truncated form (p25) by calpain in acutely injured neuronal cells.

    PubMed

    Nath, R; Davis, M; Probert, A W; Kupina, N C; Ren, X; Schielke, G P; Wang, K K

    2000-07-21

    Recently, it was shown that conversion of cdk5 activator protein p35 to a C-terminal fragment p25 promotes a deregulation of cdk5 activity, which may contribute to neurodegeneration in Alzheimer's disease. In this study, we present evidence that calpain is a protease involved in the conversion of p35 to p25. To activate calpain, rat cerebellar granule neurons were treated with maitotoxin (MTX). A C-terminus-directed anti-p35 antibody detected that p35 conversion to p25 paralleled the formation of calpain-generated alpha-spectrin (alpha-fodrin) breakdown products (SBDP's) in a maitotoxin-dose-dependent manner. Two calpain inhibitors (MDl28170 and SJA6017) reduced p35 processing but were unchanged when exposed to the caspase inhibitor carbobenzoxy-Asp-CH(2)OC(=O)-2, 6-dichlorobenzene or the proteasome inhibitors (lactacystin and Z-Ile-Glu(OtBu)Ala-Leu-CHO). p35 protein was also degraded to p25 when rat brain lysate was subjected to in vitro digestion with purified mu- and m-calpains. Additionally, in a rat temporary middle cerebral artery occlusion model, p35 processing to p25 again paralleled SBDP formation in the ischemic core. Lastly, in malonate-injured rat brains, the ipsilateral side showed a striking correlation of SBDP formation with p35 to p25 conversion and tau phosphorylation (at Ser202 and Thr205) increase. These data suggest that calpain is a major neuronal protease capable of converting p35 to p25 and might play a pathological role of activating cdk5 and its phosphorylation of tau in Alzheimer's disease.

  8. Cyclin B1–Cdk1 Activation Continues after Centrosome Separation to Control Mitotic Progression

    PubMed Central

    Lindqvist, Arne; van Zon, Wouter; Karlsson Rosenthal, Christina; Wolthuis, Rob M. F

    2007-01-01

    Activation of cyclin B1–cyclin-dependent kinase 1 (Cdk1), triggered by a positive feedback loop at the end of G2, is the key event that initiates mitotic entry. In metaphase, anaphase-promoting complex/cyclosome–dependent destruction of cyclin B1 inactivates Cdk1 again, allowing mitotic exit and cell division. Several models describe Cdk1 activation kinetics in mitosis, but experimental data on how the activation proceeds in mitotic cells have largely been lacking. We use a novel approach to determine the temporal development of cyclin B1–Cdk1 activity in single cells. By quantifying both dephosphorylation of Cdk1 and phosphorylation of the Cdk1 target anaphase-promoting complex/cyclosome 3, we disclose how cyclin B1–Cdk1 continues to be activated after centrosome separation. Importantly, we discovered that cytoplasmic cyclin B1–Cdk1 activity can be maintained even when cyclin B1 translocates to the nucleus in prophase. These experimental data are fitted into a model describing cyclin B1–Cdk1 activation in human cells, revealing a striking resemblance to a bistable circuit. In line with the observed kinetics, cyclin B1–Cdk1 levels required to enter mitosis are lower than the amount of cyclin B1–Cdk1 needed for mitotic progression. We propose that gradually increasing cyclin B1–Cdk1 activity after centrosome separation is critical to coordinate mitotic progression. PMID:17472438

  9. MDM2 and CDK4 expression in periosteal osteosarcoma.

    PubMed

    Righi, Alberto; Gambarotti, Marco; Benini, Stefania; Gamberi, Gabriella; Cocchi, Stefania; Picci, Piero; Bertoni, Franco

    2015-04-01

    Periosteal osteosarcoma is defined by the World Health Organization as an intermediate-grade, malignant, cartilaginous, and bone-forming neoplasm arising on the surface of bone. Unlike other subtypes of osteosarcoma, no data have been published about mouse double minute 2 (MDM2) and cyclin-dependent kinase 4 (CDK4) expression. For this reason, we evaluated the molecular and immunohistochemical features of MDM2 and CDK4 in 27 cases relative to 20 patients with a diagnosis of periosteal osteosarcoma, surgically treated at the Rizzoli Institute between 1981 and 2014. When possible, these results were compared with the MDM2 amplification status as determined by fluorescence in situ hybridization (FISH). All but 1 case (26/27, 96.3%) were negative for MDM2 protein using immunohistochemistry both in primary and in recurrent periosteal osteosarcoma, whereas gene amplification of MDM2 was not detected in any tumor analyzed (10 cases). The positive immunohistochemical case shows a weak/moderate focal nuclear expression of MDM2 antibody in the prevalent cartilaginous component and in the spindle cells of peripheral fibroblastic areas associated with osteoid production in a primary periosteal osteosarcoma. CDK4 immunohistochemical expression was negative in all 27 cases. This retrospective analysis has demonstrated that MDM2 and CDK4 are very rarely expressed in primary and recurrent periosteal osteosarcomas and therefore do not appear to be molecules central to the control of cancer development, growth, and progression in periosteal osteosarcoma. Therefore, when compared with low-grade central and parosteal osteosarcomas, MDM2 and CDK4 markers cannot be used diagnostically to differentiate this subtype of osteosarcoma.

  10. The clinical significance of CDK1 expression in oral squamous cell carcinoma

    PubMed Central

    Chen, Xin; Chen, Qiao-Er; Wang, Yuan-Yin; Wang, Yin-Long; He, Jia-Cai; Zhou, Jian

    2015-01-01

    Objectives: To evaluate the clinical significance of cyclin-dependent kinase 1 (CDK1) in 77 oral squamous cell carcinomas (OSCC) using immunohistochemical methods. Study Design: Immunohistochemical expression of CDK1 was compared with various clinicopathological features in 77 OSCC and 60 controlled epithelia adjacent to the tumours. In addition, correlation of CDK1 expression and prognostic and the 5-year accumulative survival rate of OSCC were investigated. Results: The CDK1 protein was expressed in 52 cases of 77 tumor tissues (67.5%), compared with 21 cases of 60 controlled (35.0%). The expression of CDK1 was significantly correlated with the histological grade of OSCC (P<0.05). The CDK1 protein was over-expressed in recurrent tumors or in those with lymph node metastasis. Statistical analysis showed a significant reduction in the 5-year accumulative survival rate in CDK1 positive cases compared with CDK1 negative cases (P<0.05). Namely, the CDK1 positive patients had poor prognosis. Conclusions: The expression of CDK1 might serve as malignant degree and prognostic markers for the survival of OSCC. Key words:Cyclin-dependent kinase 1 (CDK1), oral squamous cell carcinoma (OSCC), immunohistochemistry, cell proliferation. PMID:25129248

  11. Cdk1, PKCδ and calcineurin-mediated Drp1 pathway contributes to mitochondrial fission-induced cardiomyocyte death.

    PubMed

    Zaja, Ivan; Bai, Xiaowen; Liu, Yanan; Kikuchi, Chika; Dosenovic, Svjetlana; Yan, Yasheng; Canfield, Scott G; Bosnjak, Zeljko J

    2014-10-31

    Myocardial ischemia-reperfusion (I/R) injury is one of the leading causes of death and disability worldwide. Mitochondrial fission has been shown to be involved in cardiomyocyte death. However, molecular machinery involved in mitochondrial fission during I/R injury has not yet been completely understood. In this study we aimed to investigate molecular mechanisms of controlling activation of dynamin-related protein 1 (Drp1, a key protein in mitochondrial fission) during anoxia-reoxygenation (A/R) injury of HL1 cardiomyocytes. A/R injury induced cardiomyocyte death accompanied by the increases of mitochondrial fission, reactive oxygen species (ROS) production and activated Drp1 (pSer616 Drp1), and decrease of inactivated Drp1 (pSer637 Drp1) while mitochondrial fusion protein levels were not significantly changed. Blocking Drp1 activity with mitochondrial division inhibitor mdivi1 attenuated cell death, mitochondrial fission, and Drp1 activation after A/R. Trolox, a ROS scavenger, decreased pSer616 Drp1 level and mitochondrial fission after A/R. Immunoprecipitation assay further indicates that cyclin dependent kinase 1 (Cdk1) and protein kinase C isoform delta (PKCδ) bind Drp1, thus increasing mitochondrial fission. Inhibiting Cdk1 and PKCδ attenuated the increases in pSer616 Drp1, mitochondrial fission, and cardiomyocyte death. FK506, a calcineurin inhibitor, blocked the decrease in expression of inactivated pSer637 Drp1 and mitochondrial fission. Our findings reveal the following novel molecular mechanisms controlling mitochondrial fission during A/R injury of cardiomyocytes: (1) ROS are upstream initiators of mitochondrial fission; and (2) the increased mitochondrial fission is resulted from both increased activation and decreased inactivation of Drp1 through Cdk1, PKCδ, and calcineurin-mediated pathways, respectively.

  12. Roscovitine is a proteostasis regulator that corrects the trafficking defect of F508del-CFTR by a CDK-independent mechanism

    PubMed Central

    Norez, C; Vandebrouck, C; Bertrand, J; Noel, S; Durieu, E; Oumata, N; Galons, H; Antigny, F; Chatelier, A; Bois, P; Meijer, L; Becq, F

    2014-01-01

    Background and Purpose The most common mutation in cystic fibrosis (CF), F508del, causes defects in trafficking, channel gating and endocytosis of the CF transmembrane conductance regulator (CFTR) protein. Because CF is an orphan disease, therapeutic strategies aimed at improving mutant CFTR functions are needed to target the root cause of CF. Experimental Approach Human CF airway epithelial cells were treated with roscovitine 100 μM for 2 h before CFTR maturation, expression and activity were examined. The mechanism of action of roscovitine was explored by recording the effect of depleting endoplasmic reticulum (ER) Ca2+ on the F508del-CFTR/calnexin interaction and by measuring proteasome activity. Key Results Of the cyclin-dependent kinase (CDK) inhibitors investigated, roscovitine was found to restore the cell surface expression and defective channel function of F508del-CFTR in human CF airway epithelial cells. Neither olomoucine nor (S)-CR8, two very efficient CDK inhibitors, corrected F508del-CFTR trafficking demonstrating that the correcting effect of roscovitine was independent of CDK inhibition. Competition studies with inhibitors of the ER quality control (ERQC) indicated that roscovitine acts on the calnexin pathway and on the degradation machinery. Roscovitine was shown (i) to partially inhibit the interaction between F508del-CFTR and calnexin by depleting ER Ca2+ and (ii) to directly inhibit the proteasome activity in a Ca2+-independent manner. Conclusions and Implications Roscovitine is able to correct the defective function of F508del-CFTR by preventing the ability of the ERQC to interact with and degrade F508del-CFTR via two synergistic but CDK-independent mechanisms. Roscovitine has potential as a pharmacological therapy for CF. PMID:25065395

  13. Phenyl-1-Pyridin-2yl-Ethanone-Based Iron Chelators Increase IκB-α Expression, Modulate CDK2 and CDK9 Activities, and Inhibit HIV-1 Transcription

    PubMed Central

    Kumari, Namita; Iordanskiy, Sergey; Kovalskyy, Dmytro; Breuer, Denitra; Niu, Xiaomei; Lin, Xionghao; Xu, Min; Gavrilenko, Konstantin; Kashanchi, Fatah; Dhawan, Subhash

    2014-01-01

    HIV-1 transcription is activated by the Tat protein, which recruits CDK9/cyclin T1 to the HIV-1 promoter. CDK9 is phosphorylated by CDK2, which facilitates formation of the high-molecular-weight positive transcription elongation factor b (P-TEFb) complex. We previously showed that chelation of intracellular iron inhibits CDK2 and CDK9 activities and suppresses HIV-1 transcription, but the mechanism of the inhibition was not understood. In the present study, we tested a set of novel iron chelators for the ability to inhibit HIV-1 transcription and elucidated their mechanism of action. Novel phenyl-1-pyridin-2yl-ethanone (PPY)-based iron chelators were synthesized and examined for their effects on cellular iron, HIV-1 inhibition, and cytotoxicity. Activities of CDK2 and CDK9, expression of CDK9-dependent and CDK2-inhibitory mRNAs, NF-κB expression, and HIV-1- and NF-κB-dependent transcription were determined. PPY-based iron chelators significantly inhibited HIV-1, with minimal cytotoxicity, in cultured and primary cells chronically or acutely infected with HIV-1 subtype B, but they had less of an effect on HIV-1 subtype C. Iron chelators upregulated the expression of IκB-α, with increased accumulation of cytoplasmic NF-κB. The iron chelators inhibited CDK2 activity and reduced the amount of CDK9/cyclin T1 in the large P-TEFb complex. Iron chelators reduced HIV-1 Gag and Env mRNA synthesis but had no effect on HIV-1 reverse transcription. In addition, iron chelators moderately inhibited basal HIV-1 transcription, equally affecting HIV-1 and Sp1- or NF-κB-driven transcription. By virtue of their involvement in targeting several key steps in HIV-1 transcription, these novel iron chelators have the potential for the development of new therapeutics for the treatment of HIV-1 infection. PMID:25155598

  14. CDK2 and mTOR are direct molecular targets of isoangustone A in the suppression of human prostate cancer cell growth

    SciTech Connect

    Lee, Eunjung; Son, Joe Eun; Byun, Sanguine; Lee, Seung Joon; Kim, Yeong A; Liu, Kangdong; Kim, Jiyoung; Lim, Soon Sung; Park, Jung Han Yoon; Dong, Zigang; Lee, Ki Won; Lee, Hyong Joo

    2013-10-01

    Licorice extract which is used as a natural sweetener has been shown to possess inhibitory effects against prostate cancer, but the mechanisms responsible are poorly understood. Here, we report a compound, isoangustone A (IAA) in licorice that potently suppresses the growth of aggressive prostate cancer and sought to clarify its mechanism of action. We analyzed its inhibitory effects on the growth of PTEN-deleted human prostate cancer cells, in vitro and in vivo. Administration of IAA significantly attenuated the growth of prostate cancer cell cultures and xenograft tumors. These effects were found to be attributable to inhibition of the G1/S phase cell cycle transition and the accumulation of p27{sup kip1}. The elevated p27{sup kip1} expression levels were concurrent with the decrease of its phosphorylation at threonine 187 through suppression of CDK2 kinase activity and the reduced phosphorylation of Akt at Serine 473 by diminishing the kinase activity of the mammalian target of rapamycin (mTOR). Further analysis using recombinant proteins and immunoprecipitated cell lysates determined that IAA exerts suppressive effects against CDK2 and mTOR kinase activity by direct binding with both proteins. These findings suggested that the licorice compound IAA is a potent molecular inhibitor of CDK2 and mTOR, with strong implications for the treatment of prostate cancer. Thus, licorice-derived extracts with high IAA content warrant further clinical investigation for nutritional sources for prostate cancer patients. - Highlights: • Isoangustone A suppresses growth of PC3 and LNCaP prostate cancer cells. • Administration of isoangustone A inhibits tumor growth in mice. • Treatment of isoangustone A induces cell cycle arrest and accumulation of p27{sup kip1}. • Isoangustone A inhibits CDK2 and mTOR activity. • Isoangustone A directly binds with CDK2 and mTOR complex in prostate cancer cells.

  15. Specific overexpression of cyclin E·CDK2 in early preinvasive and primary breast tumors in female ACI rats induced by estrogen.

    PubMed

    Weroha, S John; Lingle, Wilma L; Hong, Yan; Li, Sara Antonia; Li, Jonathan J

    2010-02-01

    Overexpressed Aurora A, amplified centrosomes, and aneuploidy are salient features of estrogen-induced mammary preinvasive lesions and tumors in female August--Copenhagen Irish (ACI) rats. Intimately involved in these events are cyclins and their associated cyclin-dependent kinase (CDK) partners. Cyclin E1·CDK2 overexpression plays an important dual role in late G1/S phase of the cell cycle in cancer cells. It increases DNA replication providing growth advantage to cancer cells and facilitates aberrant centrosome duplication, generating chromosomal instability and aneuploidy leading to tumor development. Presented herein, a 24.0- and 45.0-fold elevation in cyclin E1 and CDK2 was found in 17β-estradiol (E(2))-induced ACI rat mammary tumors (MTs), respectively. Cyclin E·CDK2 positive staining was confined to the large round cells found within focal dysplasias, ductal carcinomas in situ, and invasive MTs. Co-immunoprecipitation and in vitro kinase activity of these tumors revealed that these cell cycle entities are functional. When mammary tissue derived from untreated normal, E(2)-induced hyperplasia and primary tumors were normalized to cyclin E1 levels, low molecular weight (LMW) cyclin E1 forms (33- and 45-kDa) were detected in all of these tissue groups. Moreover, increasing concentrations of protease inhibitor in tissue lysates resulted in a marked reduction of LMW forms, indicating that the presence of cyclin E1 LMW forms can be markedly reduced. Significant increases in cyclin E1 mRNA (2.1-fold) were detected in primary ACI rat E(2)-induced breast tumors, and quantitative real-time polymerase chain reaction revealed a 20% amplification of the cyclin E1 gene (CCNE1). Collectively, these results support the involvement of cyclin E1·CDK2 in centrosome overduplication during each stage of E(2)-induced mammary tumorigenesis.

  16. Filamin B Regulates Chondrocyte Proliferation and Differentiation through Cdk1 Signaling

    PubMed Central

    Lian, Gewei; Zhang, Jingping; Hecht, Jonathan L.; Sheen, Volney L.

    2014-01-01

    Humans who harbor loss of function mutations in the actin-associated filamin B (FLNB) gene develop spondylocarpotarsal syndrome (SCT), a disorder characterized by dwarfism (delayed bone formation) and premature fusion of the vertebral, carpal and tarsal bones (premature differentiation). To better understand the cellular and molecular mechanisms governing these seemingly divergent processes, we generated and characterized FlnB knockdown ATDC5 cell lines. We found that FlnB knockdown led to reduced proliferation and enhanced differentiation in chondrocytes. Within the shortened growth plate of postnatal FlnB−/− mice long bone, we observed a similarly progressive decline in the number of rapidly proliferating chondrocytes and premature differentiation characterized by an enlarged prehypertrophic zone, a widened Col2a1+/Col10a1+ overlapping region, but relatively reduced hypertrophic zone length. The reduced chondrocyte proliferation and premature differentiation were, in part, attributable to enhanced G2/M phase progression, where fewer FlnB deficient ATDC5 chondrocytes resided in the G2/M phase of the cell cycle. FlnB loss reduced Cdk1 phosphorylation (an inhibitor of G2/M phase progression) and Cdk1 inhibition in chondrocytes mimicked the null FlnB, premature differentiation phenotype, through a β1-integrin receptor- Pi3k/Akt (a key regulator of chondrocyte differentiation) mediated pathway. In this context, the early prehypertrophic differentiation provides an explanation for the premature differentiation seen in this disorder, whereas the progressive decline in proliferating chondrocytes would ultimately lead to reduced chondrocyte production and shortened bone length. These findings begin to define a role for filamin proteins in directing both cell proliferation and differentiation through indirect regulation of cell cycle associated proteins. PMID:24551245

  17. Consequences of abnormal CDK activity in S phase.

    PubMed

    Anda, Silje; Rothe, Christiane; Boye, Erik; Grallert, Beáta

    2016-01-01

    Cyclin Dependent Kinases (CDKs) are important regulators of DNA replication. In this work we have investigated the consequences of increasing or decreasing the CDK activity in S phase. To this end we identified S-phase regulators of the fission yeast CDK, Cdc2, and used appropriate mutants to modulate Cdc2 activity. In fission yeast Mik1 has been thought to be the main regulator of Cdc2 activity in S phase. However, we find that Wee1 has a major function in S phase and thus we used wee1 mutants to investigate the consequences of increased Cdc2 activity. These wee1 mutants display increased replication stress and, particularly in the absence of the S-phase checkpoint, accumulate DNA damage. Notably, more cells incorporate EdU in a wee1(-) strain as compared to wildtype, suggesting altered regulation of DNA replication. In addition, a higher number of cells contain chromatin-bound Cdc45, an indicator of active replication forks. In addition, we found that Cdc25 is required to activate Cdc2 in S phase and used a cdc25 mutant to explore a situation where Cdc2 activity is reduced. Interestingly, a cdc25 mutant has a higher tolerance for replication stress than wild-type cells, suggesting that reduced CDK activity in S phase confers resistance to at least some forms of replication stress.

  18. Cdk1, PKCδ and calcineurin-mediated Drp1 pathway contributes to mitochondrial fission-induced cardiomyocyte death

    SciTech Connect

    Zaja, Ivan; Bai, Xiaowen; Liu, Yanan; Kikuchi, Chika; Dosenovic, Svjetlana; Yan, Yasheng; Canfield, Scott G.; Bosnjak, Zeljko J.

    2014-10-31

    Highlights: • Drp1-mediated increased mitochondrial fission but not fusion is involved the cardiomyocyte death during anoxia-reoxygenation injury. • Reactive oxygen species are upstream initiators of mitochondrial fission. • Increased mitochondrial fission is resulted from Cdk1-, PKCδ-, and calcineurin-mediated Drp1 pathways. - Abstract: Myocardial ischemia–reperfusion (I/R) injury is one of the leading causes of death and disability worldwide. Mitochondrial fission has been shown to be involved in cardiomyocyte death. However, molecular machinery involved in mitochondrial fission during I/R injury has not yet been completely understood. In this study we aimed to investigate molecular mechanisms of controlling activation of dynamin-related protein 1 (Drp1, a key protein in mitochondrial fission) during anoxia-reoxygenation (A/R) injury of HL1 cardiomyocytes. A/R injury induced cardiomyocyte death accompanied by the increases of mitochondrial fission, reactive oxygen species (ROS) production and activated Drp1 (pSer616 Drp1), and decrease of inactivated Drp1 (pSer637 Drp1) while mitochondrial fusion protein levels were not significantly changed. Blocking Drp1 activity with mitochondrial division inhibitor mdivi1 attenuated cell death, mitochondrial fission, and Drp1 activation after A/R. Trolox, a ROS scavenger, decreased pSer616 Drp1 level and mitochondrial fission after A/R. Immunoprecipitation assay further indicates that cyclin dependent kinase 1 (Cdk1) and protein kinase C isoform delta (PKCδ) bind Drp1, thus increasing mitochondrial fission. Inhibiting Cdk1 and PKCδ attenuated the increases in pSer616 Drp1, mitochondrial fission, and cardiomyocyte death. FK506, a calcineurin inhibitor, blocked the decrease in expression of inactivated pSer637 Drp1 and mitochondrial fission. Our findings reveal the following novel molecular mechanisms controlling mitochondrial fission during A/R injury of cardiomyocytes: (1) ROS are upstream initiators of

  19. HIV-1 Tat-associated RNA polymerase C-terminal domain kinase, CDK2, phosphorylates CDK7 and stimulates Tat-mediated transcription.

    PubMed Central

    Nekhai, Sergei; Zhou, Meisheng; Fernandez, Anne; Lane, William S; Lamb, Ned J C; Brady, John; Kumar, Ajit

    2002-01-01

    HIV-1 gene expression is regulated by a viral transactivator protein (Tat) which induces transcriptional elongation of HIV-1 long tandem repeat (LTR). This induction requires hyperphosphorylation of the C-terminal domain (CTD) repeats of RNA polymerase II (Pol II). To achieve CTD hyperphosphorylation, Tat stimulates CTD kinases associated with general transcription factors of the promoter complex, specifically TFIIH-associated CDK7 and positive transcription factor b-associated CDK9 (cyclin-dependent kinase 9). Other studies indicate that Tat may bind an additional CTD kinase that regulates the target-specific phosphorylation of RNA Pol II CTD. We previously reported that Tat-associated T-cell-derived kinase (TTK), purified from human primary T-cells, stimulates Tat-dependent transcription of HIV-1 LTR in vivo [Nekhai, Shukla, Fernandez, Kumar and Lamb (2000) Virology 266, 246-256]. In the work presented here, we characterized the components of TTK by biochemical fractionation and the function of TTK in transcription assays in vitro. TTK uniquely co-purified with CDK2 and not with either CDK9 or CDK7. Tat induced the TTK-associated CDK2 kinase to phosphorylate CTD, specifically at Ser-2 residues. The TTK fraction restored Tat-mediated transcription activation of HIV-1 LTR in a HeLa nuclear extract immunodepleted of CDK9, but not in the HeLa nuclear extract double-depleted of CDK9 and CDK7. Direct microinjection of the TTK fraction augmented Tat transactivation of HIV-1 LTR in human primary HS68 fibroblasts. The results argue that TTK-associated CDK2 may function to maintain target-specific phosphorylation of RNA Pol II that is essential for Tat transactivation of HIV-1 promoter. They are also consistent with the observed cell-cycle-specific induction of viral gene transactivation. PMID:12049628

  20. Cigarette smoke extract alters the cell cycle via the phospholipid transfer protein/transforming growth factor-β1/CyclinD1/CDK4 pathway.

    PubMed

    Chai, Xue-Min; Li, You-Lun; Chen, Hong; Guo, Shu-Liang; Shui, Li-Li; Chen, Ya-Juan

    2016-09-05

    This study was aimed to investigate the effect of phospholipid transfer protein (PLTP) on cigarette smoke extract (CSE)-induced alteration of the cell cycle and the possible mechanism. Male Wistar rats and the rat alveolar epithelial cell line (RLE-6TN) were exposed to normal air or different concentrations of CSE. Then PLTP siRNA was transfected into cells and an inhibitor of transforming growth factor-β1 (TGF-β1) was administered prior to CSE exposure. Histological changes and cell cycle stage were recorded, as were the expression levels of PLTP, TGF-β1, CyclinD1 and CDK4. Resulting morphological changes included diffuse interstitial substance incrassation and elevated alveolar rupturing. Flow cytometry analysis revealed an increase in the number of cells in the G1 phase in a time- and dose-related manner. Both PLTP and TGF-β1 were up-regulated at protein and mRNA levels, whereas CyclinD1 and CDK4 expression was down-regulated after CSE exposure. Furthermore, PLTP siRNA significantly suppressed CSE-induced TGF-β1 expression, resulting in up-regulation of CyclinD1 and CDK4, but the TGF-β1 inhibitor was not able to abrogate CSE-induced PLTP over-expression. In conclusion, PLTP may operate upstream of the TGF-β1/CyclinD1/CDK4 pathway and may mediate the CSE-induced G1 arrest in RLE-6TN cells. Our work provides some new insight into the relation between PLTP and cell cycle progression.

  1. Activation of Cdk5/p25 and tau phosphorylation following chronic brain hypoperfusion in rats involves microRNA-195 down-regulation.

    PubMed

    Sun, Li-Hua; Ban, Tao; Liu, Cheng-Di; Chen, Qing-Xin; Wang, Xu; Yan, Mei-Ling; Hu, Xue-Ling; Su, Xiao-Lin; Bao, Ya-Nan; Sun, Lin-Lin; Zhao, Lin-Jing; Pei, Shuang-Chao; Jiang, Xue-Mei; Zong, De-Kang; Ai, Jing

    2015-09-01

    . Schematic diagram of miR-195 mediated Aβ aggregation and tau hyperphosphorylation in chronic brain hypoperfusion (CBH). First, CBH results in the elevation of nuclear factor-κB (NF-κB), which binds with the promoter sequences of miR-195 and negatively regulates the expression of miR-195. Second, down-regulated miR-195 induces up-regulation of APP and BACE1 and leads to an increase in Aβ levels. Third, some of the elevated Aβ then enter the intracellular space and activate calpain, which promotes the conversion of Cdk5/p35 to Cdk5/p25 and catalyzes the degradation of IκB; IκB is an inhibitor of NF-κB, which activates NF-κB. Cdk5/p25 directly phosphorylates Tau. Fourth, down-regulated miR-195 induces an up-regulation of p35, which provides the active substrates of p25. Our findings demonstrated that the down-regulation of miR-195 plays a key role in the increased vulnerability to dementia via the regulation of multiple targets following CBH.

  2. From quiescence to proliferation: Cdk oscillations drive the mammalian cell cycle.

    PubMed

    Gérard, Claude; Goldbeter, Albert

    2012-01-01

    We recently proposed a detailed model describing the dynamics of the network of cyclin-dependent kinases (Cdks) driving the mammalian cell cycle (Gérard and Goldbeter, 2009). The model contains four modules, each centered around one cyclin/Cdk complex. Cyclin D/Cdk4-6 and cyclin E/Cdk2 promote progression in G1 and elicit the G1/S transition, respectively; cyclin A/Cdk2 ensures progression in S and the transition S/G2, while the activity of cyclin B/Cdk1 brings about the G2/M transition. This model shows that in the presence of sufficient amounts of growth factor the Cdk network is capable of temporal self-organization in the form of sustained oscillations, which correspond to the ordered, sequential activation of the various cyclin/Cdk complexes that control the successive phases of the cell cycle. The results suggest that the switch from cellular quiescence to cell proliferation corresponds to the transition from a stable steady state to sustained oscillations in the Cdk network. The transition depends on a finely tuned balance between factors that promote or hinder progression in the cell cycle. We show that the transition from quiescence to proliferation can occur in multiple ways that alter this balance. By resorting to bifurcation diagrams, we analyze the mechanism of oscillations in the Cdk network. Finally, we show that the complexity of the detailed model can be greatly reduced, without losing its key dynamical properties, by considering a skeleton model for the Cdk network. Using such a skeleton model for the mammalian cell cycle we show that positive feedback (PF) loops enhance the amplitude and the robustness of Cdk oscillations with respect to molecular noise. We compare the relative merits of the detailed and skeleton versions of the model for the Cdk network driving the mammalian cell cycle.

  3. Oncogenic Actions of SKP2 Involves Deregulation of CDK1 Turnover Mediated by FOXM1.

    PubMed

    Krishnan, Anand; K, Dhanya; Babu P S, Saneesh; Jagadeeshan, Sankar; Prasad, Manu; Nair, S Asha

    2017-04-01

    Cyclin-dependent kinases (cdks) are central catalytic units of cell division cycle. Among the cdk family members, cdk1 has critical roles in multiple phases of the cell cycle. Aberrant expression or hyper-actions of cdk1 are tumorigenic and yet the complex oncogenic network that regulates its turnover is poorly understood. We found a hitherto unexplored functional connection between skp2 and cdk1 turn over. In vitro knockdown or overexpression of skp2 in cultured cells reduced or induced cdk1 expression indicating skp2 as a positive driver for cdk1. A partial inhibitory role for p27 was identified in this context. Interestingly, concurrent overexpression of skp2 and p27 favored cdk1 upregulation in vitro, which correlated well with similar observations in clinical tumor samples. We found that the transcription factor FOXM1 may play a central role in the skp2-cdk1 loop. Additional molecular involvement in the skp2-cdk1 loop was also explored. In conclusion, our results revealed hitherto unexplored p27 independent molecular mechanisms for skp2 driven tumor progression. Our results support the previous findings that skp2 may be a potential therapeutic target for the management of tumors. J. Cell. Biochem. 118: 797-807, 2017. © 2016 Wiley Periodicals, Inc.

  4. CDK5RAP3 is a novel repressor of p14ARF in hepatocellular carcinoma cells.

    PubMed

    Mak, Grace Wing-Yan; Lai, Wai-Lung; Zhou, Yuan; Li, Mingtao; Ng, Irene Oi-Lin; Ching, Yick-Pang

    2012-01-01

    CDK5 regulatory subunit associated protein 3 (CDK5RAP3) is a novel activator of PAK4 and processes important pro-metastatic function in hepatocarcinogenesis. However, it remains unclear if there are other mechanisms by which CDK5RAP3 promotes HCC metastasis. Here, we showed that in CDK5RAP3 stable knockdown SMMC-7721 HCC cells, p14(ARF) tumor suppressor was upregulated at protein and mRNA levels, and ectopic expression of CDK5RAP3 was found to repress the transcription of p14(ARF). Using chromatin immunoprecipitation assay, we demonstrated that CDK5RAP3 bound to p14(ARF) promoter in vivo. Furthermore, knockdown of p14(ARF) in CDK5RAP3 stable knockdown HCC cells reversed the suppression of HCC cell invasiveness mediated by knockdown of CDK5RAP3. Taken together, our findings provide the new evidence that overexpression of CDK5RAP3 promotes HCC metastasis via downregulation of p14(ARF).

  5. CDK5RAP3 Is a Novel Repressor of p14ARF in Hepatocellular Carcinoma Cells

    PubMed Central

    Mak, Grace Wing-Yan; Li, Mingtao; Ng, Irene Oi-Lin; Ching, Yick-Pang

    2012-01-01

    CDK5 regulatory subunit associated protein 3 (CDK5RAP3) is a novel activator of PAK4 and processes important pro-metastatic function in hepatocarcinogenesis. However, it remains unclear if there are other mechanisms by which CDK5RAP3 promotes HCC metastasis. Here, we showed that in CDK5RAP3 stable knockdown SMMC-7721 HCC cells, p14ARF tumor suppressor was upregulated at protein and mRNA levels, and ectopic expression of CDK5RAP3 was found to repress the transcription of p14ARF. Using chromatin immunoprecipitation assay, we demonstrated that CDK5RAP3 bound to p14ARF promoter in vivo. Furthermore, knockdown of p14ARF in CDK5RAP3 stable knockdown HCC cells reversed the suppression of HCC cell invasiveness mediated by knockdown of CDK5RAP3. Taken together, our findings provide the new evidence that overexpression of CDK5RAP3 promotes HCC metastasis via downregulation of p14ARF. PMID:22860085

  6. CDK4 deficiency promotes genomic instability and enhances Myc-driven lymphomagenesis

    PubMed Central

    Lu, Yuanzhi; Wu, Yongsheng; Feng, Xiaoling; Shen, Rulong; Wang, Jing H.; Fallahi, Mohammad; Li, Weimin; Yang, Chunying; Hankey, William; Zhao, Weiqiang; Ganju, Ramesh K.; Li, Ming O.; Cleveland, John L.; Zou, Xianghong

    2014-01-01

    The G1 kinase CDK4 is amplified or overexpressed in some human tumors and promotes tumorigenesis by inhibiting known tumor suppressors. Here, we report that CDK4 deficiency markedly accelerated lymphoma development in the Eμ-Myc transgenic mouse model of B lymphoma and that silencing or loss of CDK4 augmented the tumorigenic potential of Myc-driven mouse and human B cell lymphoma in transplant models. Accelerated disease in CDK4-deficient Eμ-Myc transgenic mice was associated with rampant genomic instability that was provoked by dysregulation of a FOXO1/RAG1/RAG2 pathway. Specifically, CDK4 phosphorylated and inactivated FOXO1, which prevented FOXO1-dependent induction of Rag1 and Rag2 transcription. CDK4-deficient Eμ-Myc B cells had high levels of the active form of FOXO1 and elevated RAG1 and RAG2. Furthermore, overexpression of RAG1 and RAG2 accelerated lymphoma development in a transplant model, with RAG1/2-expressing tumors exhibiting hallmarks of genomic instability. Evaluation of human tumor samples revealed that CDK4 expression was markedly suppressed, while FOXO1 expression was elevated, in several subtypes of human non-Hodgkin B cell lymphoma. Collectively, these findings establish a context-specific tumor suppressor function for CDK4 that prevents genomic instability, which contributes to B cell lymphoma. Furthermore, our data suggest that targeting CDK4 may increase the risk for the development and/or progression of lymphoma. PMID:24614102

  7. Cyclin A2 Mutagenesis Analysis: A New Insight into CDK Activation and Cellular Localization Requirements

    PubMed Central

    Bendris, Nawal; Lemmers, Bénédicte; Blanchard, Jean-Marie; Arsic, Nikola

    2011-01-01

    Cyclin A2 is essential at two critical points in the somatic cell cycle: during S phase, when it activates CDK2, and during the G2 to M transition when it activates CDK1. Based on the crystal structure of Cyclin A2 in association with CDKs, we generated a panel of mutants to characterize the specific amino acids required for partner binding, CDK activation and subcellular localization. We find that CDK1, CDK2, p21, p27 and p107 have overlapping but distinct requirements for association with this protein. Our data highlight the crucial importance of the N-terminal α helix, in conjunction with the α3 helix within the cyclin box, in activating CDK. Several Cyclin A2 mutants selectively bind to either CDK1 or CDK2. We demonstrate that association of Cyclin A2 to proteins such as CDK2 that was previously suggested as crucial is not a prerequisite for its nuclear localization, and we propose that the whole protein structure is involved. PMID:21829545

  8. The structure and substrate specificity of human Cdk12/Cyclin K

    PubMed Central

    Bösken, Christian A.; Farnung, Lucas; Hintermair, Corinna; Merzel Schachter, Miriam; Vogel-Bachmayr, Karin; Blazek, Dalibor; Anand, Kanchan; Fisher, Robert P.; Eick, Dirk; Geyer, Matthias

    2014-01-01

    Phosphorylation of the RNA polymerase II C-terminal domain (CTD) by cyclin-dependent kinases is important for productive transcription. Here we determine the crystal structure of Cdk12/CycK and analyse its requirements for substrate recognition. Active Cdk12/CycK is arranged in an open conformation similar to that of Cdk9/CycT but different from those of cell cycle kinases. Cdk12 contains a C-terminal extension that folds onto the N- and C-terminal lobes thereby contacting the ATP ribose. The interaction is mediated by an HE motif followed by a polybasic cluster that is conserved in transcriptional CDKs. Cdk12/CycK showed the highest activity on a CTD substrate prephosphorylated at position Ser7, whereas the common Lys7 substitution was not recognized. Flavopiridol is most potent towards Cdk12 but was still 10-fold more potent towards Cdk9. T-loop phosphorylation of Cdk12 required coexpression with a Cdk-activating kinase. These results suggest the regulation of Pol II elongation by a relay of transcriptionally active CTD kinases. PMID:24662513

  9. Cooperativity of Cdk4R24C and Ras in melanoma development.

    PubMed

    Chawla, Rachna; Procknow, Judith A; Tantravahi, Ramana V; Khurana, Jasvir S; Litvin, Judith; Reddy, E Premkumar

    2010-08-15

    The importance of the CDK4 protein in human cancer first became evident following the identification of a germ line mutation in the Cdk4 locus that predisposes humans to melanoma. This mutation results in substitution of arginine with cysteine at position 24 (R24C). In an earlier study, we introduced the R24C mutation into the Cdk4 locus of mice using Cre-loxP-mediated "knock-in" technology and observed a very low incidence of spontaneous melanomas in Cdk4(R24C/R24C) mice. This suggested that additional oncogenic mutations might be required for development of melanomas. Here we report an increased incidence of spontaneous cutaneous melanoma in mice expressing the oncogene HRAS(G12V) in melanocytes on a Cdk4(R24C) background. Treatment of Tyr-HRas:Cdk4(R24C/R24C) mice with the carcinogen, DMBA/TPA resulted in a further increase in the number of nevi and melanomas developed when compared with Tyr-HRas:Cdk4(+/+) mice. In summary, in Tyr-HRas:Cdk4(R24C/R24C) mice, we observed that activated CDK4 cooperates with the oncogenic HRAS(G12V) protein to increase the susceptibility of melanoma development in vivo.

  10. Molecular cloning and functional characterization of cyclin E and CDK2 from Penaeus monodon.

    PubMed

    Zhao, C; Fu, M J; Qiu, L H

    2016-09-16

    Reduced reproductive performance of the black tiger shrimp (Penaeus monodon) has caused economic losses and hampered the fishing industry. Detailed investigation of the molecular mechanism by which the cell cycle is regulated in this organism is needed to understand the development and maturation of ovaries and oocytes, with a view to improving reproductive capacity. Cell cycle progression is mainly determined by cyclin-dependent kinase (CDK) and cyclin complexes, the cyclin E/CDK2 complex playing a key role in G1/S transition. However, knowledge of the interplay between cyclin E and CDK2 in invertebrates remains limited. In this study, full-length P. monodon cyclin E (Pmcyclin E) and CDK2 (PmCDK2) sequences were cloned. The open reading frame of Pmcyclin E was 1263 bp in length and encoded a 47.9-kDa protein, while that of PmCDK2 was 921 bp, encoding a protein of 34.9 kDa. Recombinant cyclin E and CDK2 proteins were expressed in Escherichia coli and purified by Ni-chelating affinity chromatography. In addition, a pull-down assay was performed to identify any interaction between Pmcyclin E and PmCDK2. This research provides a basis for the study of the functional mechanisms of the cyclin E/CDK2 complex in shrimp, further enriching our knowledge of invertebrate cell cycle regulation.

  11. Waves of Cdk1 Activity in S Phase Synchronize the Cell Cycle in Drosophila Embryos.

    PubMed

    Deneke, Victoria E; Melbinger, Anna; Vergassola, Massimo; Di Talia, Stefano

    2016-08-22

    Embryos of most metazoans undergo rapid and synchronous cell cycles following fertilization. While diffusion is too slow for synchronization of mitosis across large spatial scales, waves of Cdk1 activity represent a possible process of synchronization. However, the mechanisms regulating Cdk1 waves during embryonic development remain poorly understood. Using biosensors of Cdk1 and Chk1 activities, we dissect the regulation of Cdk1 waves in the Drosophila syncytial blastoderm. We show that Cdk1 waves are not controlled by the mitotic switch but by a double-negative feedback between Cdk1 and Chk1. Using mathematical modeling and surgical ligations, we demonstrate a fundamental distinction between S phase Cdk1 waves, which propagate as active trigger waves in an excitable medium, and mitotic Cdk1 waves, which propagate as passive phase waves. Our findings show that in Drosophila embryos, Cdk1 positive feedback serves primarily to ensure the rapid onset of mitosis, while wave propagation is regulated by S phase events.

  12. G1/S phase progression is regulated by PLK1 degradation through the CDK1/βTrCP axis.

    PubMed

    Giráldez, Servando; Galindo-Moreno, María; Limón-Mortés, M Cristina; Rivas, A Cristina; Herrero-Ruiz, Joaquín; Mora-Santos, Mar; Sáez, Carmen; Japón, Miguel Á; Tortolero, Maria; Romero, Francisco

    2017-03-30

    Polo-like kinase 1 (PLK1) is a serine/threonine kinase involved in several stages of the cell cycle, including the entry and exit from mitosis, and cytokinesis. Furthermore, it has an essential role in the regulation of DNA replication. Together with cyclin A, PLK1 also promotes CDH1 phosphorylation to trigger its ubiquitination and degradation, allowing cell cycle progression. The PLK1 levels in different type of tumors are very high compared to normal tissues, which is consistent with its role in promoting proliferation. Therefore, several PLK1 inhibitors have been developed and tested for the treatment of cancer. Here, we further analyzed PLK1 degradation and found that cytoplasmic PLK1 is ubiquitinated and subsequently degraded by the SCF(βTrCP)/proteasome. This procedure is triggered when heat shock protein (HSP) 90 is inhibited with geldanamycin, which results in misfolding of PLK1. We also identified CDK1 as the major kinase involved in this degradation. Our work shows for the first time that HSP90 inhibition arrests cell cycle progression at the G1/S transition. This novel mechanism inhibits CDH1 degradation through CDK1-dependent PLK1 destruction by the SCF(βTrCP)/proteasome. In these conditions, CDH1 substrates do not accumulate and cell cycle arrests, providing a novel pathway for regulation of the cell cycle at the G1-to-S boundary.-Giráldez, S., Galindo-Moreno, M., Limón-Mortés, M. C., Rivas, A. C., Herrero-Ruiz, J., Mora-Santos, M., Sáez, C., Japón, M. Á., Tortolero, M., Romero, F. G1/S phase progression is regulated by PLK1 degradation through the CDK1/βTrCP axis.

  13. Explicit treatment of active-site waters enhances quantum mechanical/implicit solvent scoring: Inhibition of CDK2 by new pyrazolo[1,5-a]pyrimidines.

    PubMed

    Hylsová, Michaela; Carbain, Benoit; Fanfrlík, Jindřich; Musilová, Lenka; Haldar, Susanta; Köprülüoğlu, Cemal; Ajani, Haresh; Brahmkshatriya, Pathik S; Jorda, Radek; Kryštof, Vladimír; Hobza, Pavel; Echalier, Aude; Paruch, Kamil; Lepšík, Martin

    2017-01-27

    We present comprehensive testing of solvent representation in quantum mechanics (QM)-based scoring of protein-ligand affinities. To this aim, we prepared 21 new inhibitors of cyclin-dependent kinase 2 (CDK2) with the pyrazolo[1,5-a]pyrimidine core, whose activities spanned three orders of magnitude. The crystal structure of a potent inhibitor bound to the active CDK2/cyclin A complex revealed that the biphenyl substituent at position 5 of the pyrazolo[1,5-a]pyrimidine scaffold was located in a previously unexplored pocket and that six water molecules resided in the active site. Using molecular dynamics, protein-ligand interactions and active-site water H-bond networks as well as thermodynamics were probed. Thereafter, all the inhibitors were scored by the QM approach utilizing the COSMO implicit solvent model. Such a standard treatment failed to produce a correlation with the experiment (R(2) = 0.49). However, the addition of the active-site waters resulted in significant improvement (R(2) = 0.68). The activities of the compounds could thus be interpreted by taking into account their specific noncovalent interactions with CDK2 and the active-site waters. In summary, using a combination of several experimental and theoretical approaches we demonstrate that the inclusion of explicit solvent effects enhance QM/COSMO scoring to produce a reliable structure-activity relationship with physical insights. More generally, this approach is envisioned to contribute to increased accuracy of the computational design of novel inhibitors.

  14. Structure-guided discovery of cyclin-dependent kinase inhibitors

    SciTech Connect

    Fischmann, Thierry O.; Hruza, Alan; Duca, Jose S.; Ramanathan, Lata; Mayhood, Todd; Windsor, William T.; Le, Hung V.; Guzi, Timothy J.; Dwyer, Michael P.; Paruch, Kamil; Doll, Ronald J.; Lees, Emma; Parry, David; Seghezzi, Wolfgang; Madison, Vincent

    2008-10-02

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

  15. Curcumin suppresses proliferation of colon cancer cells by targeting CDK2.

    PubMed

    Lim, Tae-Gyu; Lee, Sung-Young; Huang, Zunnan; Lim, Do Young; Chen, Hanyong; Jung, Sung Keun; Bode, Ann M; Lee, Ki Won; Dong, Zigang

    2014-04-01

    Curcumin, the yellow pigment of turmeric found in Southeast Indian food, is one of the most popular phytochemicals for cancer prevention. Numerous reports have demonstrated modulation of multiple cellular signaling pathways by curcumin and its molecular targets in various cancer cell lines. To identify a new molecular target of curcumin, we used shape screening and reverse docking to screen the Protein Data Bank against curcumin. Cyclin-dependent kinase 2 (CDK2), a major cell-cycle protein, was identified as a potential molecular target of curcumin. Indeed, in vitro and ex vivo kinase assay data revealed a dramatic suppressive effect of curcumin on CDK2 kinase activity. Furthermore, curcumin induced G1 cell-cycle arrest, which is regulated by CDK2 in HCT116 cells. Although the expression levels of CDK2 and its regulatory subunit, cyclin E, were not changed, the phosphorylation of retinoblastoma (Rb), a well-known CDK2 substrate, was reduced by curcumin. Because curcumin induced cell-cycle arrest, we investigated the antiproliferative effect of curcumin on HCT116 colon cancer cells. In this experiment, curcumin suppressed HCT116 cell proliferation effectively. To determine whether CDK2 is a direct target of curcumin, CDK2 expression was knocked down in HCT116 cells. As expected, HCT116 sh-CDK2 cells exhibited G1 arrest and reduced proliferation. Because of the low levels of CDK2 in HCT116 sh-CDK2 cells, the effects of curcumin on G1 arrest and cell proliferation were not substantially relative to HCT116 sh-control cells. From these results, we identified CDK2 as a direct target of curcumin in colon cancer cells.

  16. STAR syndrome-associated CDK10/Cyclin M regulates actin network architecture and ciliogenesis.

    PubMed

    Guen, Vincent J; Gamble, Carly; Perez, Dahlia E; Bourassa, Sylvie; Zappel, Hildegard; Gärtner, Jutta; Lees, Jacqueline A; Colas, Pierre

    2016-01-01

    CDK10/CycM is a protein kinase deficient in STAR (toe Syndactyly, Telecanthus and Anogenital and Renal malformations) syndrome, which results from mutations in the X-linked FAM58A gene encoding Cyclin M. The biological functions of CDK10/CycM and etiology of STAR syndrome are poorly understood. Here, we report that deficiency of CDK10/Cyclin M promotes assembly and elongation of primary cilia. We establish that this reflects a key role for CDK10/Cyclin M in regulation of actin network organization, which is known to govern ciliogenesis. In an unbiased screen, we identified the RhoA-associated kinase PKN2 as a CDK10/CycM phosphorylation substrate. We establish that PKN2 is a bone fide regulator of ciliogenesis, acting in a similar manner to CDK10/CycM. We discovered that CDK10/Cyclin M binds and phosphorylates PKN2 on threonines 121 and 124, within PKN2's core RhoA-binding domain. Furthermore, we demonstrate that deficiencies in CDK10/CycM or PKN2, or expression of a non-phosphorylatable version of PKN2, destabilize both the RhoA protein and the actin network architecture. Importantly, we established that ectopic expression of RhoA is sufficient to override the induction of ciliogenesis resulting from CDK10/CycM knockdown, indicating that RhoA regulation is critical for CDK10/CycM's negative effect on ciliogenesis. Finally, we show that kidney sections from a STAR patient display dilated renal tubules and abnormal, elongated cilia. Altogether, these results reveal CDK10/CycM as a key regulator of actin dynamics and a suppressor of ciliogenesis through phosphorylation of PKN2 and promotion of RhoA signaling. Moreover, they suggest that STAR syndrome is a ciliopathy.

  17. Cloning of three novel neuronal Cdk5 activator binding proteins.

    PubMed

    Ching, Y P; Qi, Z; Wang, J H

    2000-01-25

    Neuronal Cdc2-like kinase (Nclk) is involved in the regulation of neuronal differentiation and neuro-cytoskeleton dynamics. The active kinase consists of a catalytic subunit, Cdk5, and a 25 kDa activator protein (p25nck5a) derived from a 35 kDa neuronal-specific protein (p35nck5a). As an extension of our previous study (Qi, Z., Tang, D., Zhu, X., Fujita, D.J., Wang, J.H., 1998. Association of neurofilament proteins with neuronal Cdk5 activator. J. Biol. Chem. 270, 2329-2335), which showed that neurofilament is one of the p35nck5a-associated proteins, we now report the isolation of three other novel p35nck5a-associated proteins using the yeast two-hybrid screen. The full-length forms of these three novel proteins, designated C42, C48 and C53, have a molecular mass of 66, 24, and 57 kDa, respectively. Northern analysis indicates that these novel proteins are widely expressed in human tissues, including the heart, brain, skeletal muscle, placenta, lung, liver, kidney and pancreas. The bacterially expressed glutathione S-transferase (GST)-fusion forms of these three proteins were able to co-precipitate p35nck5a complexed with Cdk5 from insect cell lysate. Among these three proteins, only C48 and C53 can be phosphorylated by Nclk, suggesting that they may be the substrates of Nclk. Sequence homology searches have suggested that the C48 protein is marginally related to restin protein, whereas the C42 protein has homologues of unknown function in Caenorhabditis elegans and Arabidopsis thaliana.

  18. Phosphorylation of EZH2 at T416 by CDK2 contributes to the malignancy of triple negative breast cancers

    PubMed Central

    Yang, Cheng-Chieh; LaBaff, Adam; Wei, Yongkun; Nie, Lei; Xia, Weiya; Huo, Longfei; Yamaguchi, Hirohito; Hsu, Yi-Hsin; Hsu, Jennifer L; Liu, Dongping; Lang, Jingyu; Du, Yi; Lien, Huang-Chun; Li, Long-Yuan; Deng, Rong; Chan, Li-Chuan; Yao, Jun; Kleer, Celina G; Hortobagyi, Gabriel N; Hung, Mien-Chie

    2015-01-01

    Triple-negative breast cancer (TNBC), which is closely related to basal-like breast cancer, is a highly aggressive subtype of breast cancer that initially responds to chemotherapy but eventually develops resistance. This presents a major clinical challenge as there are currently no effective targeted therapies available due to its lack of HER2 and estrogen receptor expression. Here, we show that cyclin E and the enhancer of zeste 2 (EZH2) are closely co-expressed in TNBC patients, and cyclin E/CDK2 phosphorylates EZH2 at T416 (pT416-EZH2) in vivo. Phosphorylation of EZH2 at T416 enhances the ability of EZH2 to promote TNBC cell migration/invasion, tumorsphere formation, and in vivo tumor growth. In addition, high pT416-EZH2 correlates with poorer survival in TNBC patients. These findings suggest that pT416 has the potential to serve as a therapeutic biomarker for the aggressive forms of breast cancer and provide a rationale for the use of CDK2 inhibitors to treat TNBC. PMID:26279746

  19. ClC-3 Chloride Channel Proteins Regulate the Cell Cycle by Up-regulating cyclin D1-CDK4/6 through Suppressing p21/p27 Expression in Nasopharyngeal Carcinoma Cells

    PubMed Central

    Ye, Dong; Luo, Hai; Lai, Zhouyi; Zou, Lili; Zhu, Linyan; Mao, Jianwen; Jacob, Tim; Ye, Wencai; Wang, Liwei; Chen, Lixin

    2016-01-01

    It was shown in this study that knockdown of ClC-3 expression by ClC-3 siRNA prevented the activation of hypotonicity-induced chloride currents, and arrested cells at the G0/G1 phase in nasopharyngeal carcinoma CNE-2Z cells. Reconstitution of ClC-3 expression with ClC-3 expression plasmids could rescue the cells from the cell cycle arrest caused by ClC-3 siRNA treatments. Transfection of cells with ClC-3 siRNA decreased the expression of cyclin D1, cyclin dependent kinase 4 and 6, and increased the expression of cyclin dependent kinase inhibitors (CDKIs), p21 and p27. Pretreatments of cells with p21 and p27 siRNAs depleted the inhibitory effects of ClC-3 siRNA on the expression of CDK4 and CDK6, but not on that of cyclin D1, indicating the requirement of p21 and p27 for the inhibitory effects of ClC-3 siRNA on CDK4 and CDK6 expression. ClC-3 siRNA inhibited cells to progress from the G1 phase to the S phase, but pretreatments of cells with p21 and p27 siRNAs abolished the inhibitory effects of ClC-3 siRNA on the cell cycle progress. Our data suggest that ClC-3 may regulate cell cycle transition between G0/G1 and S phases by up-regulation of the expression of CDK4 and CDK6 through suppression of p21 and p27 expression. PMID:27451945

  20. ClC-3 Chloride Channel Proteins Regulate the Cell Cycle by Up-regulating cyclin D1-CDK4/6 through Suppressing p21/p27 Expression in Nasopharyngeal Carcinoma Cells.

    PubMed

    Ye, Dong; Luo, Hai; Lai, Zhouyi; Zou, Lili; Zhu, Linyan; Mao, Jianwen; Jacob, Tim; Ye, Wencai; Wang, Liwei; Chen, Lixin

    2016-07-25

    It was shown in this study that knockdown of ClC-3 expression by ClC-3 siRNA prevented the activation of hypotonicity-induced chloride currents, and arrested cells at the G0/G1 phase in nasopharyngeal carcinoma CNE-2Z cells. Reconstitution of ClC-3 expression with ClC-3 expression plasmids could rescue the cells from the cell cycle arrest caused by ClC-3 siRNA treatments. Transfection of cells with ClC-3 siRNA decreased the expression of cyclin D1, cyclin dependent kinase 4 and 6, and increased the expression of cyclin dependent kinase inhibitors (CDKIs), p21 and p27. Pretreatments of cells with p21 and p27 siRNAs depleted the inhibitory effects of ClC-3 siRNA on the expression of CDK4 and CDK6, but not on that of cyclin D1, indicating the requirement of p21 and p27 for the inhibitory effects of ClC-3 siRNA on CDK4 and CDK6 expression. ClC-3 siRNA inhibited cells to progress from the G1 phase to the S phase, but pretreatments of cells with p21 and p27 siRNAs abolished the inhibitory effects of ClC-3 siRNA on the cell cycle progress. Our data suggest that ClC-3 may regulate cell cycle transition between G0/G1 and S phases by up-regulation of the expression of CDK4 and CDK6 through suppression of p21 and p27 expression.

  1. Prevalence of Germline BAP1, CDKN2A, and CDK4 Mutations in an Australian Population-Based Sample of Cutaneous Melanoma Cases.

    PubMed

    Aoude, Lauren G; Gartside, Michael; Johansson, Peter; Palmer, Jane M; Symmons, Judith; Martin, Nicholas G; Montgomery, Grant W; Hayward, Nicholas K

    2015-04-01

    Mutations in Cyclin-Dependent Kinase Inhibitor 2A (CDKN2A) and Cyclin-Dependent Kinase 4 (CDK4) contribute to susceptibility in approximately 40% of high-density cutaneous melanoma (CMM) families and about 2% of unselected CMM cases. BRCA-1 associated protein-1 (BAP1) has been more recently shown to predispose to CMM and uveal melanoma (UMM) in some families; however, its contribution to CMM development in the general population is unreported. We sought to determine the contribution of these genes to CMM susceptibility in a population-based sample of cases from Australia. We genotyped 1,109 probands from Queensland families and found that approximately 1.31% harbored mutations in CDKN2A, including some with novel missense mutations (p.R22W, p.G35R and p.I49F). BAP1 missense variants occurred in 0.63% of cases but no CDK4 variants were observed in the sample. This is the first estimate of the contribution of BAP1 and CDK4 to a population-based sample of CMM and supports the previously reported estimate of CDKN2A germline mutation prevalence.

  2. CDK-1 Inhibition in G2 Stabilizes Kinetochore-Microtubules in the following Mitosis

    PubMed Central

    Gayek, A. Sophia; Ohi, Ryoma

    2016-01-01

    Cell proliferation is driven by cyclical activation of cyclin-dependent kinases (CDKs), which produce distinct biochemical cell cycle phases. Mitosis (M phase) is orchestrated by CDK-1, complexed with mitotic cyclins. During M phase, chromosomes are segregated by a bipolar array of microtubules called the mitotic spindle. The essential bipolarity of the mitotic spindle is established by the kinesin-5 Eg5, but factors influencing the maintenance of spindle bipolarity are not fully understood. Here, we describe an unexpected link between inhibiting CDK-1 before mitosis and bipolar spindle maintenance. Spindles in human RPE-1 cells normally collapse to monopolar structures when Eg5 is inhibited at metaphase. However, we found that inhibition of CDK-1 in the G2 phase of the cell cycle improved the ability of RPE-1 cells to maintain spindle bipolarity without Eg5 activity in the mitosis immediately after release from CDK-1 inhibition. This improved bipolarity maintenance correlated with an increase in the stability of kinetochore-microtubules, the subset of microtubules that link chromosomes to the spindle. The improvement in bipolarity maintenance after CDK-1 inhibition in G2 required both the kinesin-12 Kif15 and increased stability of kinetochore-microtubules. Consistent with increased kinetochore-microtubule stability, we find that inhibition of CDK-1 in G2 impairs mitotic fidelity by increasing the incidence of lagging chromosomes in anaphase. These results suggest that inhibition of CDK-1 in G2 causes unpredicted effects in mitosis, even after CDK-1 inhibition is relieved. PMID:27281342

  3. Chemoprevention utility of silibinin and Cdk4 pathway inhibition in Apc−/+ mice

    PubMed Central

    2013-01-01

    Background Colorectal cancer (CRC) is the second leading cause of death from cancer in the United States. Colorectal cancers have a prolonged latency following initiation that may span decades providing ample time for implementing a chemoprevention strategy that could block or reverse the progression to CRC. Cdk4 pathway alterations have been linked to a number of cancers including CRC. In these experiments we focused on the Cdk4 pathway and its role in intestinal tumorigenesis as a possible target in chemoprevention strategies. Methods We evaluated the effect of Cdk4 blockade on the prevention of intestinal tumor formation by crossing Cdk4−/− mice to Apc−/+ mice. In addition, we tested the effect of the dietary compound silibinin on the Cdk4 pathway in Apc−/+ mice and HT-29 colon cancer cells in culture. Results Cdk4−/− mice backcrossed to Apc−/+ mice reduced intestinal adenoma formation compared to Apc−/+ controls. Silibinin effectively targeted the Cdk4 pathway causing hypophosphorylation of the retinoblastoma protein, inhibited cell growth, and induced apoptosis. As a result silibinin blocked the development of intestinal adenomas by 52% in this genetic model (Apc−/+ mice) of early events in colorectal cancer formation. No toxic abnormalities were detected in mice which received silibinin. Conclusions Modification of the Cdk4 pathway using a natural plant-derived compound such as silibinin may be a useful chemopreventive strategy for colorectal carcinomas. PMID:23530816

  4. Identification of New Substrates for Breast Tumor Specific LMW Cyclin E/CDK2 Kinase

    DTIC Science & Technology

    2012-09-01

    Cyclin E/CDK2 phosphorylation of Hbo1 does not affect the HAT activity of Hbo1 CDK1 phosphorylates Hbo1 at T85/T88 to create a docking site for polo ...values first subtracted from a water control and then normalized to the positive control, which used HeLa nuclear extract as a source of HAT

  5. 55K isoform of CDK9 associates with Ku70 and is involved in DNA repair

    SciTech Connect

    Liu, Hongbing; Herrmann, Christine H.; Chiang, Karen; Sung, Tzu-Ling; Moon, Sung-Hwan; Donehower, Lawrence A.; Rice, Andrew P.

    2010-06-25

    Positive elongation factor b (P-TEFb) is a cellular protein kinase that is required for RNA polymerase II (RNAP II) transcriptional elongation of protein coding genes. P-TEFb is a set of different molecular complexes, each containing CDK9 as the catalytic subunit. There are two isoforms of the CDK9 protein - the major 42 KDa CDK9 isoform and the minor 55KDa isoform that is translated from an in-frame mRNA that arises from an upstream transcriptional start site. We found that shRNA depletion of the 55K CDK9 protein in HeLa cells induces apoptosis and double-strand DNA breaks (DSBs). The levels of apoptosis and DSBs induced by the depletion were reduced by expression of a 55K CDK9 protein variant resistant to the shRNA, indicating that these phenotypes are the consequence of depletion of the 55K protein and not off-target effects. We also found that the 55K CDK9 protein, but not the 42K CDK9 protein, specifically associates with Ku70, a protein involved in DSB repair. Our findings suggest that the 55K CDK9 protein may function in repair of DNA through an association with Ku70.

  6. The cell cycle rallies the transcription cycle: Cdc28/Cdk1 is a cell cycle-regulated transcriptional CDK.

    PubMed

    Chymkowitch, Pierre; Enserink, Jorrit M

    2013-01-01

    In the budding yeast Saccharomyces cerevisiae, the cyclin-dependent kinases (CDKs) Kin28, Bur1 and Ctk1 regulate basal transcription by phosphorylating the carboxyl-terminal domain (CTD) of RNA polymerase II. However, very little is known about the involvement of the cell cycle CDK Cdc28 in the transcription process. We have recently shown that, upon cell cycle entry, Cdc28 kinase activity boosts transcription of a subset of genes by directly stimulating the basal transcription machinery. Here, we discuss the biological significance of this finding and give our view of the kinase-dependent role of Cdc28 in regulation of RNA polymerase II.

  7. Hcm1 integrates signals from Cdk1 and calcineurin to control cell proliferation.

    PubMed

    Arsenault, Heather E; Roy, Jagoree; Mapa, Claudine E; Cyert, Martha S; Benanti, Jennifer A

    2015-10-15

    Cyclin-dependent kinase (Cdk1) orchestrates progression through the cell cycle by coordinating the activities of cell-cycle regulators. Although phosphatases that oppose Cdk1 are likely to be necessary to establish dynamic phosphorylation, specific phosphatases that target most Cdk1 substrates have not been identified. In budding yeast, the transcription factor Hcm1 activates expression of genes that regulate chromosome segregation and is critical for maintaining genome stability. Previously we found that Hcm1 activity and degradation are stimulated by Cdk1 phosphorylation of distinct clusters of sites. Here we show that, upon exposure to environmental stress, the phosphatase calcineurin inhibits Hcm1 by specifically removing activating phosphorylations and that this regulation is important for cells to delay proliferation when they encounter stress. Our work identifies a mechanism by which proliferative signals from Cdk1 are removed in response to stress and suggests that Hcm1 functions as a rheostat that integrates stimulatory and inhibitory signals to control cell proliferation.

  8. CDK6 binds and promotes the degradation of the EYA2 protein

    PubMed Central

    Kohrt, Dawn; Crary, Jennifer; Zimmer, Marc; Patrick, Aaron N; Ford, Heide L; Hinds, Philip W; Grossel, Martha J

    2014-01-01

    Cyclin-dependent kinase 6 (Cdk6) is a D-Cyclin-activated kinase that is directly involved in driving the cell cycle through inactivation of pRB in G1 phase. Increasingly, evidence suggests that CDK6, while directly driving the cell cycle, may only be essential for proliferation of specialized cell types, agreeing with the notion that CDK6 also plays an important role in differentiation. Here, evidence is presented that CDK6 binds to and promotes degradation of the EYA2 protein. The EYA proteins are a family of proteins that activate genes essential for the development of multiple organs, regulate cell proliferation, and are misregulated in several types of cancer. This interaction suggests that CDK6 regulates EYA2 activity, a mechanism that could be important in development and in cancer. PMID:24196439

  9. Cdk2 loss accelerates precursor differentiation and remyelination in the adult central nervous system

    PubMed Central

    Caillava, Céline; Vandenbosch, Renaud; Jablonska, Beata; Deboux, Cyrille; Spigoni, Giulia; Gallo, Vittorio; Malgrange, Brigitte

    2011-01-01

    The specific functions of intrinsic regulators of oligodendrocyte progenitor cell (OPC) division are poorly understood. Type 2 cyclin-dependent kinase (Cdk2) controls cell cycle progression of OPCs, but whether it acts during myelination and repair of demyelinating lesions remains unexplored. Here, we took advantage of a viable Cdk2−/− mutant mouse to investigate the function of this cell cycle regulator in OPC proliferation and differentiation in normal and pathological conditions. During central nervous system (CNS) development, Cdk2 loss does not affect OPC cell cycle, oligodendrocyte cell numbers, or myelination. However, in response to CNS demyelination, it clearly alters adult OPC renewal, cell cycle exit, and differentiation. Importantly, Cdk2 loss accelerates CNS remyelination of demyelinated axons. Thus, Cdk2 is dispensable for myelination but is important for adult OPC renewal, and could be one of the underlying mechanisms that drive adult progenitors to differentiate and thus regenerate myelin. PMID:21502361

  10. CDK1 phosphorylates WRN at collapsed replication forks

    PubMed Central

    Palermo, Valentina; Rinalducci, Sara; Sanchez, Massimo; Grillini, Francesca; Sommers, Joshua A.; Brosh, Robert M.; Zolla, Lello; Franchitto, Annapaola; Pichierri, Pietro

    2016-01-01

    Regulation of end-processing is critical for accurate repair and to switch between homologous recombination (HR) and non-homologous end joining (NHEJ). End resection is a two-stage process but very little is known about regulation of the long-range resection, especially in humans. WRN participates in one of the two alternative long-range resection pathways mediated by DNA2 or EXO1. Here we demonstrate that phosphorylation of WRN by CDK1 is essential to perform DNA2-dependent end resection at replication-related DSBs, promoting HR, replication recovery and chromosome stability. Mechanistically, S1133 phosphorylation of WRN is dispensable for relocalization in foci but is involved in the interaction with the MRE11 complex. Loss of WRN phosphorylation negatively affects MRE11 foci formation and acts in a dominant negative manner to prevent long-range resection altogether, thereby licensing NHEJ at collapsed forks. Collectively, we unveil a CDK1-dependent regulation of the WRN-DNA2-mediated resection and identify an undescribed function of WRN as a DSB repair pathway switch. PMID:27634057

  11. Inhibition of calpain-regulated p35/cdk5 plays a central role in sildenafil-induced protection against chemical hypoxia produced by malonate.

    PubMed

    Barros-Miñones, Lucía; Martín-de-Saavedra, Dolores; Perez-Alvarez, Sergio; Orejana, Lourdes; Suquía, Verónica; Goñi-Allo, Beatriz; Hervias, Isabel; López, Manuela G; Jordan, Joaquin; Aguirre, Norberto; Puerta, Elena

    2013-06-01

    Phosphodiesterase 5 (PDE5) inhibitors have recently been reported to exert beneficial effects against ischemia-reperfusion injury in several organs but their neuroprotective effects in brain stroke models are scarce. The present study was undertaken to assess the effects of sildenafil against cell death caused by intrastriatal injection of malonate, an inhibitor of succinate dehydrogenase; which produces both energy depletion and lesions similar to those seen in cerebral ischemia. Our data demonstrate that sildenafil (1.5mg/kg by mouth (p.o.)), given 30min before malonate (1.5μmol/2μL), significantly decreased the lesion volume caused by this toxin. This protective effect can be probably related to the inhibition of excitotoxic pathways. Thus, malonate induced the activation of the calcium-dependent protease, calpain and the cyclin-dependent kinase 5, cdk5; which resulted in the hyperphosphorylation of tau and the cleavage of the protective transcription factor, myocyte enhancer factor 2, MEF2. All these effects were also significantly reduced by sildenafil pre-treatment, suggesting that sildenafil protects against malonate-induced cell death through the regulation of the calpain/p25/cdk5 signaling pathway. Similar findings were obtained using inhibitors of calpain or cdk5, further supporting our contention. Sildenafil also increased MEF2 phosphorylation and Bcl-2/Bax and Bcl-xL/Bax ratios, effects that might as well contribute to prevent cell death. Finally, sildenafil neuroprotection was extended not only to rat hippocampal slices subjected to oxygen and glucose deprivation when added at the time of reoxygenation, but also, in vivo when administered after malonate injection. Thus, the therapeutic window for sildenafil against malonate-induced hypoxia was set at 3h.

  12. Putting one step before the other: distinct activation pathways for Cdk1 and Cdk2 bring order to the mammalian cell cycle.

    PubMed

    Merrick, Karl A; Fisher, Robert P

    2010-02-15

    Eukaryotic cell division is controlled by the activity of cyclin-dependent kinases (CDKs). Cdk1 and Cdk2, which function at different stages of the mammalian cell cycle, both require cyclin-binding and phosphorylation of the activation (T-) loop for full activity, but differ with respect to the order in which the two steps occur in vivo. To form stable complexes with either of its partners-cyclins A and B-Cdk1 must be phosphorylated on its T-loop, but that phosphorylation in turn depends on the presence of cyclin. Cdk2 can follow a kinetically distinct path to activation in which T-loop phosphorylation precedes cyclin-binding, and thereby out-compete the more abundant Cdk1 for limiting amounts of cyclin A. Mathematical modeling suggests this could be a principal basis for the temporal ordering of CDK activation during S phase, which may dictate the sequence in which replication origins fire. Still to be determined are how: (1) the activation machinery discriminates between closely related CDKs, and (2) coordination of the cell cycle is affected when this mechanism of pathway insulation breaks down.

  13. MD simulation of the Tat/Cyclin T1/CDK9 complex revealing the hidden catalytic cavity within the CDK9 molecule upon Tat binding

    PubMed Central

    Asamitsu, Kaori; Hirokawa, Takatsugu; Okamoto, Takashi

    2017-01-01

    In this study, we applied molecular dynamics (MD) simulation to analyze the dynamic behavior of the Tat/CycT1/CDK9 tri-molecular complex and revealed the structural changes of P-TEFb upon Tat binding. We found that Tat could deliberately change the local flexibility of CycT1. Although the structural coordinates of the H1 and H2 helices did not substantially change, H1ʹ, H2ʹ, and H3ʹ exhibited significant changes en masse. Consequently, the CycT1 residues involved in Tat binding, namely Tat-recognition residues (TRRs), lost their flexibility with the addition of Tat to P-TEFb. In addition, we clarified the structural variation of CDK9 in complex with CycT1 in the presence or absence of Tat. Interestingly, Tat addition significantly reduced the structural variability of the T-loop, thus consolidating the structural integrity of P-TEFb. Finally, we deciphered the formation of the hidden catalytic cavity of CDK9 upon Tat binding. MD simulation revealed that the PITALRE signature sequence of CDK9 flips the inactive kinase cavity of CDK9 into the active form by connecting with Thr186, which is crucial for its activity, thus presumably recruiting the substrate peptide such as the C-terminal domain of RNA pol II. These findings provide vital information for the development of effective novel anti-HIV drugs with CDK9 catalytic activity as the target. PMID:28178316

  14. CDK5-mediated phosphorylation of p19INK4d avoids DNA damage-induced neurodegeneration in mouse hippocampus and prevents loss of cognitive functions.

    PubMed

    Ogara, María Florencia; Belluscio, Laura M; de la Fuente, Verónica; Berardino, Bruno G; Sonzogni, Silvina V; Byk, Laura; Marazita, Mariela; Cánepa, Eduardo T

    2014-07-01

    DNA damage, which perturbs genomic stability, has been linked to cognitive decline in the aging human brain, and mutations in DNA repair genes have neurological implications. Several studies have suggested that DNA damage is also increased in brain disorders such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. However, the precise mechanisms connecting DNA damage with neurodegeneration remain poorly understood. CDK5, a critical enzyme in the development of the central nervous system, phosphorylates a number of synaptic proteins and regulates dendritic spine morphogenesis, synaptic plasticity and learning. In addition to these physiological roles, CDK5 has been involved in the neuronal death initiated by DNA damage. We hypothesized that p19INK4d, a member of the cell cycle inhibitor family INK4, is involved in a neuroprotective mechanism activated in response to DNA damage. We found that in response to genotoxic injury or increased levels of intracellular calcium, p19INK4d is transcriptionally induced and phosphorylated by CDK5 which provides it with greater stability in postmitotic neurons. p19INK4d expression improves DNA repair, decreases apoptosis and increases neuronal survival under conditions of genotoxic stress. Our in vivo experiments showed that decreased levels of p19INK4d rendered hippocampal neurons more sensitive to genotoxic insult resulting in the loss of cognitive abilities that rely on the integrity of this brain structure. We propose a feedback mechanism by which the neurotoxic effects of CDK5-p25 activated by genotoxic stress or abnormal intracellular calcium levels are counteracted by the induction and stabilization of p19INK4d protein reducing the adverse consequences on brain functions.

  15. A flexible multiplex bead-based assay for detecting germline CDKN2A and CDK4 variants in melanoma-prone kindreds.

    PubMed

    Lang, Julie M; Shennan, Michael; Njauw, Jenny C-N; Luo, Su; Bishop, Julia N; Harland, Mark; Hayward, Nicholas K; Tucker, Margaret A; Goldstein, Alisa M; Landi, Maria T; Puig, Susana; Gruis, Nelleke A; Bergman, Wilma; Bianchi-Scarra, Giovanna; Ghiorzo, Paola; Hogg, David; Tsao, Hensin

    2011-02-01

    The presence of recurrent high-risk mutations in cyclin-dependent kinase inhibitor 2A/cyclin-dependent kinase 4 (CDKN2A/CDK4) among melanoma-prone families suggests that a high-throughput, multiplex assay could serve as an effective initial screening tool. To this end, we have developed a multiplex bead-based assay for high-throughput CDKN2A/CDK4 genotyping in the context of familial melanoma. Genomic DNA from 1,603 subjects (1,005 in training set and 598 in validation set) were amplified by multiplex PCR using five CDKN2A/CDK4 primer sets followed by multiplex allele-specific primer extension for 39 distinct germline variants. The products were then sorted and analyzed using the Luminex xMAP system. Genotypes were compared with previously determined sequence data. In the Toronto training cohort, all 145 samples with known variants were detected by the bead assay (100% concordance). Analysis of the 598 samples from the GenoMEL validation set led to identification of 150/155 expected variants (96.77%). Overall, the bead assay correctly genotyped 1,540/1,603 (96.07%) of all individuals in the study and 1,540/1,545 (99.68%) of individuals whose variants were represented in the probe set. Out of a total of 62,517 allelic calls, 62,512 (99.99%) were correctly assigned. The multiplex bead-based assay is an accurate method for genotyping CDKN2A/CDK4 variants and is potentially useful in genotyping low-to-moderate melanoma risk single-nucleotide polymorphisms.

  16. Prolonged induction of p21Cip1/WAF1/CDK2/PCNA complex by epidermal growth factor receptor activation mediates ligand-induced A431 cell growth inhibition

    PubMed Central

    1995-01-01

    Proliferation of some cultured human tumor cell lines bearing high numbers of epidermal growth factor (EGF) receptors is paradoxically inhibited by EGF in nanomolar concentrations. In the present study, we have investigated the biochemical mechanism of growth inhibition in A431 human squamous carcinoma cells exposed to exogenous EGF. In parallel, we studied a selected subpopulation, A431-F, which is resistant to EGF-mediated growth inhibition. We observed a marked reduction in cyclin-dependent kinase-2 (CDK2) activity when A431 and A431-F cells were cultured with 20 nM EGF for 4 h. After further continuous exposure of A431 cells to EGF, the CDK2 activity remained at a low level and was accompanied by persistent G1 arrest. In contrast, the early reduced CDK2 activity and G1 accumulation in A431-F cells was only transient. We found that, at early time points (4-8 h), EGF induces p21Cip1/WAF1 mRNA and protein expression in both EGF-sensitive A431 cells and EGF-resistant A431-F cells. But only in A431 cells, was p21Cip1/WAF1 expression sustained at a significantly increased level for up to 5 d after addition of EGF. Induction of p21Cip1/WAF1 by EGF could be inhibited by a specific EGF receptor tyrosine kinase inhibitor, tyrphostin AG1478, suggesting that p21Cip1/WAF1 induction was a consequence of receptor tyrosine kinase activation by EGF. We also demonstrated that the increased p21Cip1/WAF1 was associated with both CDK2 and proliferating cell nuclear antigen (PCNA). Taken together, our results demonstrate that p21Cip1/WAF1 is an important mediator of EGF-induced G1 arrest and growth inhibition in A431 cells. PMID:7559780

  17. Cyclin-dependent kinase inhibitors in maize endosperm and their potential role in endoreduplication.

    PubMed

    Coelho, Cintia M; Dante, Ricardo A; Sabelli, Paolo A; Sun, Yuejin; Dilkes, Brian P; Gordon-Kamm, William J; Larkins, Brian A

    2005-08-01

    Two maize (Zea mays) cyclin-dependent kinase (CDK) inhibitors, Zeama;KRP;1 and Zeama;KRP;2, were characterized and shown to be expressed in developing endosperm. Similar to the CDK inhibitors in Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum), the maize proteins contain a carboxy-terminal region related to the inhibitory domain of the mammalian Cip/Kip inhibitors. Zeama;KRP;1 is present in the endosperm between 7 and 21 d after pollination, a period that encompasses the onset of endoreduplication, while the Zeama;KRP;2 protein declines during this time. Nevertheless, Zeama;KRP;1 accounts for only part of the CDK inhibitory activity that peaks coincident with the endoreduplication phase of endosperm development. In vitro assays showed that Zeama;KRP;1 and Zeama;KRP;2 are able to inhibit endosperm Cdc2-related CKD activity that associates with p13(Suc1). They were also shown to specifically inhibit cyclin A1;3- and cyclin D5;1-associated CDK activities, but not cyclin B1;3/CDK. Overexpression of Zeama;KRP;1 in maize embryonic calli that ectopically expressed the wheat dwarf virus RepA protein, which counteracts retinoblastoma-related protein function, led to an additional round of DNA replication without nuclear division.

  18. Structure-based discovery of the first allosteric inhibitors of cyclin-dependent kinase 2.

    PubMed

    Rastelli, Giulio; Anighoro, Andrew; Chripkova, Martina; Carrassa, Laura; Broggini, Massimo

    2014-01-01

    Allosteric targeting of protein kinases via displacement of the structural αC helix with type III allosteric inhibitors is currently gaining a foothold in drug discovery. Recently, the first crystal structure of CDK2 with an open allosteric pocket adjacent to the αC helix has been described, prospecting new opportunities to design more selective inhibitors, but the structure has not yet been exploited for the structure-based design of type III allosteric inhibitors. In this work we report the results of a virtual screening campaign that resulted in the discovery of the first-in-class type III allosteric ligands of CDK2. Using a combination of docking and post-docking analyses made with our tool BEAR, 7 allosteric ligands (hit rate of 20%) with micromolar affinity for CDK2 were identified, some of them inhibiting the growth of breast cancer cell lines in the micromolar range. Competition experiments performed in the presence of the ATP-competitive inhibitor staurosporine confirmed that the 7 ligands are truly allosteric, in agreement with their design. Of these, compound 2 bound CDK2 with an EC50 value of 3 μM and inhibited the proliferation of MDA-MB231 and ZR-75-1 breast cancer cells with IC50 values of approximately 20 μM, while compound 4 had an EC50 value of 71 μM and IC50 values around 4 μM. Remarkably, the most potent compound 4 was able to selectively inhibit CDK2-mediated Retinoblastoma phosphorylation, confirming that its mechanism of action is fully compatible with a selective inhibition of CDK2 phosphorylation in cells. Finally, hit expansion through analog search of the most potent inhibitor 4 revealed an additional ligand 4g with similar in vitro potency on breast cancer cells.

  19. Growth arrest by the cyclin-dependent kinase inhibitor p27Kip1 is abrogated by c-Myc.

    PubMed Central

    Vlach, J; Hennecke, S; Alevizopoulos, K; Conti, D; Amati, B

    1996-01-01

    We show here that c-Myc antagonizes the cyclin-dependent kinase (CDK) inhibitor p27Kip1. p27 expressed from recombinant retroviruses in Rat1 cells associated with and inhibited cyclin E/CDK2 complexes, induced accumulation of the pRb and p130 proteins in their hypophosphorylated forms, and arrested cells in G1. Prior expression of c-Myc prevented inactivation of cyclin E/CDK2 as well as dephosphorylation of pRb and p130, and allowed continuous cell proliferation in the presence of p27. This effect did not require ubiquitin-mediated degradation of p27. Myc altered neither the susceptibility of cyclin E/CDK2 to inhibition by p27, nor the intrinsic CDK-inhibitory activity of p27, but induced sequestration of p27 in a form unable to bind cyclin E/CDK2. Neither Myc itself nor other G1-cyclin/CDK complexes were directly responsible for p27 sequestration. Retroviral expression of G1 cyclins (D1-3, E or A) or of the Cdc25A phosphatase did not overcome p27-induced arrest. Growth rescue by Myc required dimerization with Max, DNA binding and an intact transcriptional activation domain, as previously shown for cellular transformation. We propose that this activity is mediated by the product of an as yet unknown Myc-Max target gene(s) and represents an essential aspect of Myc's mitogenic and oncogenic functions. Images PMID:8978686

  20. Preclinical Therapeutic Synergy of MEK1/2 and CDK4/6 Inhibition in Neuroblastoma.

    PubMed

    Hart, Lori S; Rader, JulieAnn; Raman, Pichai; Batra, Vandana; Russell, Mike R; Tsang, Matthew; Gagliardi, Maria; Chen, Lucy; Martinez, Daniel; Li, Yimei; Wood, Andrew; Kim, Sunkyu; Parasuraman, Sudha; Delach, Scott; Cole, Kristina A; Krupa, Shiva; Boehm, Markus; Peters, Malte; Caponigro, Giordano; Maris, John M

    2017-04-01

    Purpose: Neuroblastoma is treated with aggressive multimodal therapy, yet more than 50% of patients experience relapse. We recently showed that relapsed neuroblastomas frequently harbor mutations leading to hyperactivated ERK signaling and sensitivity to MEK inhibition therapy. Here we sought to define a synergistic therapeutic partner to potentiate MEK inhibition.Experimental Design: We first surveyed 22 genetically annotated human neuroblastoma-derived cell lines (from 20 unique patients) for sensitivity to the MEK inhibitor binimetinib. After noting an inverse correlation with sensitivity to ribociclib (CDK4/6 inhibitor), we studied the combinatorial effect of these two agents using proliferation assays, cell-cycle analysis, Ki67 immunostaining, time-lapse microscopy, and xenograft studies.Results: Sensitivity to binimetinib and ribociclib was inversely related (r = -0.58, P = 0.009). MYCN amplification status and expression were associated with ribociclib sensitivity and binimetinib resistance, whereas increased MAPK signaling was the main determinant of binimetinib sensitivity and ribociclib resistance. Treatment with both compounds resulted in synergistic or additive cellular growth inhibition in all lines tested and significant inhibition of tumor growth in three of four xenograft models of neuroblastoma. The augmented growth inhibition was attributed to diminished cell-cycle progression that was reversible upon removal of drugs.Conclusions: Here we demonstrate that combined binimetinib and ribociclib treatment shows therapeutic synergy across a broad panel of high-risk neuroblastoma preclinical models. These data support testing this combination therapy in relapsed high-risk neuroblastoma patients, with focus on cases with hyperactivated RAS-MAPK signaling. Clin Cancer Res; 23(7); 1785-96. ©2016 AACR.

  1. Differential Regulation of Progesterone Receptor-Mediated Transcription by CDK2 and DNA-PK.

    PubMed

    Treviño, Lindsey S; Bolt, Michael J; Grimm, Sandra L; Edwards, Dean P; Mancini, Michael A; Weigel, Nancy L

    2016-02-01

    Progesterone receptor (PR) function is altered by cell signaling, but the mechanisms of kinase-specific regulation are not well defined. To examine the role of cell signaling in the regulation of PR transcriptional activity, we have utilized a previously developed mammalian-based estrogen-response element promoter array cell model and automated cell imaging and analysis platform to visualize and quantify effects of specific kinases on different mechanistic steps of PR-mediated target gene activation. For these studies, we generated stable estrogen-response element array cell lines expressing inducible chimeric PR that contains a swap of the estrogen receptor-α DNA-binding domain for the DNA-binding domain of PR. We have focused on 2 kinases important for steroid receptor activity: cyclin-dependent kinase 2 and DNA-dependent protein kinase. Treatment with either a Cdk1/2 inhibitor (NU6102) or a DNA-dependent protein kinase inhibitor (NU7441) decreased hormone-mediated chromatin decondensation and transcriptional activity. Further, we observed a quantitative reduction in the hormone-mediated recruitment of select coregulator proteins with NU6102 that is not observed with NU7441. In parallel, we determined the effect of kinase inhibition on hormone-mediated induction of primary and mature transcripts of endogenous genes in T47D breast cancer cells. Treatment with NU6102 was much more effective than NU7441, in inhibiting induction of PR target genes that exhibit a rapid increase in primary transcript expression in response to hormone. Taken together, these results indicate that the 2 kinases regulate PR transcriptional activity by distinct mechanisms.

  2. Amygdalin blocks bladder cancer cell growth in vitro by diminishing cyclin A and cdk2.

    PubMed

    Makarević, Jasmina; Rutz, Jochen; Juengel, Eva; Kaulfuss, Silke; Reiter, Michael; Tsaur, Igor; Bartsch, Georg; Haferkamp, Axel; Blaheta, Roman A

    2014-01-01

    Amygdalin, a natural compound, has been used by many cancer patients as an alternative approach to treat their illness. However, whether or not this substance truly exerts an anti-tumor effect has never been settled. An in vitro study was initiated to investigate the influence of amygdalin (1.25-10 mg/ml) on the growth of a panel of bladder cancer cell lines (UMUC-3, RT112 and TCCSUP). Tumor growth, proliferation, clonal growth and cell cycle progression were investigated. The cell cycle regulating proteins cdk1, cdk2, cdk4, cyclin A, cyclin B, cyclin D1, p19, p27 as well as the mammalian target of rapamycin (mTOR) related signals phosphoAkt, phosphoRaptor and phosphoRictor were examined. Amygdalin dose-dependently reduced growth and proliferation in all three bladder cancer cell lines, reflected in a significant delay in cell cycle progression and G0/G1 arrest. Molecular evaluation revealed diminished phosphoAkt, phosphoRictor and loss of Cdk and cyclin components. Since the most outstanding effects of amygdalin were observed on the cdk2-cyclin A axis, siRNA knock down studies were carried out, revealing a positive correlation between cdk2/cyclin A expression level and tumor growth. Amygdalin, therefore, may block tumor growth by down-modulating cdk2 and cyclin A. In vivo investigation must follow to assess amygdalin's practical value as an anti-tumor drug.

  3. CDK14 expression is down-regulated by cigarette smoke in vivo and in vitro

    PubMed Central

    Pollack, Daniel; Xiao, Yuxuan; Shrivasatava, Vibha; Levy, Avi; Andrusier, Miriam; D’Armiento, Jeanine; Holz, Marina K.; Vigodner, Margarita

    2016-01-01

    In this study, DNA arrays have been employed to monitor gene expression patterns in testis of mice exposed to tobacco smoke for 24 weeks and compared to control animals. The results of the analysis revealed significant changes in expression of several genes that may have a role in spermatogenesis. Cdk14 was chosen for further characterization because of a suggested role in the testis and in regulation of Wnt signaling. RT-PCR analysis confirmed down regulation of Cdk14 in mice exposed to cigarette smoke (CS). Cdk14 is expressed in all testicular cells; spermatogonia- and Sertoli-derived cell lines treated with cigarette smoke extract (CSE) in vitro showed down-regulation of CDK14 mRNA and protein levels as well as down-regulation of β-catenin levels. CS-induced down-regulation of CDK14 mRNA and protein levels was also observed in several lung epithelium-derived cell lines including primary normal human bronchial epithelial cells (NHBE), suggesting that the effect is not restricted to the testis. Similar to testicular cells, CS-induced down-regulation of CDK14 in lung cells correlated with decreased levels of β-catenin, a finding suggesting impaired Wnt signaling. In the lungs, CDK14 was localized to the alveolar and bronchial epithelium. PMID:25680692

  4. Meier-Gorlin syndrome mutations disrupt an Orc1 CDK inhibitory domain and cause centrosome reduplication.

    PubMed

    Hossain, Manzar; Stillman, Bruce

    2012-08-15

    Like DNA replication, centrosomes are licensed to duplicate once per cell division cycle to ensure genetic stability. In addition to regulating DNA replication, the Orc1 subunit of the human origin recognition complex controls centriole and centrosome copy number. Here we report that Orc1 harbors a PACT centrosome-targeting domain and a separate domain that differentially inhibits the protein kinase activities of Cyclin E-CDK2 and Cyclin A-CDK2. A cyclin-binding motif (Cy motif) is required for Orc1 to bind Cyclin A and inhibit Cyclin A-CDK2 kinase activity but has no effect on Cyclin E-CDK2 kinase activity. In contrast, Orc1 inhibition of Cyclin E-CDK2 kinase activity occurs by a different mechanism that is affected by Orc1 mutations identified in Meier-Gorlin syndrome patients. The cyclin/CDK2 kinase inhibitory domain of Orc1, when tethered to the PACT domain, localizes to centrosomes and blocks centrosome reduplication. Meier-Gorlin syndrome mutations that disrupt Cyclin E-CDK2 kinase inhibition also allow centrosome reduplication. Thus, Orc1 contains distinct domains that control centrosome copy number and DNA replication. We suggest that the Orc1 mutations present in some Meier-Gorlin syndrome patients contribute to the pronounced microcephaly and dwarfism observed in these individuals by altering centrosome duplication in addition to DNA replication defects.

  5. Cdc14 phosphatases preferentially dephosphorylate a subset of cyclin-dependent kinase (Cdk) sites containing phosphoserine.

    PubMed

    Bremmer, Steven C; Hall, Hana; Martinez, Juan S; Eissler, Christie L; Hinrichsen, Thomas H; Rossie, Sandra; Parker, Laurie L; Hall, Mark C; Charbonneau, Harry

    2012-01-13

    Mitotic cell division is controlled by cyclin-dependent kinases (Cdks), which phosphorylate hundreds of protein substrates responsible for executing the division program. Cdk inactivation and reversal of Cdk-catalyzed phosphorylation are universal requirements for completing and exiting mitosis and resetting the cell cycle machinery. Mechanisms that define the timing and order of Cdk substrate dephosphorylation remain poorly understood. Cdc14 phosphatases have been implicated in Cdk inactivation and are thought to be generally specific for Cdk-type phosphorylation sites. We show that budding yeast Cdc14 possesses a strong and unusual preference for phosphoserine over phosphothreonine at Pro-directed sites in vitro. Using serine to threonine substitutions in the Cdk consensus sites of the Cdc14 substrate Acm1, we demonstrate that phosphoserine specificity exists in vivo. Furthermore, it appears to be a conserved property of all Cdc14 family phosphatases. An invariant active site residue was identified that sterically restricts phosphothreonine binding and is largely responsible for phosphoserine selectivity. Optimal Cdc14 substrates also possessed a basic residue at the +3 position relative to the phosphoserine, whereas substrates lacking this basic residue were not effectively hydrolyzed. The intrinsic selectivity of Cdc14 may help establish the order of Cdk substrate dephosphorylation during mitotic exit and contribute to roles in other cellular processes.

  6. Amygdalin Blocks Bladder Cancer Cell Growth In Vitro by Diminishing Cyclin A and cdk2

    PubMed Central

    Makarević, Jasmina; Rutz, Jochen; Juengel, Eva; Kaulfuss, Silke; Reiter, Michael; Tsaur, Igor; Bartsch, Georg; Haferkamp, Axel; Blaheta, Roman A.

    2014-01-01

    Amygdalin, a natural compound, has been used by many cancer patients as an alternative approach to treat their illness. However, whether or not this substance truly exerts an anti-tumor effect has never been settled. An in vitro study was initiated to investigate the influence of amygdalin (1.25–10 mg/ml) on the growth of a panel of bladder cancer cell lines (UMUC-3, RT112 and TCCSUP). Tumor growth, proliferation, clonal growth and cell cycle progression were investigated. The cell cycle regulating proteins cdk1, cdk2, cdk4, cyclin A, cyclin B, cyclin D1, p19, p27 as well as the mammalian target of rapamycin (mTOR) related signals phosphoAkt, phosphoRaptor and phosphoRictor were examined. Amygdalin dose-dependently reduced growth and proliferation in all three bladder cancer cell lines, reflected in a significant delay in cell cycle progression and G0/G1 arrest. Molecular evaluation revealed diminished phosphoAkt, phosphoRictor and loss of Cdk and cyclin components. Since the most outstanding effects of amygdalin were observed on the cdk2-cyclin A axis, siRNA knock down studies were carried out, revealing a positive correlation between cdk2/cyclin A expression level and tumor growth. Amygdalin, therefore, may block tumor growth by down-modulating cdk2 and cyclin A. In vivo investigation must follow to assess amygdalin's practical value as an anti-tumor drug. PMID:25136960

  7. Cdk1 plays matchmaker for the Polo-like kinase and its activator SPAT-1/Bora.

    PubMed

    Tavernier, Nicolas; Panbianco, Costanza; Gotta, Monica; Pintard, Lionel

    2015-08-03

    Mitosis is orchestrated by several protein kinases including Cdks, Plks and Aurora kinases. Despite considerable progress toward understanding the individual function of these protein kinases, how their activity is coordinated in space and time during mitosis is less well understood. In a recent article published in the Journal of Cell Biology, we show that CDK-1 regulates PLK-1 activity during mitosis in C. elegans embryos through multisite phosphorylation of the PLK-1 activator SPAT-1 (Aurora Borealis, Bora in human). SPAT-1 variants mutated on CDK-1 phosphorylation sites results in severe delays in mitotic entry, mimicking embryos lacking spat-1 or plk-1 function. We further show that SPAT-1 phosphorylation by CDK-1 promotes its binding to PLK-1 and stimulates PLK-1 phosphorylation on its activator T-loop by Aurora A kinase in vitro. Likewise, we find that phosphorylation of Bora by Cdk1 promotes phosphorylation of human Plk1 by Aurora A suggesting that this mechanism is conserved in humans. These results indicate that Cdk1 regulates Plk1 by boosting its kinase activity. Here we discuss these recent findings and open questions regarding the regulation of Plk1/PLK-1 by Cdk1/CDK-1 and Bora/SPAT-1.

  8. Cdk1 plays matchmaker for the Polo-like kinase and its activator SPAT-1/Bora

    PubMed Central

    Tavernier, Nicolas; Panbianco, Costanza; Gotta, Monica; Pintard, Lionel

    2015-01-01

    Mitosis is orchestrated by several protein kinases including Cdks, Plks and Aurora kinases. Despite considerable progress toward understanding the individual function of these protein kinases, how their activity is coordinated in space and time during mitosis is less well understood. In a recent article published in the Journal of Cell Biology, we show that CDK-1 regulates PLK-1 activity during mitosis in C. elegans embryos through multisite phosphorylation of the PLK-1 activator SPAT-1 (Aurora Borealis, Bora in human). SPAT-1 variants mutated on CDK-1 phosphorylation sites results in severe delays in mitotic entry, mimicking embryos lacking spat-1 or plk-1 function. We further show that SPAT-1 phosphorylation by CDK-1 promotes its binding to PLK-1 and stimulates PLK-1 phosphorylation on its activator T-loop by Aurora A kinase in vitro. Likewise, we find that phosphorylation of Bora by Cdk1 promotes phosphorylation of human Plk1 by Aurora A suggesting that this mechanism is conserved in humans. These results indicate that Cdk1 regulates Plk1 by boosting its kinase activity. Here we discuss these recent findings and open questions regarding the regulation of Plk1/PLK-1 by Cdk1/CDK-1 and Bora/SPAT-1. PMID:26038951

  9. Genetic inactivation of Cdk7 leads to cell cycle arrest and induces premature aging due to adult stem cell exhaustion

    PubMed Central

    Ganuza, Miguel; Sáiz-Ladera, Cristina; Cañamero, Marta; Gómez, Gonzalo; Schneider, Ralph; Blasco, María A; Pisano, David; Paramio, Jesús M; Santamaría, David; Barbacid, Mariano

    2012-01-01

    Cyclin-dependent kinase (Cdk)7, the catalytic subunit of the Cdk-activating kinase (CAK) complex has been implicated in the control of cell cycle progression and of RNA polymerase II (RNA pol II)-mediated transcription. Genetic inactivation of the Cdk7 locus revealed that whereas Cdk7 is completely dispensable for global transcription, is essential for the cell cycle via phosphorylation of Cdk1 and Cdk2. In vivo, Cdk7 is also indispensable for cell proliferation except during the initial stages of embryonic development. Interestingly, widespread elimination of Cdk7 in adult tissues with low proliferative indexes had no phenotypic consequences. However, ablation of conditional Cdk7 alleles in tissues with elevated cellular turnover led to the efficient repopulation of these tissues with Cdk7-expressing cells most likely derived from adult stem cells that may have escaped the inactivation of their targeted Cdk7 alleles. This process, a physiological attempt to maintain tissue homeostasis, led to the attrition of adult stem cell pools and to the appearance of age-related phenotypes, including telomere shortening and early death. PMID:22505032

  10. S-nitrosylation of Cdk5: potential implications in amyloid-β-related neurotoxicity in Alzheimer disease.

    PubMed

    Qu, Jing; Nakamura, Tomohiro; Holland, Emily A; McKercher, Scott R; Lipton, Stuart A

    2012-01-01

    Aberrant activation of Cdk5 has been implicated in the process of neurodegenerative diseases such as Alzheimer's disease (AD). We recently reported that S-nitrosylation of Cdk5 (forming SNO-Cdk5) at specific cysteine residues results in excessive activation of Cdk5, contributing to mitochondrial dysfunction, synaptic damage, and neuronal cell death in models of AD. Furthermore, SNO-Cdk5 acts as a nascent S-nitrosylase, transnitrosylating the mitochondrial fission protein Drp1 and enhancing excessive mitochondrial fission in dendritic spines. However, a molecular mechanism that leads to the formation of SNO-Cdk5 in neuronal cells remained obscure. Here, we demonstrate that neuronal nitric oxide synthase (NOS1) interacts with Cdk5 and that the close proximity of the two proteins facilitates the formation of SNO-Cdk5. Interestingly, as a negative feedback mechanism, Cdk5 phosphorylates and suppresses NOS1 activity. Thus, together with our previous report, these findings delineate an S-nitrosylation pathway wherein Cdk5/NOS1 interaction enhances SNO-Cdk5 formation, mediating mitochondrial dysfunction and synaptic loss during the etiology of AD.

  11. Cdk5 at crossroads of protein oligomerization in neurodegenerative diseases: facts and hypotheses.

    PubMed

    Wilkaniec, Anna; Czapski, Grzegorz A; Adamczyk, Agata

    2016-01-01

    Cyclin-dependent kinase 5 (Cdk5) is involved in proper neurodevelopment and brain function and serves as a switch between neuronal survival and death. Overactivation of Cdk5 is associated with many neurodegenerative disorders such as Alzheimer's or Parkinson's diseases. It is believed that in those diseases Cdk5 may be an important link between disease-initiating factors and cell death effectors. A common hallmark of neurodegenerative disorders is incorrect folding of specific proteins, thus leading to their intra- and extracellular accumulation in the nervous system. Abnormal Cdk5 signaling contributes to dysfunction of individual proteins and has a substantial role in either direct or indirect interactions of proteins common to, and critical in, different neurodegenerative diseases. While the roles of Cdk5 in α-synuclein (ASN) - tau or β-amyloid peptide (Aβ) - tau interactions are well documented, its contribution to many other pertinent interactions, such as that of ASN with Aβ, or interactions of the Aβ - ASN - tau triad with prion proteins, did not get beyond plausible hypotheses and remains to be proven. Understanding of the exact position of Cdk5 in the deleterious feed-forward loop critical for development and progression of neurodegenerative diseases may help designing successful therapeutic strategies of several fatal neurodegenerative diseases. Cyclin-dependent kinase 5 (Cdk5) is associated with many neurodegenerative disorders such as Alzheimer's or Parkinson's diseases. It is believed that in those diseases Cdk5 may be an important factor involved in protein misfolding, toxicity and interaction. We suggest that Cdk5 may contribute to the vicious circle of neurotoxic events involved in the pathogenesis of different neurodegenerative diseases.

  12. Expression of CRM1 and CDK5 shows high prognostic accuracy for gastric cancer

    PubMed Central

    Sun, Yu-Qin; Xie, Jian-Wei; Xie, Hong-Teng; Chen, Peng-Chen; Zhang, Xiu-Li; Zheng, Chao-Hui; Li, Ping; Wang, Jia-Bin; Lin, Jian-Xian; Cao, Long-Long; Huang, Chang-Ming; Lin, Yao

    2017-01-01

    AIM To evaluate the predictive value of the expression of chromosomal maintenance (CRM)1 and cyclin-dependent kinase (CDK)5 in gastric cancer (GC) patients after gastrectomy. METHODS A total of 240 GC patients who received standard gastrectomy were enrolled in the study. The expression level of CRM1 and CDK5 was detected by immunohistochemistry. The correlations between CRM1 and CDK5 expression and clinicopathological factors were explored. Univariate and multivariate survival analyses were used to identify prognostic factors for GC. Receiver operating characteristic analysis was used to compare the accuracy of the prediction of clinical outcome by the parameters. RESULTS The expression of CRM1 was significantly related to size of primary tumor (P = 0.005), Borrmann type (P = 0.006), degree of differentiation (P = 0.004), depth of invasion (P = 0.008), lymph node metastasis (P = 0.013), TNM stage (P = 0.002) and distant metastasis (P = 0.015). The expression of CDK5 was significantly related to sex (P = 0.048) and Lauren’s classification (P = 0.011). Multivariate Cox regression analysis identified that CRM1 and CDK5 co-expression status was an independent prognostic factor for overall survival (OS) of patients with GC. Integration of CRM1 and CDK5 expression could provide additional prognostic value for OS compared with CRM1 or CDK5 expression alone (P = 0.001). CONCLUSION CRM1 and CDK5 co-expression was an independent prognostic factors for GC. Combined CRM1 and CDK5 expression could provide a prognostic model for OS of GC. PMID:28373767

  13. A synthetic lethal siRNA screen identifying genes mediating sensitivity to a PARP inhibitor.

    PubMed

    Turner, Nicholas C; Lord, Christopher J; Iorns, Elizabeth; Brough, Rachel; Swift, Sally; Elliott, Richard; Rayter, Sydonia; Tutt, Andrew N; Ashworth, Alan

    2008-05-07

    Inhibitors of poly (ADP-ribose)-polymerase-1 (PARP) are highly lethal to cells with deficiencies in BRCA1, BRCA2 or other components of the homologous recombination pathway. This has led to PARP inhibitors entering clinical trials as a potential therapy for cancer in carriers of BRCA1 and BRCA2 mutations. To discover new determinants of sensitivity to these drugs, we performed a PARP-inhibitor synthetic lethal short interfering RNA (siRNA) screen. We identified a number of kinases whose silencing strongly sensitised to PARP inhibitor, including cyclin-dependent kinase 5 (CDK5), MAPK12, PLK3, PNKP, STK22c and STK36. How CDK5 silencing mediates sensitivity was investigated. Previously, CDK5 has been suggested to be active only in a neuronal context, but here we show that CDK5 is required in non-neuronal cells for the DNA-damage response and, in particular, intra-S and G(2)/M cell-cycle checkpoints. These results highlight the potential of synthetic lethal siRNA screens with chemical inhibitors to define new determinants of sensitivity and potential therapeutic targets.

  14. CDK4/6 or MAPK blockade enhances efficacy of EGFR inhibition in oesophageal squamous cell carcinoma

    PubMed Central

    Zhou, Jin; Wu, Zhong; Wong, Gabrielle; Pectasides, Eirini; Nagaraja, Ankur; Stachler, Matthew; Zhang, Haikuo; Chen, Ting; Zhang, Haisheng; Liu, Jie Bin; Xu, Xinsen; Sicinska, Ewa; Sanchez-Vega, Francisco; Rustgi, Anil K.; Diehl, J. Alan; Wong, Kwok-Kin; Bass, Adam J.

    2017-01-01

    Oesophageal squamous cell carcinoma is a deadly disease where systemic therapy has relied upon empiric chemotherapy despite the presence of genomic alterations pointing to candidate therapeutic targets, including recurrent amplification of the gene encoding receptor tyrosine kinase epidermal growth factor receptor (EGFR). Here, we demonstrate that EGFR-targeting small-molecule inhibitors have efficacy in EGFR-amplified oesophageal squamous cell carcinoma (ESCC), but may become quickly ineffective. Resistance can occur following the emergence of epithelial–mesenchymal transition and by reactivation of the mitogen-activated protein kinase (MAPK) pathway following EGFR blockade. We demonstrate that blockade of this rebound activation with MEK (mitogen-activated protein kinase kinase) inhibition enhances EGFR inhibitor-induced apoptosis and cell cycle arrest, and delays resistance to EGFR monotherapy. Furthermore, genomic profiling shows that cell cycle regulators are altered in the majority of EGFR-amplified tumours and a combination of cyclin-dependent kinase 4/6 (CDK4/6) and EGFR inhibitors prevents the emergence of resistance in vitro and in vivo. These data suggest that upfront combination strategies targeting EGFR amplification, guided by adaptive pathway reactivation or by co-occurring genomic alterations, should be tested clinically. PMID:28059068

  15. Action of the p53 Effector, p21, on its Targets: Cyclin-cdk and PCNA

    DTIC Science & Technology

    2001-10-01

    Lineweaver - Burke analysis of the inhibition of cyclin E-cdk2 by intact p21 and by p21 without the cyclin binding site. Task 2: Months 24-36: Determination...the peptide sequence was the critical feature of a Cy motif. Task 1: Months 1-24: Lineweaver - Burke analysis of the inhibition of cyclin E-cdk2 by...intact p21 and by p21 without the cyclin binding site. p21 without a Cy motif was ineffective in inhibiting cyclin E-cdk2 [2]. Lineweaver - Burke analysis

  16. Low Expression of CDK5 and p27 Are Associated with Poor Prognosis in Patients with Gastric Cancer

    PubMed Central

    Sun, Yu-Qin; Xie, Jian-Wei; Chen, Peng-Chen; Zheng, Chao-Hui; Li, Ping; Wang, Jia-Bin; Lin, Jian-Xian; Lu, Jun; Chen, Qi-Yue; Cao, Long-Long; Lin, Mi; Tu, Ru-Hong; Lin, Yao; Huang, Chang-Ming

    2016-01-01

    Several previous studies have demonstrated that CDK5 or p27 expression in gastric cancer are associated with overall survival. We have previously reported that tumor suppressive function of CDK5 is related to p27. The aim of this study was to investigate correlation between the clinicopathological parameters and overall survival with different CDK5/p27 expression statuses in 244 gastric cancer patients using immunohistochemistry. Low CDK5 expression was detected in 93 cases (38.11%) and low p27 in 157 cases (64.34%). The expression of CDK5 was significantly related to sex (P = 0.034) and Lauren's classification (P = 0.013). The expression of p27 was significantly related to sex (P = 0.012), differentiation (P = 0.003), TNM stage (P = 0.013) and lymph node metastasis (P = 0.001). Based on the combined expression of CDK5 and p27, we classified the patients into four subtypes: CDK5 Low/p27 Low (n = 69), CDK5 High/p27 Low (n = 88), CDK5 Low/p27 High (n = 24) and CDK5 High/p27 High (n = 63). The CDK5 Low/p27 Low expression was closely related to female (P = 0.026), diffuse type (P = 0.027) and lymph node metastasis (P = 0.010). The CDK5 Low/p27 Low patients displayed poorer survival in comparison with the rest of the patients in Kaplan-Meier analysis. No significant overall survival difference was observed among the patients with CDK5 High and/or p27 High expression. In the multivariate analysis, CDK5 and p27 co-expression status was identified as an independent prognostic factor for patients with gastric cancer. PMID:27326247

  17. Nuclear Import of Cdk/Cyclin Complexes: Identification of Distinct Mechanisms for Import of Cdk2/Cyclin E and Cdc2/Cyclin B1

    PubMed Central

    Moore, Jonathan D.; Yang, Jing; Truant, Ray; Kornbluth, Sally

    1999-01-01

    Reversible phosphorylation of nuclear proteins is required for both DNA replication and entry into mitosis. Consequently, most cyclin-dependent kinase (Cdk)/cyclin complexes are localized to the nucleus when active. Although our understanding of nuclear transport processes has been greatly enhanced by the recent identification of nuclear targeting sequences and soluble nuclear import factors with which they interact, the mechanisms used to target Cdk/cyclin complexes to the nucleus remain obscure; this is in part because these proteins lack obvious nuclear localization sequences. To elucidate the molecular mechanisms responsible for Cdk/cyclin transport, we examined nuclear import of fluorescent Cdk2/cyclin E and Cdc2/cyclin B1 complexes in digitonin-permeabilized mammalian cells and also examined potential physical interactions between these Cdks, cyclins, and soluble import factors. We found that the nuclear import machinery recognizes these Cdk/cyclin complexes through direct interactions with the cyclin component. Surprisingly, cyclins E and B1 are imported into nuclei via distinct mechanisms. Cyclin E behaves like a classical basic nuclear localization sequence–containing protein, binding to the α adaptor subunit of the importin-α/β heterodimer. In contrast, cyclin B1 is imported via a direct interaction with a site in the NH2 terminus of importin-β that is distinct from that used to bind importin-α. PMID:9922449

  18. Preclinical Activity of Simvastatin Induces Cell Cycle Arrest in G1 via Blockade of Cyclin D-Cdk4 Expression in Non-Small Cell Lung Cancer (NSCLC)

    PubMed Central

    Liang, Yu-Wei; Chang, Chi-Chang; Hung, Chao-Ming; Chen, Tzu-Yu; Huang, Tzuu-Yuan; Hsu, Yi-Chiang

    2013-01-01

    Lung cancer is the most common cause of cancer-related death. Nonetheless, a decrease in overall incidence and mortality has been observed in the last 30 years due to prevention strategies and improvements in the use of chemotherapeutic agents. In recent studies, Simvastatin (SIM) has demonstrated anti-tumor activity, as well as potent chemopreventive action. As an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA), SIM has been shown to stimulate apoptotic cell death. In this study, an MTT assay revealed the cytotoxic activity of SIM against human large cell lung cancer (Non-small cell lung cancer; NSCLC) cells (NCI-H460); however, induced apoptosis was not observed in NCI-H460 cells. Protein expression levels of cell cycle regulating proteins Cdk4, Cyclin D1, p16 and p27 were markedly altered by SIM. Collectively, our results indicate that SIM inhibits cell proliferation and arrests NCI-H460 cell cycle progression via inhibition of cyclin-dependent kinases and cyclins and the enhancement of CDK inhibitors p16 and p27. Our findings suggest that, in addition to the known effects on hypercholesterolemia therapy, SIM may also provide antitumor activity in established NSCLC. PMID:23481641

  19. CDK4/6-dependent activation of DUB3 regulates cancer metastasis through SNAIL1.

    PubMed

    Liu, Tongzheng; Yu, Jia; Deng, Min; Yin, Yujiao; Zhang, Haoxing; Luo, Kuntian; Qin, Bo; Li, Yunhui; Wu, Chenming; Ren, Tao; Han, Yang; Yin, Peng; Kim, JungJin; Lee, SeungBaek; Lin, Jing; Zhang, Lizhi; Zhang, Jun; Nowsheen, Somaira; Wang, Liewei; Boughey, Judy; Goetz, Matthew P; Yuan, Jian; Lou, Zhenkun

    2017-01-09

    Tumour metastasis, the spread of cancer cells from the original tumour site followed by growth of secondary tumours at distant organs, is the primary cause of cancer-related deaths and remains poorly understood. Here we demonstrate that inhibition of CDK4/6 blocks breast tumour metastasis in the triple-negative breast cancer model, without affecting tumour growth. Mechanistically, we identify a deubiquitinase, DUB3, as a target of CDK4/6; CDK4/6-mediated activation of DUB3 is essential to deubiquitinate and stabilize SNAIL1, a key factor promoting epithelial-mesenchymal transition and breast cancer metastasis. Overall, our study establishes the CDK4/6-DUB3 axis as an important regulatory mechanism of breast cancer metastasis and provides a rationale for potential therapeutic interventions in the treatment of breast cancer metastasis.

  20. CDK1 Enhances Mitochondrial Bioenergetics for Radiation-Induced DNA Repair

    PubMed Central

    Qin, Lili; Fan, Ming; Candas, Demet; Jiang, Guochun; Papadopoulos, Stelios; Tian, Lin; Woloschak, Gayle; Grdina, David J.; Li, Jian Jian

    2015-01-01

    SUMMARY Nuclear DNA repair capacity is a critical determinant of cell fate under genotoxic stress conditions. DNA repair is a well-defined energy consuming process; however, it is unclear how DNA repair is fueled and whether mitochondrial energy production contributes to nuclear DNA repair. Here, we report a dynamic enhancement of oxygen consumption and mitochondrial ATP generation in irradiated normal cells, paralleled with increased mitochondrial relocation of cell cycle kinase CDK1 and nuclear DNA repair. The basal and radiation-induced mitochondrial ATP generation is significantly reduced in cells harboring CDK1 phosphorylation deficient mutant complex I subunits. Similarly, mitochondrial ATP generation and nuclear DNA repair are also severely compromised in cells harboring mitochondrial-targeted kinase deficient CDK1. These results demonstrate a mechanism governing the communication between mitochondria and nucleus, by which CDK1 boosts mitochondrial bioenergetics to meet the increased cellular fuel demand for DNA repair and cell survival under genotoxic stress. PMID:26670043

  1. CDK4/6-dependent activation of DUB3 regulates cancer metastasis through SNAIL1

    PubMed Central

    Liu, Tongzheng; Yu, Jia; Deng, Min; Yin, Yujiao; Zhang, Haoxing; Luo, Kuntian; Qin, Bo; Li, Yunhui; Wu, Chenming; Ren, Tao; Han, Yang; Yin, Peng; Kim, JungJin; Lee, SeungBaek; Lin, Jing; Zhang, Lizhi; Zhang, Jun; Nowsheen, Somaira; Wang, Liewei; Boughey, Judy; Goetz, Matthew P.; Yuan, Jian; Lou, Zhenkun

    2017-01-01

    Tumour metastasis, the spread of cancer cells from the original tumour site followed by growth of secondary tumours at distant organs, is the primary cause of cancer-related deaths and remains poorly understood. Here we demonstrate that inhibition of CDK4/6 blocks breast tumour metastasis in the triple-negative breast cancer model, without affecting tumour growth. Mechanistically, we identify a deubiquitinase, DUB3, as a target of CDK4/6; CDK4/6-mediated activation of DUB3 is essential to deubiquitinate and stabilize SNAIL1, a key factor promoting epithelial–mesenchymal transition and breast cancer metastasis. Overall, our study establishes the CDK4/6–DUB3 axis as an important regulatory mechanism of breast cancer metastasis and provides a rationale for potential therapeutic interventions in the treatment of breast cancer metastasis. PMID:28067227

  2. Fimbrin phosphorylation by metaphase Cdk1 regulates actin cable dynamics in budding yeast

    PubMed Central

    Miao, Yansong; Han, Xuemei; Zheng, Liangzhen; Xie, Ying; Mu, Yuguang; Yates, John R.; Drubin, David G.

    2016-01-01

    Actin cables, composed of actin filament bundles nucleated by formins, mediate intracellular transport for cell polarity establishment and maintenance. We previously observed that metaphase cells preferentially promote actin cable assembly through cyclin-dependent kinase 1 (Cdk1) activity. However, the relevant metaphase Cdk1 targets were not known. Here we show that the highly conserved actin filament crosslinking protein fimbrin is a critical Cdk1 target for actin cable assembly regulation in budding yeast. Fimbrin is specifically phosphorylated on threonine 103 by the metaphase cyclin–Cdk1 complex, in vivo and in vitro. On the basis of conformational simulations, we suggest that this phosphorylation stabilizes fimbrin's N-terminal domain, and modulates actin filament binding to regulate actin cable assembly and stability in cells. Overall, this work identifies fimbrin as a key target for cell cycle regulation of actin cable assembly in budding yeast, and suggests an underlying mechanism. PMID:27068241

  3. CDK8-Cyclin C Mediates Nutritional Regulation of Developmental Transitions through the Ecdysone Receptor in Drosophila

    PubMed Central

    Xie, Xiao-Jun; Hsu, Fu-Ning; Gao, Xinsheng; Xu, Wu; Ni, Jian-Quan; Xing, Yue; Huang, Liying; Hsiao, Hao-Ching; Zheng, Haiyan; Wang, Chenguang; Zheng, Yani; Xiaoli, Alus M.; Yang, Fajun; Bondos, Sarah E.; Ji, Jun-Yuan

    2015-01-01

    The steroid hormone ecdysone and its receptor (EcR) play critical roles in orchestrating developmental transitions in arthropods. However, the mechanism by which EcR integrates nutritional and developmental cues to correctly activate transcription remains poorly understood. Here, we show that EcR-dependent transcription, and thus, developmental timing in Drosophila, is regulated by CDK8 and its regulatory partner Cyclin C (CycC), and the level of CDK8 is affected by nutrient availability. We observed that cdk8 and cycC mutants resemble EcR mutants and EcR-target genes are systematically down-regulated in both mutants. Indeed, the ability of the EcR-Ultraspiracle (USP) heterodimer to bind to polytene chromosomes and the promoters of EcR target genes is also diminished. Mass spectrometry analysis of proteins that co-immunoprecipitate with EcR and USP identified multiple Mediator subunits, including CDK8 and CycC. Consistently, CDK8-CycC interacts with EcR-USP in vivo; in particular, CDK8 and Med14 can directly interact with the AF1 domain of EcR. These results suggest that CDK8-CycC may serve as transcriptional cofactors for EcR-dependent transcription. During the larval–pupal transition, the levels of CDK8 protein positively correlate with EcR and USP levels, but inversely correlate with the activity of sterol regulatory element binding protein (SREBP), the master regulator of intracellular lipid homeostasis. Likewise, starvation of early third instar larvae precociously increases the levels of CDK8, EcR and USP, yet down-regulates SREBP activity. Conversely, refeeding the starved larvae strongly reduces CDK8 levels but increases SREBP activity. Importantly, these changes correlate with the timing for the larval–pupal transition. Taken together, these results suggest that CDK8-CycC links nutrient intake to developmental transitions (EcR activity) and fat metabolism (SREBP activity) during the larval–pupal transition. PMID:26222308

  4. Phosphorylation of HIV-1 Tat by CDK2 in HIV-1 transcription

    PubMed Central

    Ammosova, Tatyana; Berro, Reem; Jerebtsova, Marina; Jackson, Angela; Charles, Sharroya; Klase, Zachary; Southerland, William; Gordeuk, Victor R; Kashanchi, Fatah; Nekhai, Sergei

    2006-01-01

    Background Transcription of HIV-1 genes is activated by HIV-1 Tat protein, which induces phosphorylation of RNA polymerase II (RNAPII) C-terminal domain (CTD) by CDK9/cyclin T1. Earlier we showed that CDK2/cyclin E phosphorylates HIV-1 Tat in vitro. We also showed that CDK2 induces HIV-1 transcription in vitro and that inhibition of CDK2 expression by RNA interference inhibits HIV-1 transcription and viral replication in cultured cells. In the present study, we analyzed whether Tat is phosphorylated in cultured cells by CDK2 and whether Tat phosphorylation has a regulatory effect on HIV-1 transcription. Results We analyzed HIV-1 Tat phosphorylation by CDK2 in vitro and identified Ser16 and Ser46 residues of Tat as potential phosphorylation sites. Tat was phosphorylated in HeLa cells infected with Tat-expressing adenovirus and metabolically labeled with 32P. CDK2-specific siRNA reduced the amount and the activity of cellular CDK2 and significantly decreased phosphorylation of Tat. Tat co-migrated with CDK2 on glycerol gradient and co-immunoprecipitated with CDK2 from the cellular extracts. Tat was phosphorylated on serine residues in vivo, and mutations of Ser16 and Ser46 residues of Tat reduced Tat phosphorylation in vivo. Mutation of Ser16 and Ser46 residues of Tat reduced HIV-1 transcription in transiently transfected cells. The mutations of Tat also inhibited HIV-1 viral replication and Tat phosphorylation in the context of the integrated HIV-1 provirus. Analysis of physiological importance of the S16QP(K/R)19 and S46YGR49 sequences of Tat showed that Ser16 and Ser46 and R49 residues are highly conserved whereas mutation of the (K/R)19 residue correlated with non-progression of HIV-1 disease. Conclusion Our results indicate for the first time that Tat is phosphorylated in vivo; Tat phosphorylation is likely to be mediated by CDK2; and phosphorylation of Tat is important for HIV-1 transcription. PMID:17083724

  5. Identification of New Substrates for Breast Tumor-Specific LMW Cyclin E/CDk2 Kinase

    DTIC Science & Technology

    2011-09-01

    cyclin EL or cyclin E-LMW and CDK2 (F80A) and CDK2 (F80G) from insect cells and carried out a similar     8   Rb kinase assay to test their...Multani, A. S., Wingate , H. F., Pathak, S., Zhang, N., Tucker, S. L., Chang, S., and Keyomarsi, K. (2004). Tumor-specific low molecular weight forms of

  6. Cdc25 Phosphatases Are Required for Timely Assembly of CDK1-Cyclin B at the G2/M Transition*

    PubMed Central

    Timofeev, Oleg; Cizmecioglu, Onur; Settele, Florian; Kempf, Tore; Hoffmann, Ingrid

    2010-01-01

    Progression through mitosis requires the coordinated regulation of Cdk1 kinase activity. Activation of Cdk1 is a multistep process comprising binding of Cdk1 to cyclin B, relocation of cyclin-kinase complexes to the nucleus, activating phosphorylation of Cdk1 on Thr161 by the Cdk-activating kinase (CAK; Cdk7 in metazoans), and removal of inhibitory Thr14 and Tyr15 phosphorylations. This dephosphorylation is catalyzed by the dual specific Cdc25 phosphatases, which occur in three isoforms in mammalian cells, Cdc25A, -B, and -C. We find that expression of Cdc25A leads to an accelerated G2/M phase transition. In Cdc25A-overexpressing cells, Cdk1 exhibits high kinase activity despite being phosphorylated on Tyr15. In addition, Tyr15-phosphorylated Cdk1 binds more cyclin B in Cdc25A-overexpressing cells compared with control cells. Consistent with this observation, we demonstrate that in human transformed cells, Cdc25A and Cdc25B, but not Cdc25C phosphatases have an effect on timing and efficiency of cyclin-kinase complex formation. Overexpression of Cdc25A or Cdc25B promotes earlier assembly and activation of Cdk1-cyclin B complexes, whereas repression of these phosphatases by short hairpin RNA has a reverse effect, leading to a substantial decrease in amounts of cyclin B-bound Cdk1 in G2 and mitosis. Importantly, we find that Cdc25A overexpression leads to an activation of Cdk7 and increase in Thr161 phosphorylation of Cdk1. In conclusion, our data suggest that complex assembly and dephosphorylation of Cdk1 at G2/M is tightly coupled and regulated by Cdc25 phosphatases. PMID:20360007

  7. Roles of the CDK phosphorylation sites of yeast Cdc6 in chromatin binding and rereplication.

    PubMed

    Honey, Sangeet; Futcher, Bruce

    2007-04-01

    The Saccharomyces cerevisiae Cdc6 protein is crucial for DNA replication. In the absence of cyclin-dependent kinase (CDK) activity, Cdc6 binds to replication origins, and loads Mcm proteins. In the presence of CDK activity, Cdc6 does not bind to origins, and this helps prevent rereplication. CDK activity affects Cdc6 function by multiple mechanisms: CDK activity affects transcription of CDC6, degradation of Cdc6, nuclear import of Cdc6, and binding of Cdc6 to Clb2. Here we examine some of these mechanisms individually. We find that when Cdc6 is forced into the nucleus during late G1 or S, it will not substantially reload onto chromatin no matter whether its CDK sites are present or not. In contrast, at a G2/M nocodazole arrest, Cdc6 will reload onto chromatin if and only if its CDK sites have been removed. Trace amounts of nonphosphorylatable Cdc6 are dominant lethal in strains bearing nonphosphorylatable Orc2 and Orc6, apparently because of rereplication. This synthetic dominant lethality occurs even in strains with wild-type MCM genes. Nonphosphorylatable Cdc6, or Orc2 and Orc6, sensitize cells to rereplication caused by overexpression of various replication initiation proteins such as Dpb11 and Sld2.

  8. Regulation of a transcription factor network by Cdk1 coordinates late cell cycle gene expression.

    PubMed

    Landry, Benjamin D; Mapa, Claudine E; Arsenault, Heather E; Poti, Kristin E; Benanti, Jennifer A

    2014-05-02

    To maintain genome stability, regulators of chromosome segregation must be expressed in coordination with mitotic events. Expression of these late cell cycle genes is regulated by cyclin-dependent kinase (Cdk1), which phosphorylates a network of conserved transcription factors (TFs). However, the effects of Cdk1 phosphorylation on many key TFs are not known. We find that elimination of Cdk1-mediated phosphorylation of four S-phase TFs decreases expression of many late cell cycle genes, delays mitotic progression, and reduces fitness in budding yeast. Blocking phosphorylation impairs degradation of all four TFs. Consequently, phosphorylation-deficient mutants of the repressors Yox1 and Yhp1 exhibit increased promoter occupancy and decreased expression of their target genes. Interestingly, although phosphorylation of the transcriptional activator Hcm1 on its N-terminus promotes its degradation, phosphorylation on its C-terminus is required for its activity, indicating that Cdk1 both activates and inhibits a single TF. We conclude that Cdk1 promotes gene expression by both activating transcriptional activators and inactivating transcriptional repressors. Furthermore, our data suggest that coordinated regulation of the TF network by Cdk1 is necessary for faithful cell division.

  9. Cdk5 Modulates Long-Term Synaptic Plasticity and Motor Learning in Dorsolateral Striatum

    PubMed Central

    Hernandez, Adan; Tan, Chunfeng; Mettlach, Gabriel; Pozo, Karine; Plattner, Florian; Bibb, James A.

    2016-01-01

    The striatum controls multiple cognitive aspects including motivation, reward perception, decision-making and motor planning. In particular, the dorsolateral striatum contributes to motor learning. Here we define an approach for investigating synaptic plasticity in mouse dorsolateral cortico-striatal circuitry and interrogate the relative contributions of neurotransmitter receptors and intracellular signaling components. Consistent with previous studies, we show that long-term potentiation (LTP) in cortico-striatal circuitry is facilitated by dopamine, and requires activation of D1-dopamine receptors, as well as NMDA receptors (NMDAR) and their calcium-dependent downstream effectors, including CaMKII. Moreover, we assessed the contribution of the protein kinase Cdk5, a key neuronal signaling molecule, in cortico-striatal LTP. Pharmacological Cdk5 inhibition, brain-wide Cdk5 conditional knockout, or viral-mediated dorsolateral striatal-specific loss of Cdk5 all impaired dopamine-facilitated LTP or D1-dopamine receptor-facilitated LTP. Selective loss of Cdk5 in dorsolateral striatum increased locomotor activity and attenuated motor learning. Taken together, we report an approach for studying synaptic plasticity in mouse dorsolateral striatum and critically implicate D1-dopamine receptor, NMDAR, Cdk5, and CaMKII in cortico-striatal plasticity. Furthermore, we associate striatal plasticity deficits with effects upon behaviors mediated by this circuitry. This approach should prove useful for the study of the molecular basis of plasticity in the dorsolateral striatum. PMID:27443506

  10. Microarray expression analysis of MYCN-amplified neuroblastoma cells after inhibition of CDK2.

    PubMed

    Song, H; Wu, F; Li, S; Wang, Z; Liu, X; Cui, Y; Lin, C

    2017-03-03

    The study was aimed to explore the underlying molecular mechanisms of CDK2 inhibition in neuroblastoma by bioinformatics analysis. Gene expression profile GSE16480 was downloaded from the Gene Expression Omnibus. The differentially expressed genes (DEGs) were identified from IMR32 between each time point and average expression of all time points. Gene significance was calculated using dSVDsig algorithm of dnet package. Protein-protein interaction (PPI) network was built. Then, integrated with gene significance, a core PPI network was detected by dNetPipeline algorithm in dnet package. Finally, pathway enrichment analysis was performed for genes in network. Total 1524 DEGs were identified. CCNA2 (cyclin A2), EXO1 (exonuclease 1), RAD51AP1 (RAD51 associated protein 1), TOP2A (topoisomerase (DNA) II alpha) and CDK1 (cyclin-dependent kinase 1) were selected as DEGs with higher connectivity after PPI network analysis. In the network, CCNA2, CDK1, BUB1B (BUB1 mitotic checkpoint serine/threonine kinase B) and CCNB1 (cyclin B1) were involved in cell cycle pathway. Additionally, CCNB1, CDK1, CCNE2 (Cyclin E2), and RRM2B (ribonucleotide reductase subunit M2B) were involved in p53 signaling pathway. Cell cycle and p53 signaling pathway were closely associated with neuroblastoma after CDK2 inhibition. The DEGs, such as CCNA2, CCNB1, CDK1 and RRM2B may be the potential targets for neuroblastoma.

  11. Early Intervention with Cdk9 Inhibitors to Prevent Post-Traumatic Osteoarthritis

    DTIC Science & Technology

    2013-10-01

    administration of Flavopiridol reduces synovial hyperplasia, but does not induce apoptosis, and result in preventing the development of rheumatoid arthritis in...Synovial fluid from rheumatoid arthritis patients contains sufficient levels of IL-1 beta and IL-6 to promote production of serum amyloid A by Hep3B...Miyake, H. Hirai, M. Yoshida, N. Miyasaka, et al. 2008. Successful treatment of animal models of rheumatoid arthritis with small-molecule cyclin

  12. Early Intervention with cdk9 Inhibitors to Prevent Post-Traumatic Osteoarthritis

    DTIC Science & Technology

    2014-10-01

    atherosclerosis with a novel, protease-activatable fluorescence sensor. Circu- lation 2007;115:2292e8. 18. Jaffer FA, Libby P, Weissleder R. Optical...and multimodality molecular imaging: insights into atherosclerosis . Arterioscler Thromb Vasc Biol 2009;29:1017e24. 19. Razansky D, Harlaar NJ

  13. Structure-Based Design of Cdk4/6-Specific Inhibitors

    DTIC Science & Technology

    2006-10-01

    jbc.M208061200 Ravichandran N. Venkataramani‡§, Timothy K. MacLachlan¶, Xiaomei Chai‡, Wafik S. El-Deiry¶, and Ronen Marmorstein‡§** From the ‡The Wistar...Domaille, P. J., Smith, B. O., Owen, D., Brotherton, D. H., Raine, A. R. C., Xu , X., Brizuela, L., Brenner, S. L., and Laue, E. D. (1997) Nature 389...Brotherton, D., Dhanaraj, V., Wick, S., Brizuela, L., Domaille, P. J., Volyanik, E., Xu , X., Parisini, E., Smith, B. O., Archer, S. J., Serrano, M

  14. Structure-based Design of Cdk4/6-Specific Inhibitors

    DTIC Science & Technology

    2005-10-01

    Timothy K. MacLachlan¶, Xiaomei Chait, Wafik S. El-Deiryl, and Ronen Marmorstein*§II** From the +The Wistar Institute, the ¶Howard Hughes Medical...Modeling of a glutamine suggests 16. Luh, F. Y., Archer, S. J., Domaille, P. J., Smith, B. 0., Owen, D., Brotherton, D. H., Raine, A. R. C., Xu , X...E., Xu , X., Parisini, E., Smith, B. 0., Archer, S. J., Serrano, M., Brennen, thermal stability of these mutants relative to the wild type S. L

  15. Role of p53 in cdk Inhibitor VMY-1-103-Induced Apoptosis in Prostate Cancer

    DTIC Science & Technology

    2012-09-01

    ultrasonic disruption at 4°C and immediately assayed for kinase activity. For each lysate, parallel assays were per- formed for 12 min at 33°C in the...ONE 2008; 3:3088; PMID:18769486; DOI:10.1371/ journal.pone.0003088. 5. Northcott PA, Korshunov A, Witt H, Hielscher T, Eberhart CG, Mack S, et al

  16. CDK inhibitor p57Kip2 is negatively regulated by COP9 signalosome subunit 6

    PubMed Central

    Chen, Bo; Zhao, Ruiying; Su, Chun-Hui; Linan, Monica; Tseng, Chieh; Phan, Liem; Fang, Lekuan; Yang, Heng-Yin; Yang, Huiling; Wang, Wenqian; Xu, Xiaoyin; Jiang, Nan; Cai, Shouliang; Jin, Feng; Yeung, Sai-Ching J.; Lee, Mong-Hong

    2012-01-01

    Subunit 6 of the COP9 signalosome complex, CSN6, is known to be critical to the regulation of the MDM2-p53 axis for cell proliferation and anti-apoptosis, but its many targets remain unclear. Here we show that p57Kip2 is a target of CSN6, and that CSN6 is a negative regulator of p57Kip2. CSN6 associates with p57Kip2, and its overexpression can decrease the steady-state expression of p57Kip2; accordingly, CSN6 deficiency leads to p57Kip2 stabilization. Mechanistic studies show that CSN6 associates with p57Kip2 and Skp2, a component of the E3 ligase, which, in turn, facilitates Skp2-mediated protein ubiquitination of p57Kip2. Loss of Skp2 compromised CSN6-mediated p57Kip2 destabilization, suggesting collaboration between Skp2 and CSN6 in degradation of p57Kip2. CSN6’s negative impact on p57Kip2 elevation translates into cell growth promotion, cell cycle deregulation and potentiated transformational activity. Significantly, univariate Kaplan-Meier analysis of tumor samples demonstrates that high CSN6 expression or low p57 expression is associated with poor overall survival. These data suggest that CSN6 is an important negative regulator of p57Kip2, and that overexpression of CSN6 in many types of cancer could lead to decreased expression of p57Kip2 and result in promoted cancer cell growth. PMID:23187808

  17. Methods Of Using Chemical Libraries To Search For New Kinase Inhibitors

    DOEpatents

    Gray, Nathanael S. , Schultz, Peter , Wodicka, Lisa , Meijer, Laurent , Lockhart, David J.

    2003-06-03

    The generation of selective inhibitors for specific protein kinases would provide new tools for analyzing signal transduction pathways and possibly new therapeutic agents. We have invented an approach to the development of selective protein kinase inhibitors based on the unexpected binding mode of 2,6,9-trisubstituted purines to the ATP binding site of human CDK2. The most potent inhibitor, purvalanol B (IC.sub.50 =6 nM), binds with a 30-fold greater affinity than the known CDK2 inhibitor, flavopiridol. The cellular effects of this class of compounds were examined and compared to those of flavopiridol by monitoring changes in mRNA expression levels for all genes in treated cells of Saccharomyces cerevisiae using high-density oligonucleotide probe arrays.

  18. A large Norwegian family with inherited malignant melanoma, multiple atypical nevi, and CDK4 mutation.

    PubMed

    Molven, Anders; Grimstvedt, Magne B; Steine, Solrun J; Harland, Mark; Avril, Marie-Françoise; Hayward, Nicholas K; Akslen, Lars A

    2005-09-01

    Mutations in two loci encoding cell-cycle-regulatory proteins have been shown to cause familial malignant melanoma. About 20% of melanoma-prone families bear a mutation in the CDKN2A locus, which encodes two unrelated proteins, p16INK4A and p14ARF. Mutations in the other locus, CDK4, are much rarer and have been linked to the disease in only three families worldwide. In the 1960s, a large Norwegian pedigree with multiple atypical nevi and malignant melanomas was identified. Subsequently, six generations and more than 100 family members were traced and 20 cases of melanoma verified. In this article, we report that CDK4 codon 24 is mutated from CGT to CAT (Arg24His) in this unusually large melanoma kindred. Intriguingly, one of the family members had ocular melanoma, but the CDK4 mutation could not be detected in archival tissue samples from this subject. Thus, the case of ocular melanoma in this family was sporadic, suggesting an etiology different from that of the skin tumors. The CDK4 mutation in the Norwegian family was identical to that in melanoma families in France, Australia, and England. Haplotype analysis using microsatellite markers flanking the CDK4 gene and single-nucleotide polymorphisms within the gene did not support the possibility that there was a common founder, but rather indicated at least two independent mutational events. All CDK4 melanoma families known to date have a substitution of amino acid 24. In addition to resulting from selection pressure, this observation may be explained by the CG dinucleotide of codon 24 representing a mutational hot spot in the CDK4 gene.

  19. An essential role for Ink4 and Cip/Kip cell-cycle inhibitors in preventing replicative stress

    PubMed Central

    Quereda, V; Porlan, E; Cañamero, M; Dubus, P; Malumbres, M

    2016-01-01

    Cell-cycle inhibitors of the Ink4 and Cip/Kip families are involved in cellular senescence and tumor suppression. These inhibitors are individually dispensable for the cell cycle and inactivation of specific family members results in increased proliferation and enhanced susceptibility to tumor development. We have now analyzed the consequences of eliminating a substantial part of the cell-cycle inhibitory activity in the cell by generating a mouse model, which combines the absence of both p21Cip1 and p27Kip1 proteins with the endogenous expression of a Cdk4 R24C mutant insensitive to Ink4 inhibitors. Pairwise combination of Cdk4 R24C, p21-null and p27-null alleles results in frequent hyperplasias and tumors, mainly in cells of endocrine origin such as pituitary cells and in mesenchymal tissues. Interestingly, complete abrogation of p21Cip1 and p27Kip1 in Cdk4 R24C mutant mice results in a different phenotype characterized by perinatal death accompanied by general hypoplasia in most tissues. This phenotype correlates with increased replicative stress in developing tissues such as the nervous system and subsequent apoptotic cell death. Partial inhibition of Cdk4/6 rescues replicative stress signaling as well as p53 induction in the absence of cell-cycle inhibitors. We conclude that one of the major physiological activities of cell-cycle inhibitors is to prevent replicative stress during development. PMID:26292757

  20. A homologue of Cdk8 is required for spore cell differentiation in Dictyostelium.

    PubMed

    Lin, Hsiu-Hsu Sophia; Khosla, Meenal; Huang, Hao-Jen; Hsu, Duen-Wei; Michaelis, Christine; Weeks, Gerald; Pears, Catherine

    2004-07-01

    The Cdk8 proteins are kinases which phosphorylate the carboxy terminal domain (CTD) of RNA polymerase II (Pol II) as well as some transcription factors and, therefore, are involved in the regulation of transcription. Here, we report that a Cdk8 homologue from Dictyostelium discoideum is localized in the nucleus where it forms part of a high molecular weight complex that has CTD kinase activity. Insertional mutagenesis was used to abrogate gene function, and analysis of the null strain revealed that the DdCdk8 protein plays an important role in spore formation during late development. As previously reported [Dev. Growth Differ. 44 (2002) 213] Ddcdk8- cells also exhibit impaired aggregation, although we report that the severity of the defect depends upon experimental conditions. When aggregation occurs, Ddcdk8- cells form abnormal terminally differentiated structures within which the Ddcdk8- cells differentiate into stalk cells but fail to form spores, indicating a role for DdCdk8 in cell differentiation. When Ddcdk8 is expressed from its own promoter, the protein is able to rescue both the late developmental defect and the impaired aggregation. However, when expressed from an heterologous promoter, only the impaired aggregation is rescued. This result demonstrates that the defect during late development is not a consequence of impaired aggregation and indicates a direct role for DdCdk8 in spore formation.

  1. Znhit1 causes cell cycle arrest and down-regulates CDK6 expression

    SciTech Connect

    Yang, Zhengmin; Cao, Yonghao; Zhu, Xiaoyan; Huang, Ying; Ding, Yuqiang; Liu, Xiaolong

    2009-08-14

    Cyclin-dependent kinase 6 (CDK6) is the key element of the D-type cyclin holoenzymes which has been found to function in the regulation of G1-phase of the cell cycle and is presumed to play important roles in T cell function. In this study, Znhit1, a member of a new zinc finger protein family defined by a conserved Zf-HIT domain, induced arrest in the G1-phase of the cell cycle in NIH/3T3 cells. Of the G1 cell cycle factors examined, the expression of CDK6 was found to be strongly down-regulated by Znhit1 via transcriptional repression. This effect may have correlations with the decreased acetylation level of histone H4 in the CDK6 promoter region. In addition, considering that CDK6 expression predominates in T cells, the negative regulatory role of Znhit1 in TCR-induced T cell proliferation was validated using transgenic mice. These findings identified Znhit1 as a CDK6 regulator that plays an important role in cell proliferation.

  2. miR-1299 suppresses cell proliferation of hepatocellular carcinoma (HCC) by targeting CDK6.

    PubMed

    Zhu, Huaqiang; Wang, Guangchuan; Zhou, Xu; Song, Xie; Gao, Hengjun; Ma, Chaoqun; Chang, Hong; Li, Hongguang; Liu, Fang-Feng; Lu, Jun; Ma, Jinben

    2016-10-01

    microRNA (miRNA) plays critical role in HCC initiation and development, many miRNAs have been reported to regulate HCC progression. In this study, we studied the role of miR-1299 in cell proliferation of HCC. We found miR-1299 was significantly downregulated in HCC cells and tissues. miR-1299 overexpression inhibited cell proliferation and arrested cell cycle in G0/G1 phase analyzed by MTT assay, soft agar assay, BrdU cell proliferation assay and cell cycle assay, while miR-1299 knockdown promoted cell proliferation and accelerated G1/S transition. Further analysis suggested the key regulator of G1/S transition, cyclin-dependent kinase 6 (CDK6) was the target of miR-1299, miR-1299 inhibited CDK6 expression and bound to the 3'UTR of CDK6. When double knockdown of miR-1299 and CDK6 promoted cell proliferation copied the phenotype caused by miR-1299 overexpression, suggesting miR-1299 inhibits cell proliferation by targeting CDK6. In summary, our data revealed miR-1299 inhibits cell proliferation, and might be a target for HCC therapy.

  3. Cdk5rap2 regulates centrosome function and chromosome segregation in neuronal progenitors

    PubMed Central

    Lizarraga, Sofia B.; Margossian, Steven P.; Harris, Marian H.; Campagna, Dean R.; Han, An-Ping; Blevins, Sherika; Mudbhary, Raksha; Barker, Jane E.; Walsh, Christopher A.; Fleming, Mark D.

    2010-01-01

    Microcephaly affects ∼1% of the population and is associated with mental retardation, motor defects and, in some cases, seizures. We analyzed the mechanisms underlying brain size determination in a mouse model of human microcephaly. The Hertwig's anemia (an) mutant shows peripheral blood cytopenias, spontaneous aneuploidy and a predisposition to hematopoietic tumors. We found that the an mutation is a genomic inversion of exon 4 of Cdk5rap2, resulting in an in-frame deletion of exon 4 from the mRNA. The finding that CDK5RAP2 human mutations cause microcephaly prompted further analysis of Cdk5rap2an/an mice and we demonstrated that these mice exhibit microcephaly comparable to that of the human disease, resulting from striking neurogenic defects that include proliferative and survival defects in neuronal progenitors. Cdk5rap2an/an neuronal precursors exit the cell cycle prematurely and many undergo apoptosis. These defects are associated with impaired mitotic progression coupled with abnormal mitotic spindle pole number and mitotic orientation. Our findings suggest that the reduction in brain size observed in humans with mutations in CDK5RAP2 is associated with impaired centrosomal function and with changes in mitotic spindle orientation during progenitor proliferation. PMID:20460369

  4. Substrate specificity of the cdk-activating kinase (CAK) is altered upon association with TFIIH.

    PubMed Central

    Rossignol, M; Kolb-Cheynel, I; Egly, J M

    1997-01-01

    The transcription/DNA repair factor TFIIH consists of nine subunits, several exhibiting known functions: helicase/ATPase, kinase activity and DNA binding. Three subunits of TFIIH, cdk7, cyclin H and MAT1, form a ternary complex, cdk-activating kinase (CAK), found either on its own or as part of TFIIH. In the present work, we demonstrate that purified human CAK complex (free CAK) and recombinant CAK (rCAK) produced in insect cells exhibit a strong preference for the cyclin-dependent kinase 2 (cdk2) over a ctd oligopeptide substrate (which mimics the carboxy-terminal domain of the RNA polymerase II). In contrast, TFIIH preferentially phosphorylates the ctd as well as TFIIE alpha, but not cdk2. TFIIH was resolved into four subcomplexes: the kinase complex composed of cdk7, cyclin H and MAT1; the core TFIIH which contains XPB, p62, p52, p44 and p34; and two other subcomplexes in which XPD is found associated with either the kinase complex or with the core TFIIH. Using these fractions, we demonstrate that TFIIH lacking the CAK subcomplex completely recovers its transcriptional activity in the presence of free CAK. Furthermore, studies examining the interactions between TFIIH subunits provide evidence that CAK is integrated within TFIIH via XPB and XPD. PMID:9130708

  5. CDK-Dependent Hsp70 Phosphorylation Controls G1 Cyclin Abundance and Cell-Cycle Progression

    PubMed Central

    Truman, Andrew W.; Kristjansdottir, Kolbrun; Wolfgeher, Donald; Hasin, Naushaba; Polier, Sigrun; Zhang, Hong; Perrett, Sarah; Prodromou, Chrisostomos; Jones, Gary W.; Kron, Stephen J.

    2012-01-01

    Summary In budding yeast, the essential functions of Hsp70 chaperones Ssa1–4 are regulated through expression level, isoform specificity, and cochaperone activity. Suggesting a novel regulatory paradigm, we find that phosphorylation of Ssa1 T36 within a cyclin-dependent kinase (CDK) consensus site conserved among Hsp70 proteins alters cochaperone and client interactions. T36 phosphorylation triggers displacement of Ydj1, allowing Ssa1 to bind the G1 cyclin Cln3 and promote its degradation. The stress CDK Pho85 phosphorylates T36 upon nitrogen starvation or pheromone stimulation, destabilizing Cln3 to delay onset of S phase. In turn, the mitotic CDK Cdk1 phosphorylates T36 to block Cln3 accumulation in G2/M. Suggesting broad conservation from yeast to human, CDK-dependent phosphorylation of Hsc70 T38 similarly regulates Cyclin D1 binding and stability. These results establish an active role for Hsp70 chaperones as signal transducers mediating growth control of G1 cyclin abundance and activity. PMID:23217712

  6. Cdk11-cyclinL controls the assembly of the RNA polymerase II mediator complex.

    PubMed

    Drogat, Julie; Migeot, Valérie; Mommaerts, Elise; Mullier, Caroline; Dieu, Marc; van Bakel, Harm; Hermand, Damien

    2012-11-29

    The large Mediator (L-Mediator) is a general coactivator of RNA polymerase II transcription and is formed by the reversible association of the small Mediator (S-Mediator) and the kinase-module-harboring Cdk8. It is not known how the kinase module association/dissociation is regulated. We describe the fission yeast Cdk11-L-type cyclin pombe (Lcp1) complex and show that its inactivation alters the global expression profile in a manner very similar to that of mutations of the kinase module. Cdk11 is broadly distributed onto chromatin and phosphorylates the Med27 and Med4 Mediator subunits on conserved residues. The association of the kinase module and the S-Mediator is strongly decreased by the inactivation of either Cdk11 or the mutation of its target residues on the Mediator. These results show that Cdk11-Lcp1 regulates the association of the kinase module and the S-Mediator to form the L-Mediator complex.

  7. TNFα Signaling Regulates Cystic Epithelial Cell Proliferation through Akt/mTOR and ERK/MAPK/Cdk2 Mediated Id2 Signaling

    PubMed Central

    Zhou, Julie X.; Fan, Lucy X.; Li, Xiaoyan; Calvet, James P.; Li, Xiaogang

    2015-01-01

    Tumor necrosis factor alpha (TNFα) is present in cyst fluid and promotes cyst growth in autosomal dominant polycystic kidney disease (ADPKD). However, the cross-talk between TNFα and PKD associated signaling pathways remains elusive. In this study, we found that stimulation of renal epithelial cells with TNFα or RANKL (receptor activator of NF-κB ligand), a member of the TNFα cytokine family, activated either the PI3K pathway, leading to AKT and mTOR mediated the increase of Id2 protein, or MAPK and Cdk2 to induce Id2 nuclear translocation. The effects of TNFα/RANKL on increasing Id2 protein and its nuclear translocation caused significantly decreased mRNA and protein levels of the Cdk inhibitor p21, allowing increased cell proliferation. TNFα levels increase in cystic kidneys in response to macrophage infiltration and thus might contribute to cyst growth and enlargement during the progression of disease. As such, this study elucidates a novel mechanism for TNFα signaling in regulating cystic renal epithelial cell proliferation in ADPKD. PMID:26110849

  8. Identification of a common protein association region in the neuronal Cdk5 activator.

    PubMed

    Wang, X; Ching, Y P; Lam, W H; Qi, Z; Zhang, M; Wang, J H

    2000-10-13

    Cyclin-dependent protein kinase 5 (Cdk5) depends on the association with neuronal Cdk5 activator (Nck5a) for kinase activity. A variety of cellular proteins have been shown to undergo high affinity association with Nck5a, including three novel proteins, C42, C48, and C53 found by a yeast two-hybrid screen (Ching, Y. P., Qi, Z., and Wang, J. H. (2000) Gene 242, 285-294). The three proteins show competitive binding to Nck5a suggesting that they bind at a common site. The binding site has been mapped to a region of 26 amino acid residues (residues 145 to 170) at the N-terminal boundary of the kinase activation domain of Nck5a. This region of Nck5a contains an amphipathic alpha-helix whose hydrophobic face is involved in Cdk5 activation (Chin, K. T., Ohki, S, Tang, D., Cheng, H. C., Wang, J. H. , and Zhang, M. (1999) J. Biol. Chem. 274, 7120-7127). Several lines of evidence suggest that Nck5a interacts with the binding proteins at the hydrophilic face of the amphipathic alpha-helix. First, the Nck5a-(145-170) peptide can bind Cdk5 and Nck5a-binding proteins simultaneously. Second, the association of Nck5a-(145-170) to C48 can be markedly reduced by high ionic strength whereas the interaction between Nck5a and Cdk5 is not affected. Third, substitution of Glu(157) by glutamine in Nck5a-(145-170) abolishes the peptide's ability to bind to the three Nck5a-binding proteins without diminishing its Cdk5 binding activity.

  9. CDK4-mediated MnSOD activation and mitochondrial homeostasis in radioadaptive protection

    PubMed Central

    Jin, Cuihong; Qin, Lili; Shi, Yan; Candas, Demet; Fan, Ming; Lu, Chung-Ling; Vaughan, Andrew T. M.; Shen, Rulong; Wu, Larry S.; Liu, Rui; Li, Robert F.; Murley, Jeffrey S.; Gayle, Woloschak; Grdina, David J.; Li, Jian Jian

    2015-01-01

    Mammalian cells are able to sense environmental oxidative and genotoxic conditions such as the environmental low dose ionizing radiation (LDIR) present naturally on earth surface. The stressed cells then can induce a so-called radioadaptive response with an enhanced cellular homeostasis and repair capacity against subsequent similar genotoxic conditions such as a high dose radiation. MnSOD, a primary mitochondrial antioxidant in mammals, has long been known to play a crucial role in the radioadaptive protection through detoxifying O2·- generated by mitochondrial oxidative phosphorylation. Contrasted to the well-studied mechanisms of SOD2 gene regulation, the mechanisms underlying post-translational regulation of MnSOD for radioprotection remain to be defined. Herein, we demonstrate that Cyclin D1-cyclin-dependent kinase 4 (CDK4) serves as the messenger to deliver the stress signal to mitochondria to boost mitochondrial homeostasis in human skin keratinocytes under LDIR adaptive radioprotection. Cyclin D1/CDK4 is found to relocate to mitochondria at the same time as MnSOD enzymatic activation peaks without significant changes of total MnSOD protein level. The mitochondrial-localized CDK4 directly phosphorylates MnSOD at Serine 106 (S106), causing enhanced MnSOD enzymatic activity and mitochondrial respiration. Expression of mitochondria-targeted dominant negative CDK4 or the MnSOD-S106A mutant reverses LDIR-induced mitochondrial enhancement and adaptive protection. The CDK4-mediated MnSOD activation and mitochondrial metabolism boost are also detected in skin tissues of mice receiving in vivo whole body LDIR. These results demonstrate a unique CDK4-mediated mitochondrial communication that allows cells to sense environmental genotoxic stress and boost mitochondrial homeostasis via enhancing phosphorylation and activation of MnSOD. PMID:25578653

  10. CDK4-mediated MnSOD activation and mitochondrial homeostasis in radioadaptive protection.

    PubMed

    Jin, Cuihong; Qin, Lili; Shi, Yan; Candas, Demet; Fan, Ming; Lu, Chung-Ling; Vaughan, Andrew T M; Shen, Rulong; Wu, Larry S; Liu, Rui; Li, Robert F; Murley, Jeffrey S; Woloschak, Gayle; Grdina, David J; Li, Jian Jian

    2015-04-01

    Mammalian cells are able to sense environmental oxidative and genotoxic conditions such as the environmental low-dose ionizing radiation (LDIR) present naturally on the earth's surface. The stressed cells then can induce a so-called radioadaptive response with an enhanced cellular homeostasis and repair capacity against subsequent similar genotoxic conditions such as a high dose radiation. Manganese superoxide dismutase (MnSOD), a primary mitochondrial antioxidant in mammals, has long been known to play a crucial role in radioadaptive protection by detoxifying O2(•-) generated by mitochondrial oxidative phosphorylation. In contrast to the well-studied mechanisms of SOD2 gene regulation, the mechanisms underlying posttranslational regulation of MnSOD for radioprotection remain to be defined. Herein, we demonstrate that cyclin D1/cyclin-dependent kinase 4 (CDK4) serves as the messenger to deliver the stress signal to mitochondria to boost mitochondrial homeostasis in human skin keratinocytes under LDIR-adaptive radioprotection. Cyclin D1/CDK4 relocates to mitochondria at the same time as MnSOD enzymatic activation peaks without significant changes in total MnSOD protein level. The mitochondrial-localized CDK4 directly phosphorylates MnSOD at serine-106 (S106), causing enhanced MnSOD enzymatic activity and mitochondrial respiration. Expression of mitochondria-targeted dominant negative CDK4 or the MnSOD-S106 mutant reverses LDIR-induced mitochondrial enhancement and adaptive protection. The CDK4-mediated MnSOD activation and mitochondrial metabolism boost are also detected in skin tissues of mice receiving in vivo whole-body LDIR. These results demonstrate a unique CDK4-mediated mitochondrial communication that allows cells to sense environmental genotoxic stress and boost mitochondrial homeostasis by enhancing phosphorylation and activation of MnSOD.

  11. MDM2 and CDK4 amplifications are rare events in salivary duct carcinomas

    PubMed Central

    Grünewald, Inga; Trautmann, Marcel; Busch, Alina; Bauer, Larissa; Huss, Sebastian; Schweinshaupt, Petra; Vollbrecht, Claudia; Odenthal, Margarete; Quaas, Alexander; Büttner, Reinhard; Meyer, Moritz F.; Beutner, Dirk; Hüttenbrink, Karl-Bernd; Wardelmann, Eva; Stenner, Markus; Hartmann, Wolfgang

    2016-01-01

    Salivary duct carcinoma (SDC) is an aggressive adenocarcinoma of the salivary glands associated with poor clinical outcome. SDCs are known to carry TP53 mutations in about 50%, however, only little is known about alternative pathogenic mechanisms within the p53 regulatory network. Particularly, data on alterations of the oncogenes MDM2 and CDK4 located in the chromosomal region 12q13-15 are limited in SDC, while genomic rearrangements of the adjacent HMGA2 gene locus are well documented in subsets of SDCs. We here analyzed the mutational status of the TP53 gene, genomic amplification of MDM2, CDK4 and HMGA2 rearrangement/amplification as well as protein expression of TP53 (p53), MDM2 and CDK4 in 51 de novo and ex pleomorphic adenoma SDCs. 25 of 51 cases were found to carry TP53 mutations, associated with extreme positive immunohistochemical p53 staining levels in 13 cases. Three out of 51 tumors had an MDM2 amplification, one of them coinciding with a CDK4 amplification and two with a HMGA2 rearrangement/amplification. Two of the MDM2 amplifications occurred in the setting of a TP53 mutation. Two out of 51 cases showed a CDK4 amplification, one synchronously being MDM2 amplified and the other one displaying concurrent low copy number increases of both, MDM2 and HMGA2. In summary, we here show that subgroups of SDCs display genomic amplifications of MDM2 and/or CDK4, partly in association with TP53 mutations and rearrangement/amplification of HMGA2. Further research is necessary to clarify the role of chromosomal region 12q13-15 alterations in SDC tumorigenesis and their potential prognostic and therapeutic relevance. PMID:27662657

  12. CDK5RAP3 acts as a tumor suppressor in gastric cancer through inhibition of β-catenin signaling.

    PubMed

    Wang, Jia-Bin; Wang, Zu-Wei; Li, Yun; Huang, Chao-Qun; Zheng, Chao-Hui; Li, Ping; Xie, Jian-Wei; Lin, Jian-Xian; Lu, Jun; Chen, Qi-Yue; Cao, Long-Long; Lin, Mi; Tu, Ru-Hong; Lin, Yao; Huang, Chang-Ming

    2017-01-28

    CDK5RAP3 was isolated as a binding protein of the Cdk5 activator p35. Although CDK5RAP3 has been implicated in cancer progression, its expression and function have not been investigated in gastric cancer. Our study demonstrated that the mRNA and protein levels of CDK5RAP3 were markedly decreased in gastric tumor tissues when compared with respective adjacent non-tumor tissues. CDK5RAP3 in gastric cancer cells significantly reduced cell proliferation, migration, invasion and tumor xenograft growth through inhibition of β-catenin. Secondly, CDK5RAP3 was found to suppress the phosphorylation of GSK-3β (Ser9), leading to the phosphorylation (Ser37/Thr41) and subsequent degradation of β-catenin. Lastly, the prognostic value of CDK5RAP3 for overall survival was found to be dependent on β-catenin cytoplasm/nucleus localization in human gastric cancer samples. Collectively, our results demonstrated that CDK5RAP3 negatively regulates the β-catenin signaling pathway by repressing GSK-3β phosphorylation and could be a potential therapeutic target for gastric cancer.

  13. Essential role of the Cdk2 activator RingoA in meiotic telomere tethering to the nuclear envelope

    PubMed Central

    Mikolcevic, Petra; Isoda, Michitaka; Shibuya, Hiroki; del Barco Barrantes, Ivan; Igea, Ana; Suja, José A.; Shackleton, Sue; Watanabe, Yoshinori; Nebreda, Angel R.

    2016-01-01

    Cyclin-dependent kinases (CDKs) play key roles in cell cycle regulation. Genetic analysis in mice has revealed an essential role for Cdk2 in meiosis, which renders Cdk2 knockout (KO) mice sterile. Here we show that mice deficient in RingoA, an atypical activator of Cdk1 and Cdk2 that has no amino acid sequence homology to cyclins, are sterile and display meiotic defects virtually identical to those observed in Cdk2 KO mice including non-homologous chromosome pairing, unrepaired double-strand breaks, undetectable sex-body and pachytene arrest. Interestingly, RingoA is required for Cdk2 targeting to telomeres and RingoA KO spermatocytes display severely affected telomere tethering as well as impaired distribution of Sun1, a protein essential for the attachment of telomeres to the nuclear envelope. Our results identify RingoA as an important activator of Cdk2 at meiotic telomeres, and provide genetic evidence for a physiological function of mammalian Cdk2 that is not dependent on cyclins. PMID:27025256

  14. Dual control by Cdk1 phosphorylation of the budding yeast APC/C ubiquitin ligase activator Cdh1

    PubMed Central

    Höckner, Sebastian; Neumann-Arnold, Lea; Seufert, Wolfgang

    2016-01-01

    The antagonism between cyclin-dependent kinases (Cdks) and the ubiquitin ligase APC/C-Cdh1 is central to eukaryotic cell cycle control. APC/C-Cdh1 targets cyclin B and other regulatory proteins for degradation, whereas Cdks disable APC/C-Cdh1 through phosphorylation of the Cdh1 activator protein at multiple sites. Budding yeast Cdh1 carries nine Cdk phosphorylation sites in its N-terminal regulatory domain, most or all of which contribute to inhibition. However, the precise role of individual sites has remained unclear. Here, we report that the Cdk phosphorylation sites of yeast Cdh1 are organized into autonomous subgroups and act through separate mechanisms. Cdk sites 1–3 had no direct effect on the APC/C binding of Cdh1 but inactivated a bipartite nuclear localization sequence (NLS) and thereby controlled the partitioning of Cdh1 between cytoplasm and nucleus. In contrast, Cdk sites 4–9 did not influence the cell cycle–regulated localization of Cdh1 but prevented its binding to the APC/C. Cdk sites 4–9 reside near two recently identified APC/C interaction motifs in a pattern conserved with the human Cdh1 orthologue. Thus a Cdk-inhibited NLS goes along with Cdk-inhibited APC/C binding sites in yeast Cdh1 to relay the negative control by Cdk1 phosphorylation of the ubiquitin ligase APC/C-Cdh1. PMID:27226481

  15. Dual control by Cdk1 phosphorylation of the budding yeast APC/C ubiquitin ligase activator Cdh1.

    PubMed

    Höckner, Sebastian; Neumann-Arnold, Lea; Seufert, Wolfgang

    2016-07-15

    The antagonism between cyclin-dependent kinases (Cdks) and the ubiquitin ligase APC/C-Cdh1 is central to eukaryotic cell cycle control. APC/C-Cdh1 targets cyclin B and other regulatory proteins for degradation, whereas Cdks disable APC/C-Cdh1 through phosphorylation of the Cdh1 activator protein at multiple sites. Budding yeast Cdh1 carries nine Cdk phosphorylation sites in its N-terminal regulatory domain, most or all of which contribute to inhibition. However, the precise role of individual sites has remained unclear. Here, we report that the Cdk phosphorylation sites of yeast Cdh1 are organized into autonomous subgroups and act through separate mechanisms. Cdk sites 1-3 had no direct effect on the APC/C binding of Cdh1 but inactivated a bipartite nuclear localization sequence (NLS) and thereby controlled the partitioning of Cdh1 between cytoplasm and nucleus. In contrast, Cdk sites 4-9 did not influence the cell cycle-regulated localization of Cdh1 but prevented its binding to the APC/C. Cdk sites 4-9 reside near two recently identified APC/C interaction motifs in a pattern conserved with the human Cdh1 orthologue. Thus a Cdk-inhibited NLS goes along with Cdk-inhibited APC/C binding sites in yeast Cdh1 to relay the negative control by Cdk1 phosphorylation of the ubiquitin ligase APC/C-Cdh1.

  16. Expression of NPAT, a novel substrate of cyclin E–CDK2, promotes S-phase entry

    PubMed Central

    Zhao, Jiyong; Dynlacht, Brian; Imai, Takashi; Hori, Tada-aki; Harlow, Ed

    1998-01-01

    To understand the mechanisms by which CDKs regulate cell cycle progression, it is necessary to identify and characterize the physiological substrates of these kinases. We have developed a screening method to identify novel CDK substrates. One of the cDNAs identified in the screen is identical to the recently isolated NPAT gene. Here we show that NPAT associates with cyclin E–CDK2 in vivo and can be phosphorylated by this CDK. The protein level of NPAT peaks at the G1/S boundary. Overexpression of NPAT accelerates S-phase entry, and this effect is enhanced by coexpression of cyclin E–CDK2. These results suggest that NPAT is a substrate of cyclin E–CDK2 and plays a role in S-phase entry. PMID:9472014

  17. CDK11{sup p58} represses vitamin D receptor-mediated transcriptional activation through promoting its ubiquitin-proteasome degradation

    SciTech Connect

    Chi, Yayun; Hong, Yi; Zong, Hongliang; Wang, Yanlin; Zou, Weiying; Yang, Junwu; Kong, Xiangfei; Yun, Xiaojing; Gu, Jianxin

    2009-08-28

    Vitamin D receptor (VDR) is a member of the nuclear receptor superfamily and regulates transcription of target genes. In this study, we identified CDK11{sup p58} as a novel protein involved in the regulation of VDR. CDK11{sup p58}, a member of the large family of p34cdc2-related kinases, is associated with cell cycle progression, tumorigenesis, and apoptotic signaling. Our study demonstrated that CDK11{sup p58} interacted with VDR and repressed VDR-dependent transcriptional activation. Furthermore, overexpression of CDK11{sup p58} decreased the stability of VDR through promoting its ubiquitin-proteasome-mediated degradation. Taken together, these results suggest that CDK11{sup p58} is involved in the negative regulation of VDR.

  18. Involvement of cyclin D1/CDK4 and pRb mediated by PI3K/AKT pathway activation in Pb{sup 2+}-induced neuronal death in cultured hippocampal neurons

    SciTech Connect

    Li Chenchen Xing Tairan Tang Mingliang Yong Wu Yan Dan Deng Hongmin Wang Huili Wang Ming Chen Jutao Ruan Diyun

    2008-06-15

    Lead (Pb) is widely recognized as a neurotoxicant. One of the suggested mechanisms of lead neurotoxicity is apoptotic cell death. And the mechanism by which Pb{sup 2+} causes neuronal death is not well understood. The present study sought to examine the obligate nature of cyclin D1/cyclin-dependent kinase 4 (CDK4), phosphorylation of its substrate retinoblastoma protein (pRb) and its select upstream signal phosphoinositide 3-kinase (PI3K)/AKT pathway in the death of primary cultured rat hippocampal neurons evoked by Pb{sup 2+}. Our data showed that lead treatment of primary hippocampal cultures results in dose-dependent cell death. Inhibition of CDK4 prevented Pb{sup 2+}-induced neuronal death significantly but was incomplete. In addition, we demonstrated that the levels of cyclin D1 and pRb/p107 were increased during Pb{sup 2+} treatment. These elevated expression persisted up to 48 h, returning to control levels after 72 h. We also presented pharmacological and morphological evidences that cyclin D1/CDK4 and pRb/p107 were required for such kind of neuronal death. Addition of the PI3K inhibitor LY294002 (30 {mu}M) or wortmannin (100 nM) significantly rescued the cultured hippocampal neurons from death caused by Pb{sup 2+}. And that Pb{sup 2+}-elicited phospho-AKT (Ser473) participated in the induction of cyclin D1 and partial pRb/p107 expression. These results provide evidences that cell cycle elements play a required role in the death of neurons evoked by Pb{sup 2+} and suggest that certain signaling elements upstream of cyclin D1/CDK4 are modified and/or required for this form of neuronal death.

  19. The Human CDK8 Subcomplex Is a Histone Kinase That Requires Med12 for Activity and Can Function Independently of Mediator▿

    PubMed Central

    Knuesel, Matthew T.; Meyer, Krista D.; Donner, Aaron J.; Espinosa, Joaquin M.; Taatjes, Dylan J.

    2009-01-01

    The four proteins CDK8, cyclin C, Med12, and Med13 can associate with Mediator and are presumed to form a stable “CDK8 subcomplex” in cells. We describe here the isolation and enzymatic activity of the 600-kDa CDK8 subcomplex purified directly from human cells and also via recombinant expression in insect cells. Biochemical analysis of the recombinant CDK8 subcomplex identifies predicted (TFIIH and RNA polymerase II C-terminal domain [Pol II CTD]) and novel (histone H3, Med13, and CDK8 itself) substrates for the CDK8 kinase. Notably, these novel substrates appear to be metazoan-specific. Such diverse targets imply strict regulation of CDK8 kinase activity. Along these lines, we observe that Mediator itself enables CDK8 kinase activity on chromatin, and we identify Med12—but not Med13—to be essential for activating the CDK8 kinase. Moreover, mass spectrometry analysis of the endogenous CDK8 subcomplex reveals several associated factors, including GCN1L1 and the TRiC chaperonin, that may help control its biological function. In support of this, electron microscopy analysis suggests TRiC sequesters the CDK8 subcomplex and kinase assays reveal the endogenous CDK8 subcomplex—unlike the recombinant submodule—is unable to phosphorylate the Pol II CTD. PMID:19047373

  20. Mesenchymal Stem Cells Loaded with p5, Derived from CDK5 Activator p35, Inhibit Calcium-Induced CDK5 Activation in Endothelial Cells

    PubMed Central

    Kumar, Shant; McDowell, Garry; Krupinski, Jurek; Olah, Peter; Al-Baradie, Raid Saleem; Al-Rukban, Mohammad Othman; Petcu, Eugene Bogdan

    2016-01-01

    The potential use of stem cells as therapeutics in disease has gained momentum over the last few years and recently phase-I clinical trials have shown favourable results in treatment of a small cohort of acute stroke patients. Similarly, they have been used in preclinical models drug-loaded for the effective treatment of solid tumours. Here we have characterized uptake and release of a novel p5-cyclin-dependent kinase 5 (CDK5) inhibitory peptide by mesenchymal stem cells and showed release levels capable of blocking aberrant cyclin-dependent kinase 5 (CDK5) signaling pathways, through phosphorylation of cyclin-dependent kinase 5 (CDK5) and p53. These pathways represent the major acute mechanism stimulating apoptosis after stroke and hence its modulation could benefit patient recovery. This work indicates a potential use for drug-loaded stem cells as delivery vehicles for stroke therapeutics and in addition as anticancer receptacles particularly, if a targeting and/or holding mechanism can be defined. PMID:27651795

  1. CDK1-Cyclin B1 Activates RNMT, Coordinating mRNA Cap Methylation with G1 Phase Transcription

    PubMed Central

    Aregger, Michael; Kaskar, Aneesa; Varshney, Dhaval; Fernandez-Sanchez, Maria Elena; Inesta-Vaquera, Francisco A.; Weidlich, Simone; Cowling, Victoria H.

    2016-01-01

    Summary The creation of translation-competent mRNA is dependent on RNA polymerase II transcripts being modified by addition of the 7-methylguanosine (m7G) cap. The factors that mediate splicing, nuclear export, and translation initiation are recruited to the transcript via the cap. The cap structure is formed by several activities and completed by RNMT (RNA guanine-7 methyltransferase), which catalyzes N7 methylation of the cap guanosine. We report that CDK1-cyclin B1 phosphorylates the RNMT regulatory domain on T77 during G2/M phase of the cell cycle. RNMT T77 phosphorylation activates the enzyme both directly and indirectly by inhibiting interaction with KPNA2, an RNMT inhibitor. RNMT T77 phosphorylation results in elevated m7G cap methyltransferase activity at the beginning of G1 phase, coordinating mRNA capping with the burst of transcription that occurs following nuclear envelope reformation. RNMT T77 phosphorylation is required for the production of cohort of proteins, and inhibiting T77 phosphorylation reduces the cell proliferation rate. PMID:26942677

  2. Targeted Epigenetic Remodeling of the Cdk5 Gene in Nucleus Accumbens Regulates Cocaine- and Stress-Evoked Behavior

    PubMed Central

    Hamilton, Peter J.; Burek, Dominika D.; Lombroso, Sonia I.; Peña, Catherine J.; Neve, Rachael L.; Nestler, Eric J.

    2016-01-01

    Recent studies have implicated epigenetic remodeling in brain reward regions following psychostimulant or stress exposure. It has only recently become possible to target a given type of epigenetic remodeling to a single gene of interest, and to probe the functional relevance of such regulation to neuropsychiatric disease. We sought to examine the role of histone modifications at the murine Cdk5 (cyclin-dependent kinase 5) locus, given growing evidence of Cdk5 expression in nucleus accumbens (NAc) influencing reward-related behaviors. Viral-mediated delivery of engineered zinc finger proteins (ZFP) targeted histone H3 lysine 9/14 acetylation (H3K9/14ac), a transcriptionally active mark, or histone H3 lysine 9 dimethylation (H3K9me2), which is associated with transcriptional repression, specifically to the Cdk5 locus in NAc in vivo. We found that Cdk5-ZFP transcription factors are sufficient to bidirectionally regulate Cdk5 gene expression via enrichment of their respective histone modifications. We examined the behavioral consequences of this epigenetic remodeling and found that Cdk5-targeted H3K9/14ac increased cocaine-induced locomotor behavior, as well as resilience to social stress. Conversely, Cdk5-targeted H3K9me2 attenuated both cocaine-induced locomotor behavior and conditioned place preference, but had no effect on stress-induced social avoidance behavior. The current study provides evidence for the causal role of Cdk5 epigenetic remodeling in NAc in Cdk5 gene expression and in the control of reward and stress responses. Moreover, these data are especially compelling given that previous work demonstrated opposite behavioral phenotypes compared with those reported here upon Cdk5 overexpression or knockdown, demonstrating the importance of targeted epigenetic remodeling tools for studying more subtle molecular changes that contribute to neuropsychiatric disease. SIGNIFICANCE STATEMENT Addiction and depression are highly heritable diseases, yet it has been

  3. Novel plant-specific cyclin-dependent kinase inhibitors induced by biotic and abiotic stresses.

    PubMed

    Peres, Adrian; Churchman, Michelle L; Hariharan, Srivaidehirani; Himanen, Kristiina; Verkest, Aurine; Vandepoele, Klaas; Magyar, Zoltan; Hatzfeld, Yves; Van Der Schueren, Els; Beemster, Gerrit T S; Frankard, Valerie; Larkin, John C; Inzé, Dirk; De Veylder, Lieven

    2007-08-31

    The EL2 gene of rice (Oryza sativa), previously classified as early response gene against the potent biotic elicitor N-acetylchitoheptaose and encoding a short polypeptide with unknown function, was identified as a novel cell cycle regulatory gene related to the recently reported SIAMESE (SIM) gene of Arabidopsis thaliana. Iterative two-hybrid screens, in vitro pull-down assays, and fluorescence resonance energy transfer analyses showed that Orysa; EL2 binds the cyclin-dependent kinase (CDK) CDKA1;1 and D-type cyclins. No interaction was observed with the plant-specific B-type CDKs. The amino acid motif ELERFL was identified to be essential for cyclin, but not for CDK binding. Orysa;EL2 impaired the ability of Orysa; CYCD5;3 to complement a budding yeast (Saccharomyces cerevisiae) triple CLN mutant, whereas recombinant protein inhibited CDK activity in vitro. Moreover, Orysa;EL2 was able to rescue the multicellular trichome phenotype of sim mutants of Arabidopsis, unequivocally demonstrating that Orysa;EL2 operates as a cell cycle inhibitor. Orysa;EL2 mRNA levels were induced by cold, drought, and propionic acid. Our data suggest that Orysa;EL2 encodes a new type of plant CDK inhibitor that links cell cycle progression with biotic and abiotic stress responses.

  4. Balance of calcineurin Aα and CDK5 activities sets release probability at nerve terminals

    PubMed Central

    Kim, Sung Hyun; Ryan, Timothy A.

    2013-01-01

    The control of neurotransmitter release at nerve terminals is of profound importance for neurological function and provides a powerful control system in neural networks. We show that the balance of enzymatic activities of the alpha isoform of the phosphatase calcineurin (CNAα) and the kinase CDK5 has a dramatic influence over single AP-driven exocytosis at nerve terminals. Acute or chronic loss of these enzymatic activities results in a 7-fold impact on single action potential-driven exocytosis. We demonstrate that this control is mediated almost entirely through Cav2.2 (N-type) voltage-gated calcium channels as blocking these channels with a peptide toxin eliminates modulation by these enzymes. We found that a fraction of nerve terminals are kept in a presynaptically silent state with no measurable Ca2+ influx driven by single AP stimuli due to the balance of CNAα and CDK5 activities as blockade of either CNAα or CDK5 activity changes the proportion of presynaptically silent nerve terminals. Thus CNAα and CDK5 enzymatic activities are key determinants of release probability. PMID:23699505

  5. Evaluating the Significance of CDK2-PELP1 Axis in Tumorigenesis and Hormone Therapy Resistance

    DTIC Science & Technology

    2011-02-01

    Rambabu Challa1, Bramanandam Manavathi3, nee Yew2, Rakesh Kumar4, Rajeshwar Rao Tekmal1, and Ratna K. Vadlamudi1ract Estr influen recept cancer genesi the...breast cancer progression. Cancer Res; 70(18); 7166–75. ©2010 AACR.CDK2 of tum emerg stream crucia Estr prolife glands gressio cycle ductio is prop

  6. Regulation of Exit from Quiescence by p27 and Cyclin D1-CDK4

    PubMed Central

    Ladha, Mohamed H.; Lee, Kwang Y.; Upton, Todd M.; Reed, Michael F.; Ewen, Mark E.

    1998-01-01

    The synthesis of cyclin D1 and its assembly with cyclin-dependent kinase 4 (CDK4) to form an active complex is a rate-limiting step in progression through the G1 phase of the cell cycle. Using an activated allele of mitogen-activated protein kinase kinase 1 (MEK1), we show that this kinase plays a significant role in positively regulating the expression of cyclin D1. This was found both in quiescent serum-starved cells and in cells expressing dominant-negative Ras. Despite the observation that cyclin D1 is a target of MEK1, in cycling cells, activated MEK1, but not cyclin D1, is capable of overcoming a G1 arrest induced by Ras inactivation. Either wild-type or catalytically inactive CDK4 cooperates with cyclin D1 in reversing the G1 arrest induced by inhibition of Ras activity. In quiescent NIH 3T3 cells expressing either ectopic cyclin D1 or activated MEK1, cyclin D1 is able to efficiently associate with CDK4; however, the complex is inactive. A significant percentage of the cyclin D1-CDK4 complexes are associated with p27 in serum-starved activated MEK1 or cyclin D1 cell lines. Reduction of p27 levels by expression of antisense p27 allows for S-phase entry from quiescence in NIH 3T3 cells expressing ectopic cyclin D1, but not in parental cells. PMID:9774675

  7. Cdk1 Restrains NHEJ through Phosphorylation of XRCC4-like Factor Xlf1

    PubMed Central

    Hentges, Pierre; Waller, Helen; Reis, Clara C.; Ferreira, Miguel Godinho; Doherty, Aidan J.

    2014-01-01

    Summary Eukaryotic cells use two principal mechanisms for repairing DNA double-strand breaks (DSBs): homologous recombination (HR) and nonhomologous end-joining (NHEJ). DSB repair pathway choice is strongly regulated during the cell cycle. Cyclin-dependent kinase 1 (Cdk1) activates HR by phosphorylation of key recombination factors. However, a mechanism for regulating the NHEJ pathway has not been established. Here, we report that Xlf1, a fission yeast XLF ortholog, is a key regulator of NHEJ activity in the cell cycle. We show that Cdk1 phosphorylates residues in the C terminus of Xlf1 over the course of the cell cycle. Mutation of these residues leads to the loss of Cdk1 phosphorylation, resulting in elevated levels of NHEJ repair in vivo. Together, these data establish that Xlf1 phosphorylation by Cdc2Cdk1 provides a molecular mechanism for downregulation of NHEJ in fission yeast and indicates that XLF is a key regulator of end-joining processes in eukaryotic organisms. PMID:25533340

  8. Class 3 semaphorin mediates dendrite growth in adult newborn neurons through Cdk5/FAK pathway.

    PubMed

    Ng, Teclise; Ryu, Jae Ryun; Sohn, Jae Ho; Tan, Terence; Song, Hongjun; Ming, Guo-Li; Goh, Eyleen L K

    2013-01-01

    Class 3 semaphorins are well-known axonal guidance cues during the embryonic development of mammalian nervous system. However, their activity on postnatally differentiated neurons in neurogenic regions of adult brains has not been characterized. We found that silencing of semaphorin receptors neuropilins (NRP) 1 or 2 in neural progenitors at the adult mouse dentate gyrus resulted in newly differentiated neurons with shorter dendrites and simpler branching in vivo. Tyrosine phosphorylation (Tyr 397) and serine phosphorylation (Ser 732) of FAK were essential for these effects. Semaphorin 3A and 3F mediate serine phosphorylation of FAK through the activation of Cdk5. Silencing of either Cdk5 or FAK in newborn neurons phenocopied the defects in dendritic development seen upon silencing of NRP1 or NRP2. Furthermore, in vivo overexpression of Cdk5 or FAK rescued the dendritic phenotypes seen in NRP1 and NRP2 deficient neurons. These results point to a novel role for class 3 semaphorins in promoting dendritic growth and branching during adult hippocampal neurogenesis through the activation of Cdk5-FAK signaling pathway.

  9. CDK1 Inactivation Regulates Anaphase Spindle Dynamics and Cytokinesis In Vivo

    PubMed Central

    Wheatley, Sally P.; Hinchcliffe, Edward H.; Glotzer, Michael; Hyman, Anthony A.; Sluder, Greenfield; Wang, Yu-li

    1997-01-01

    Through association with CDK1, cyclin B accumulation and destruction govern the G2/M/G1 transitions in eukaryotic cells. To identify CDK1 inactivation-dependent events during late mitosis, we expressed a nondestructible form of cyclin B (cyclin BΔ90) by microinjecting its mRNA into prometaphase normal rat kidney cells. The injection inhibited chromosome decondensation and nuclear envelope formation. Chromosome disjunction occurred normally, but anaphase-like movement persisted until the chromosomes reached the cell periphery, whereupon they often somersaulted and returned to the cell center. Injection of rhodamine-tubulin showed that this movement occurred in the absence of a central anaphase spindle. In 82% of cells cytokinesis was inhibited; the remainder split themselves into two parts in a process reminiscent of Dictyostelium cytofission. In all cells injected, F-actin and myosin II were diffusely localized with no detectable organization at the equator. Our results suggest that a primary effect of CDK1 inactivation is on spindle dynamics that regulate chromosome movement and cytokinesis. Prolonged CDK1 activity may prevent cytokinesis through inhibiting midzone microtubule formation, the behavior of proteins such as TD60, or through the phosphorylation of myosin II regulatory light chain. PMID:9230080

  10. Cyclin/CDK Regulates the Nucleocytoplasmic Localization of the Human Papillomavirus E1 DNA Helicase

    PubMed Central

    Deng, Wentao; Lin, Biing Yuan; Jin, Ge; Wheeler, Crystal G.; Ma, Tianlin; Harper, J. Wade; Broker, Thomas R.; Chow, Louise T.

    2004-01-01

    Cyclin-dependent kinases (CDKs) play key roles in eukaryotic DNA replication and cell cycle progression. Phosphorylation of components of the preinitiation complex activates replication and prevents reinitiation. One mechanism is mediated by nuclear export of critical proteins. Human papillomavirus (HPV) DNA replication requires cellular machinery in addition to the viral replicative DNA helicase E1 and origin recognition protein E2. E1 phosphorylation by cyclin/CDK is critical for efficient viral DNA replication. We now show that E1 is phosphorylated by CDKs in vivo and that phosphorylation regulates its nucleocytoplasmic localization. We identified a conserved regulatory region for localization which contains a dominant leucine-rich nuclear export sequence (NES), the previously defined cyclin binding motif, three serine residues that are CDK substrates, and a putative bipartite nuclear localization sequence. We show that E1 is exported from the nucleus by a CRM1-dependent mechanism unless the NES is inactivated by CDK phosphorylation. Replication activities of E1 phosphorylation site mutations are reduced and correlate inversely with their increased cytoplasmic localization. Nuclear localization and replication activities of most of these mutations are enhanced or restored by mutations in the NES. Collectively, our data demonstrate that CDK phosphorylation controls E1 nuclear localization to support viral DNA amplification. Thus, HPV adopts and adapts the cellular regulatory mechanism to complete its reproductive program. PMID:15564503

  11. Synthesis and evaluation of pyrazolo[1,5-b]pyridazines as selective cyclin dependent kinase inhibitors

    SciTech Connect

    Stevens, Kirk L.; Reno, Michael J.; Alberti, Jennifer B.; Price, Daniel J.; Kane-Carson, Laurie S.; Knick, Victoria B.; Shewchuk, Lisa M.; Hassell, Anne M.; Veal, James M.; Davis, Stephen T.; Griffin, Robert J.; Peel, Michael R.

    2010-10-01

    A novel series of pyrazolo[1,5-b]pyridazines have been synthesized and identified as cyclin dependant kinase inhibitors potentially useful for the treatment of solid tumors. Modification of the hinge-binding amine or the C(2)- and C(6)-substitutions on the pyrazolopyridazine core provided potent inhibitors of CDK4 and demonstrated enzyme selectivity against VEGFR-2 and GSK3{beta}.

  12. Synthesis and biological evaluation of tetrahydro[1,4]diazepino[1,2-a]indol-1-ones as cyclin-dependent kinase inhibitors.

    PubMed

    Putey, Aurélien; Fournet, Guy; Lozach, Olivier; Perrin, Lionel; Meijer, Laurent; Joseph, Benoît

    2014-08-18

    New series of 2,3,4,5-tetrahydro[1,4]diazepino[1,2-a]indol-1-ones and 3,4,5,10-tetrahydro-2H-diazepino[3,4-b]indol-1-ones have been synthesized through an iodolactonisation/lactone-to-lactam rearrangement sequence. These compounds were evaluated as potential protein kinase inhibitors (CDK1, CDK5 and GSK-3). 11-Iodo-2,3,4,5-tetrahydro[1,4]diazepino[1,2-a]indol-1-one derivatives exhibited sub-micromolar inhibitory activity against cyclin-dependent kinases. Docking studies were realized to determine the binding mode of the inhibitors into the ATP binding domain of the CDK5 catalytic site. Our result highlighted two weak Van-der-Waals bonding interactions established between the iodine atom and both phenyl group of Phe 80 and ammonium end of Lys 33.

  13. Drosophila cdk5 is needed for locomotive behavior and NMJ elaboration, but seems dispensable for synaptic transmission.

    PubMed

    Kissler, Alexander E; Pettersson, Nina; Frölich, Andreas; Sigrist, Stephan J; Suter, Beat

    2009-05-01

    Cyclin-dependent kinase 5 (Cdk5) functions in postmitotic neuronal cells and play roles in cell differentiation, cell migration, axonal guidance, and synaptic function. Here, we demonstrate that Drosophila cdk5 is dispensable for adult viability and fertility, a feature that allows us to study its physiological function in the whole animal model. For the adult, cdk5 is needed for proper locomotion and flight performance. Larvae lacking cdk5 in the presynaptic tissue display abnormal crawling motion, and their neuromuscular junctions (NMJ) are elongated and contain a higher number of boutons that are smaller. As a result of these two counteracting effects, the total synaptic area/NMJ is similar to wild type, leading to normal synaptic transmission, indicating that a compensatory mechanism is capable of correcting the problem caused by the lack of cdk5. futsch, the Drosophila MAP1B homolog, is also involved in NMJ morphogenesis, and analysis of the NMJ phenotype of the double mutant futsch(K68); cdk5(-) indicates that cdk5 is epistatic to futsch in this process.

  14. G1 phase arrest induced by Wilms tumor protein WT1 is abrogated by cyclin/CDK complexes.

    PubMed Central

    Kudoh, T; Ishidate, T; Moriyama, M; Toyoshima, K; Akiyama, T

    1995-01-01

    WT1, the Wilms tumor-suppressor gene, maps to the human chromosomal region 11p13 and encodes a transcriptional repressor, WT1, implicated in controlling normal urogenital development. Microinjection of the WT1 cDNA into quiescent cells or cells in early to mid G1 phase blocked serum-induced cell cycle progression into S phase. The activity of WT1 varied significantly depending on the presence or absence of an alternatively spliced region located upstream of the zinc finger domain. The inhibitory activity of WT1 was abrogated by the overexpression of cyclin E/CDK2 as well as cyclin D1/CDK4. Furthermore, both CDK4- and CDK2-associated kinase activities were downregulated in cells overexpressing WT1, whereas the levels of CDK4, CDK2, and cyclin D1 expression were unchanged. These findings suggest that inhibition of the activity of cyclin/CDK complexes may be involved in mediating the WT1-induced cell cycle block. Images Fig. 1 Fig. 2 PMID:7753836

  15. Enrichment of Cdk1-cyclins at DNA double-strand breaks stimulates Fun30 phosphorylation and DNA end resection.

    PubMed

    Chen, Xuefeng; Niu, Hengyao; Yu, Yang; Wang, Jingjing; Zhu, Shuangyi; Zhou, Jianjie; Papusha, Alma; Cui, Dandan; Pan, Xuewen; Kwon, Youngho; Sung, Patrick; Ira, Grzegorz

    2016-04-07

    DNA double-strand breaks (DSBs) are one of the most cytotoxic types of DNA lesion challenging genome integrity. The activity of cyclin-dependent kinase Cdk1 is essential for DSB repair by homologous recombination and for DNA damage signaling. Here we identify the Fun30 chromatin remodeler as a new target of Cdk1. Fun30 is phosphorylated by Cdk1 on Serine 28 to stimulate its functions in DNA damage response including resection of DSB ends. Importantly, Cdk1-dependent phosphorylation of Fun30-S28 increases upon DNA damage and requires the recruitment of Fun30 to DSBs, suggesting that phosphorylation increases in situ at the DNA damage. Consistently, we find that Cdk1 and multiple cyclins become highly enriched at DSBs and that the recruitment of Cdk1 and cyclins Clb2 and Clb5 ensures optimal Fun30 phosphorylation and checkpoint activation. We propose that the enrichment of Cdk1-cyclin complexes at DSBs serves as a mechanism for enhanced targeting and modulating of the activity of DNA damage response proteins.

  16. CDK13, a Kinase Involved in Pre-mRNA Splicing, Is a Component of the Perinucleolar Compartment

    PubMed Central

    Fayet, Claire; Genevière, Anne-Marie

    2016-01-01

    The perinucleolar compartment (PNC) is a subnuclear stucture forming predominantly in cancer cells; its prevalence positively correlates with metastatic capacity. Although several RNA-binding proteins have been characterized in PNC, the molecular function of this compartment remains unclear. Here we demonstrate that the cyclin–dependent kinase 13 (CDK13) is a newly identified constituent of PNC. CDK13 is a kinase involved in the regulation of gene expression and whose overexpression was found to alter pre-mRNA processing. In this study we show that CDK13 is enriched in PNC and co-localizes all along the cell cycle with the PNC component PTB. In contrast, neither the cyclins K and L, known to associate with CDK13, nor the potential kinase substrates accumulate in PNC. We further show that CDK13 overexpression increases PNC prevalence suggesting that CDK13 may be determinant for PNC formation. This result linked to the finding that CDK13 gene is amplified in different types of cancer indicate that this kinase can contribute to cancer development in human. PMID:26886422

  17. Novel alterations in CDK1/cyclin B1 kinase complex formation occur during the acquisition of a polyploid DNA content.

    PubMed Central

    Datta, N S; Williams, J L; Caldwell, J; Curry, A M; Ashcraft, E K; Long, M W

    1996-01-01

    The pathways that regulate the S-phase events associated with the control of DNA replication are poorly understood. The bone marrow megakaryocytes are unique in that they leave the diploid (2C) state to differentiate, synthesizing 4 to 64 times the normal DNA content within a single nucleus, a process known as endomitosis. Human erythroleukemia (HEL) cells model this process, becoming polyploid during phorbol diester-induced megakaryocyte differentiation. The mitotic arrest occurring in these polyploid cells involves novel alterations in the cdk1/cyclin B1 complex: a marked reduction in cdk1 protein levels, and an elevated and sustained expression of cyclin B1. Endomitotic cells thus lack cdk1/cyclin B1-associated H1-histone kinase activity. Constitutive over-expression of cdk1 in endomitotic cells failed to re-initiate normal mitotic events even though cdk1 was present in a 10-fold excess. This was due to an inability of cyclin-B1 to physically associate with cdk1. Nonetheless, endomitotic cyclin B1 possesses immunoprecipitable H1-histone kinase activity, and specifically translocates to the nucleus. We conclude that mitosis is abrogated during endomitosis due to the absence of cdk1 and the failure to form M-phase promoting factor, resulting in a disassociation of mitosis from the completion of S-phase. Further studies on cyclin and its interacting proteins should be informative in understanding endomitosis and cell cycle control. Images PMID:8688553

  18. Functional Interaction between the Bovine Papillomavirus Virus Type 1 Replicative Helicase E1 and Cyclin E-Cdk2†

    PubMed Central

    Cueille, Nathalie; Nougarede, Romain; Mechali, Francisca; Philippe, Michel; Bonne-Andrea, Catherine

    1998-01-01

    We have found that the replicative helicase E1 of bovine papillomavirus type 1 (BPV-1) interacts with a key cell cycle regulator of S phase, the cyclin E-Cdk2 kinase. The E1 helicase, which interacts with cyclin E and not with Cdk2, presents the highest affinity for catalytically active kinase complexes. In addition, E1, cyclin E, and Cdk2 expressed in Xenopus egg extracts are quantitatively coimmunoprecipitated from crude extracts by either anti-Cdk2 or anti-E1 antibodies. E1 protein is also a substrate of the cyclin E-Cdk2 kinase in vitro. Using the viral components required for in vitro BPV-1 replication and free-membrane cytosol from Xenopus eggs, we show that efficient replication of BPV plasmids is dependent on the addition of E1-cyclin E-Cdk2 complexes. Thus, the BPV initiator of replication and cyclin E-Cdk2 are likely to function together as a protein complex which may be the key to the cell cycle regulation of papillomavirus replication. PMID:9696820

  19. Functional interaction between the bovine papillomavirus virus type 1 replicative helicase E1 and cyclin E-Cdk2.

    PubMed

    Cueille, N; Nougarede, R; Mechali, F; Philippe, M; Bonne-Andrea, C

    1998-09-01

    We have found that the replicative helicase E1 of bovine papillomavirus type 1 (BPV-1) interacts with a key cell cycle regulator of S phase, the cyclin E-Cdk2 kinase. The E1 helicase, which interacts with cyclin E and not with Cdk2, presents the highest affinity for catalytically active kinase complexes. In addition, E1, cyclin E, and Cdk2 expressed in Xenopus egg extracts are quantitatively coimmunoprecipitated from crude extracts by either anti-Cdk2 or anti-E1 antibodies. E1 protein is also a substrate of the cyclin E-Cdk2 kinase in vitro. Using the viral components required for in vitro BPV-1 replication and free-membrane cytosol from Xenopus eggs, we show that efficient replication of BPV plasmids is dependent on the addition of E1-cyclin E-Cdk2 complexes. Thus, the BPV initiator of replication and cyclin E-Cdk2 are likely to function together as a protein complex which may be the key to the cell cycle regulation of papillomavirus replication.

  20. Applications of the InChI in cheminformatics with the CDK and Bioclipse

    PubMed Central

    2013-01-01

    Background The InChI algorithms are written in C++ and not available as Java library. Integration into software written in Java therefore requires a bridge between C and Java libraries, provided by the Java Native Interface (JNI) technology. Results We here describe how the InChI library is used in the Bioclipse workbench and the Chemistry Development Kit (CDK) cheminformatics library. To make this possible, a JNI bridge to the InChI library was developed, JNI-InChI, allowing Java software to access the InChI algorithms. By using this bridge, the CDK project packages the InChI binaries in a module and offers easy access from Java using the CDK API. The Bioclipse project packages and offers InChI as a dynamic OSGi bundle that can easily be used by any OSGi-compliant software, in addition to the regular Java Archive and Maven bundles. Bioclipse itself uses the InChI as a key component and calculates it on the fly when visualizing and editing chemical structures. We demonstrate the utility of InChI with various applications in CDK and Bioclipse, such as decision support for chemical liability assessment, tautomer generation, and for knowledge aggregation using a linked data approach. Conclusions These results show that the InChI library can be used in a variety of Java library dependency solutions, making the functionality easily accessible by Java software, such as in the CDK. The applications show various ways the InChI has been used in Bioclipse, to enrich its functionality. PMID:23497723

  1. Pesticide Roundup provokes cell division dysfunction at the level of CDK1/cyclin B activation.

    PubMed

    Marc, Julie; Mulner-Lorillon, Odile; Boulben, Sandrine; Hureau, Dorothée; Durand, Gaël; Bellé, Robert

    2002-03-01

    To assess human health risk from environmental chemicals, we have studied the effect on cell cycle regulation of the widely used glyphosate-containing pesticide Roundup. As a model system we have used sea urchin embryonic first divisions following fertilization, which are appropriate for the study of universal cell cycle regulation without interference with transcription. We show that 0.8% Roundup (containing 8 mM glyphosate) induces a delay in the kinetic of the first cell cleavage of sea urchin embryos. The delay is dependent on the concentration of Roundup. The delay in the cell cycle could be induced using increasing glyphosate concentrations (1-10 mM) in the presence of a subthreshold concentration of Roundup 0.2%, while glyphosate alone was ineffective, thus indicating synergy between glyphosate and Roundup formulation products. The effect of Roundup was not lethal and involved a delay in entry into M-phase of the cell cycle, as judged cytologically. Since CDK1/cyclin B regulates universally the M-phase of the cell cycle, we analyzed CDK1/cyclin B activation during the first division of early development. Roundup delayed the activation of CDK1/cyclin B in vivo. Roundup inhibited also the global protein synthetic rate without preventing the accumulation of cyclin B. In summary, Roundup affects cell cycle regulation by delaying activation of the CDK1/cyclin B complex, by synergic effect of glyphosate and formulation products. Considering the universality among species of the CDK1/cyclin B regulator, our results question the safety of glyphosate and Roundup on human health.

  2. Discovery of [4-Amino-2-(1-methanesulfonylpiperidin-4-ylamino)pyrimidin-5-yl](2,3-difluoro-6-methoxyphenyl)methanone (R547), A Potent and Selective Cyclin-Dependent Kinase Inhibitor with Significiant in Vivo Antitumor Activity

    SciTech Connect

    Chu,X.; DePinto, W.; Bartkovitz, D.; So, S.; Vu, B.; Packman, K.; Lukacs, C.; Ding, Q.; Jiang, N.; et al.

    2006-01-01

    The cyclin-dependent kinases (CDKs) and their cyclin partners are key regulators of the cell cycle. Since deregulation of CDKs is found with high frequency in many human cancer cells, pharmacological inhibition of CDKs with small molecules has the potential to provide an effective strategy for the treatment of cancer. The 2,4-diamino-5-ketopyrimidines 6 reported here represent a novel class of potent and ATP-competitive inhibitors that selectively target the cyclin-dependent kinase family. This diaminopyrimidine core with a substituted 4-piperidine moiety on the C2-amino position and 2-methoxybenzoyl at the C5 position has been identified as the critical structure responsible for the CDK inhibitory activity. Further optimization has led to a good number of analogues that show potent inhibitory activities against CDK1, CDK2, and CDK4 but are inactive against a large panel of serine/threonine and tyrosine kinases (K{sub i} > 10 {mu}M). As one of these representative analogues, compound 39 (R547) has the best CDK inhibitory activities (K{sub i} = 0.001, 0.003, and 0.001 M for CDK1, CDK2, and CDK4, respectively) and excellent in vitro cellular potency, inhibiting the growth of various human tumor cell lines including an HCT116 cell line (IC{sub 50} = 0.08 {mu}M). An X-ray crystal structure of 39 bound to CDK2 has been determined in this study, revealing a binding mode that is consistent with our SAR. Compound 39 demonstrates significant in vivo efficacy in the HCT116 human colorectal tumor xenograft model in nude mice with up to 95% tumor growth inhibition. On the basis of its superior overall profile, 39 was chosen for further evaluation and has progressed into Phase I clinical trial for the treatment of cancer.

  3. Cyclin B1/Cdk1 Phosphorylation of Mitochondrial p53 Induces Anti-Apoptotic Response

    PubMed Central

    Nantajit, Danupon; Fan, Ming; Duru, Nadire; Wen, Yunfei; Reed, John C.; Li, Jian Jian

    2010-01-01

    The pro-apoptotic function of p53 has been well defined in preventing genomic instability and cell transformation. However, the intriguing fact that p53 contributes to a pro-survival advantage of tumor cells under DNA damage conditions raises a critical question in radiation therapy for the 50% human cancers with intact p53 function. Herein, we reveal an anti-apoptotic role of mitochondrial p53 regulated by the cell cycle complex cyclin B1/Cdk1 in irradiated human colon cancer HCT116 cells with p53+/+ status. Steady-state levels of p53 and cyclin B1/Cdk1 were identified in the mitochondria of many human and mouse cells, and their mitochondrial influx was significantly enhanced by radiation. The mitochondrial kinase activity of cyclin B1/Cdk1 was found to specifically phosphorylate p53 at Ser-315 residue, leading to enhanced mitochondrial ATP production and reduced mitochondrial apoptosis. The improved mitochondrial function can be blocked by transfection of mutant p53 Ser-315-Ala, or by siRNA knockdown of cyclin B1 and Cdk1 genes. Enforced translocation of cyclin B1 and Cdk1 into mitochondria with a mitochondrial-targeting-peptide increased levels of Ser-315 phosphorylation on mitochondrial p53, improved ATP production and decreased apoptosis by sequestering p53 from binding to Bcl-2 and Bcl-xL. Furthermore, reconstitution of wild-type p53 in p53-deficient HCT116 p53−/− cells resulted in an increased mitochondrial ATP production and suppression of apoptosis. Such phenomena were absent in the p53-deficient HCT116 p53−/− cells reconstituted with the mutant p53. These results demonstrate a unique anti-apoptotic function of mitochondrial p53 regulated by cyclin B1/Cdk1-mediated Ser-315 phosphorylation in p53-wild-type tumor cells, which may provide insights for improving the efficacy of anti-cancer therapy, especially for tumors that retain p53. PMID:20808790

  4. Sirtuin inhibitors, EX527 and AGK2, suppress cell migration by inhibiting HSF1 protein stability.

    PubMed

    Kim, Hyun-Woo; Kim, Soo-A; Ahn, Sang-Gun

    2016-01-01

    The histone deacetylases (HDACs), Sirtuin 1 (Sirt1) and Sirt2, play crucial roles in many biological processes, including cell proliferation, differentiation and apoptosis. HDAC inhibitors have been considered as a potential therapeutic approach for various types of cancers. Here, we demonstrated that the Sirt1 and Sirt2 inhibitors EX527 and AGK2 suppressed cell growth and caused G1 phase arrest by inhibiting the expression of Cdk6 and/or Cdk4. An agar colony formation assay revealed that EX527 and AGK2 decreased colony formation in soft agar. Furthermore, EX527 and AGK2 pretreatment inhibited the expression of HSF1 and HSP27 and induced HSF1 ubiquitination. Sirt1 overexpression increased HSF1 expression and/or stabilization and induced cell migration in a scratch assay. Overall, these results indicate that EX527 and AGK2 suppress cell growth and migration by inhibiting HSF1 protein stability.

  5. CDK10/cyclin M is a protein kinase that controls ETS2 degradation and is deficient in STAR syndrome

    PubMed Central

    Guen, Vincent J.; Gamble, Carly; Flajolet, Marc; Unger, Sheila; Thollet, Aurélie; Ferandin, Yoan; Superti-Furga, Andrea; Cohen, Pascale A.; Meijer, Laurent; Colas, Pierre

    2013-01-01

    Cyclin-dependent kinases (CDKs) regulate a variety of fundamental cellular processes. CDK10 stands out as one of the last orphan CDKs for which no activating cyclin has been identified and no kinase activity revealed. Previous work has shown that CDK10 silencing increases ETS2 (v-ets erythroblastosis virus E26 oncogene homolog 2)-driven activation of the MAPK pathway, which confers tamoxifen resistance to breast cancer cells. The precise mechanisms by which CDK10 modulates ETS2 activity, and more generally the functions of CDK10, remain elusive. Here we demonstrate that CDK10 is a cyclin-dependent kinase by identifying cyclin M as an activating cyclin. Cyclin M, an orphan cyclin, is the product of FAM58A, whose mutations cause STAR syndrome, a human developmental anomaly whose features include toe syndactyly, telecanthus, and anogenital and renal malformations. We show that STAR syndrome-associated cyclin M mutants are unable to interact with CDK10. Cyclin M silencing phenocopies CDK10 silencing in increasing c-Raf and in conferring tamoxifen resistance to breast cancer cells. CDK10/cyclin M phosphorylates ETS2 in vitro, and in cells it positively controls ETS2 degradation by the proteasome. ETS2 protein levels are increased in cells derived from a STAR patient, and this increase is attributable to decreased cyclin M levels. Altogether, our results reveal an additional regulatory mechanism for ETS2, which plays key roles in cancer and development. They also shed light on the molecular mechanisms underlying STAR syndrome. PMID:24218572

  6. Robustness of CDK2 in Triggering Cellular Senescence based on Probability of DNA-damaged Cells Passing G1/S Checkpoint

    NASA Astrophysics Data System (ADS)

    Ling, Hong; Samarasinghe, Sandhya; Kulasiri, Don

    2011-06-01

    Recent experiments have shown that cellular senescence, a mechanism employed by cells for thwarting cell proliferation, plays an important role in protecting cells against cancer; therefore, a deeper understanding of cellular senescence can lead to effective cancer treatment. Inhibition of CDK2 is thought to be the critical trigger for cellular senescence. In this study, we first implement a mathematical model of G1/S transition involving the DNA-damage pathway and show that cellular senescence can be achieved by lowering CDK2. The robustness of CDK2 in triggering cellular senescence is determined from the probability (β) of DNA-damaged cells passing G1/S checkpoint for normal CDK2 and CDK2-deficient situations based on different thresholds of the peak time of two important biomarkers, CycE and E2F. The comparison of the values of β under the normal CDK2 and lower CDK2 levels reveals that reducing CDK2 levels can decrease the percentage of damaged cells passing G1/S checkpoint; more importantly, 50% reduction of CDK2 achieves 65% reduction in the percentage of damaged cells passing the G1/S checkpoint. These results point out that the developed model can highlight the possibility of lowering the bar for cellular senescence by reducing CDK2 levels. The results of investigation of β for the different thresholds of the peak times of other biomarkers show that β is insensitive to these perturbations of the peak time indicating that CDK2 activity is robust in lowering the senescence bar for low and high levels of DNA-damage. Furthermore, a mathematical formulation of robustness indicates that the robustness of CDK2-triggered senescence increases with decreasing levels of CDK2, and is slightly greater for low-level DNA damage condition.

  7. Phosphorylation of EB2 by Aurora B and CDK1 ensures mitotic progression and genome stability

    PubMed Central

    Iimori, Makoto; Watanabe, Sugiko; Kiyonari, Shinichi; Matsuoka, Kazuaki; Sakasai, Ryo; Saeki, Hiroshi; Oki, Eiji; Kitao, Hiroyuki; Maehara, Yoshihiko

    2016-01-01

    Temporal regulation of microtubule dynamics is essential for proper progression of mitosis and control of microtubule plus-end tracking proteins by phosphorylation is an essential component of this regulation. Here we show that Aurora B and CDK1 phosphorylate microtubule end-binding protein 2 (EB2) at multiple sites within the amino terminus and a cluster of serine/threonine residues in the linker connecting the calponin homology and end-binding homology domains. EB2 phosphorylation, which is strictly associated with mitotic entry and progression, reduces the binding affinity of EB2 for microtubules. Expression of non-phosphorylatable EB2 induces stable kinetochore microtubule dynamics and delays formation of bipolar metaphase plates in a microtubule binding-dependent manner, and leads to aneuploidy even in unperturbed mitosis. We propose that Aurora B and CDK1 temporally regulate the binding affinity of EB2 for microtubules, thereby ensuring kinetochore microtubule dynamics, proper mitotic progression and genome stability. PMID:27030108

  8. CDK8-Mediated STAT1-S727 Phosphorylation Restrains NK Cell Cytotoxicity and Tumor Surveillance

    PubMed Central

    Putz, Eva Maria; Gotthardt, Dagmar; Hoermann, Gregor; Csiszar, Agnes; Wirth, Silvia; Berger, Angelika; Straka, Elisabeth; Rigler, Doris; Wallner, Barbara; Jamieson, Amanda M.; Pickl, Winfried F.; Zebedin-Brandl, Eva Maria; Müller, Mathias; Decker, Thomas; Sexl, Veronika

    2013-01-01

    Summary The transcription factor STAT1 is important in natural killer (NK) cells, which provide immediate defense against tumor and virally infected cells. We show that mutation of a single phosphorylation site (Stat1-S727A) enhances NK cell cytotoxicity against a range of tumor cells, accompanied by increased expression of perforin and granzyme B. Stat1-S727A mice display significantly delayed disease onset in NK cell-surveilled tumor models including melanoma, leukemia, and metastasizing breast cancer. Constitutive phosphorylation of S727 depends on cyclin-dependent kinase 8 (CDK8). Inhibition of CDK8-mediated STAT1-S727 phosphorylation may thus represent a therapeutic strategy for stimulating NK cell-mediated tumor surveillance. PMID:23933255

  9. Melatonin attenuates MPTP-induced neurotoxicity via preventing CDK5-mediated autophagy and SNCA/α-synuclein aggregation.

    PubMed

    Su, Ling-Yan; Li, Hao; Lv, Li; Feng, Yue-Mei; Li, Guo-Dong; Luo, Rongcan; Zhou, He-Jiang; Lei, Xiao-Guang; Ma, Liang; Li, Jia-Li; Xu, Lin; Hu, Xin-Tian; Yao, Yong-Gang

    2015-01-01

    Autophagy is involved in the pathogenesis of neurodegenerative diseases including Parkinson disease (PD). However, little is known about the regulation of autophagy in neurodegenerative process. In this study, we characterized aberrant activation of autophagy induced by neurotoxin 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) and demonstrated that melatonin has a protective effect on neurotoxicity. We found an excessive activation of autophagy in monkey brain tissues and C6 cells, induced by MPTP, which is mediated by CDK5 (cyclin-dependent kinase 5). MPTP treatment significantly reduced total dendritic length and dendritic complexity of cultured primary cortical neurons and melatonin could reverse this effect. Decreased TH (tyrosine hydroxylase)-positive cells and dendrites of dopaminergic neurons in the substantia nigra pars compacta (SNc) were observed in MPTP-treated monkeys and mice. Along with decreased TH protein level, we observed an upregulation of CDK5 and enhanced autophagic activity in the striatum of mice with MPTP injection. These changes could be salvaged by melatonin treatment or knockdown of CDK5. Importantly, melatonin or knockdown of CDK5 reduced MPTP-induced SNCA/α-synuclein aggregation in mice, which is widely thought to trigger the pathogenesis of PD. Finally, melatonin or knockdown of CDK5 counteracted the PD phenotype in mice induced by MPTP. Our findings uncover a potent role of CDK5-mediated autophagy in the pathogenesis of PD, and suggest that control of autophagic pathways may provide an important clue for exploring potential target for novel therapeutics of PD.

  10. Triple negative breast cancer therapy with CDK1 siRNA delivered by cationic lipid assisted PEG-PLA nanoparticles.

    PubMed

    Liu, Yang; Zhu, Yan-Hua; Mao, Cheng-Qiong; Dou, Shuang; Shen, Song; Tan, Zi-Bin; Wang, Jun

    2014-10-28

    There is no effective clinical therapy yet for triple-negative breast cancer (TNBC) without particular human epidermal growth factor receptor-2, estrogen and progesterone receptor expression. In this study, we report a molecularly targeted and synthetic lethality-based siRNA therapy for TNBC treatment, using cationic lipid assisted poly(ethylene glycol)-b-poly(d,l-lactide) (PEG-PLA) nanoparticles as the siRNA carrier. It is demonstrated that only in c-Myc overexpressed TNBC cells, while not in normal mammary epithelial cells, delivery of siRNA targeting cyclin-dependent kinase 1 (CDK1) with the nanoparticle carrier (NPsiCDK1) induces cell viability decreasing and cell apoptosis through RNAi-mediated CDK1 expression inhibition, indicating the synthetic lethality between c-Myc with CDK1 in TNBC cells. Moreover, systemic delivery of NPsiCDK1 is able to suppress tumor growth in mice bearing SUM149 and BT549 xenograft and cause no systemic toxicity or activate the innate immune response, suggesting the therapeutic promise with such nanoparticles carrying siCDK1 for c-Myc overexpressed triple negative breast cancer.

  11. CDK9 and its repressor LARP7 modulate cardiomyocyte proliferation and response to injury in the zebrafish heart

    PubMed Central

    Matrone, Gianfranco; Wilson, Kathryn S.; Maqsood, Sana; Mullins, John J.; Tucker, Carl S.; Denvir, Martin A.

    2015-01-01

    ABSTRACT Cyclin dependent kinase (Cdk)9 acts through the positive transcription elongation factor-b (P-TEFb) complex to activate and expand transcription through RNA polymerase II. It has also been shown to regulate cardiomyocyte hypertrophy, with recent evidence linking it to cardiomyocyte proliferation. We hypothesised that modification of CDK9 activity could both impair and enhance the cardiac response to injury by modifying cardiomyocyte proliferation. Cdk9 expression and activity were inhibited in the zebrafish (Danio rerio) embryo. We show that dephosphorylation of residue Ser2 on the C-terminal domain of RNA polymerase II is associated with impaired cardiac structure and function, and cardiomyocyte proliferation and also results in impaired functional recovery following cardiac laser injury. In contrast, de-repression of Cdk9 activity, through knockdown of La-related protein (Larp7) increases phosphorylation of Ser2 in RNA polymerase II and increases cardiomyocyte proliferation. Larp7 knockdown rescued the structural and functional phenotype associated with knockdown of Cdk9. The balance of Cdk9 and Larp7 plays a key role in cardiomyocyte proliferation and response to injury. Larp7 represents a potentially novel therapeutic target to promote cardiomyocyte proliferation and recovery from injury. PMID:26542022

  12. MicroRNA-191 triggers keratinocytes senescence by SATB1 and CDK6 downregulation

    SciTech Connect

    Lena, A.M.; Mancini, M.; Rivetti di Val Cervo, P. [University of 'Tor Vergata', Department of Experimental Medicine and Biochemical Sciences, Via Montpellier 1, Rome 00133; Istituto Dermopatico dell'Immacolata-Istituto di Ricovero e Cura a Carattere Scientifico , Laboratory of Biochemistry c Saintigny, G.; Mahe, C. [CHANEL Parfums Beaute, 135 av. Charles de Gaulle, F 92521, Neuilly Melino, G. [University of 'Tor Vergata', Department of Experimental Medicine and Biochemical Sciences, Via Montpellier 1, Rome 00133; Istituto Dermopatico dell'Immacolata-Istituto di Ricovero e Cura a Carattere Scientifico , Laboratory of Biochemistry c Association Cell Death and Differentiation c and others

    2012-07-06

    Highlights: Black-Right-Pointing-Pointer miR-191 expression is upregulated in senescencent human epidermal keratinocytes. Black-Right-Pointing-Pointer miR-191 overexpression is sufficient per se to induce senescence in keratinocytes. Black-Right-Pointing-Pointer SATB1 and CDK6 are downregulated in senescence and are direct miR-191 targets. Black-Right-Pointing-Pointer SATB1 and CDK6 silencing by siRNA triggers senescence in HEKn cells. -- Abstract: Keratinocyte replicative senescence has an important role in time-dependent changes of the epidermis, a tissue with high turnover. Senescence encompasses growth arrest during which cells remain metabolically active but acquire a typical enlarged, vacuolar and flattened morphology. It is also accompanied by the expression of endogenous senescence-associated-{beta}-galactosidase and specific gene expression profiles. MicroRNAs levels have been shown to be modulated during keratinocytes senescence, playing key roles in inhibiting proliferation and in the acquisition of senescent markers. Here, we identify miR-191 as an anti-proliferative and replicative senescence-associated miRNA in primary human keratinocytes. Its overexpression is sufficient per se to induce senescence, as evaluated by induction of several senescence-associated markers. We show that SATB1 and CDK6 3 Prime UTRs are two miR-191 direct targets involved in this pathway. Cdk6 and Satb1 protein levels decrease during keratinocytes replicative senescence and their silencing by siRNA is able to induce a G1 block in cell cycle, accompanied by an increase in senescence-associated markers.

  13. Structural Basis of CDK4 Inhibition by p18INK4

    DTIC Science & Technology

    1999-05-01

    structure database (Accession Number: IIHB). The crystal structure reveals an elongated molecule comprised of five ankyrin repeat units. Each ankyrin repeat...Marmorstein, Crystal structure of the CDK4/6 inhibitory protein p]8(INK4c) provides insights into ankyrin -like repeat structure/function and tumor... ankyrin -like repeat structure/function and tumor-derived p16(INK4) mutations. Nature Structural Biology, 1998. 5(1): p. 74-81. 2. Presentation

  14. Feasibility of using molecular docking-based virtual screening for searching dual target kinase inhibitors.

    PubMed

    Zhou, Shunye; Li, Youyong; Hou, Tingjun

    2013-04-22

    Multitarget agents have been extensively explored for solving limited efficacies, poor safety, and resistant profiles of an individual target. Theoretical approaches for searching and designing multitarget agents are critically useful. Here, the performance of molecular docking to search dual-target inhibitors for four kinase pairs (CDK2-GSK3B, EGFR-Src, Lck-Src, and Lck-VEGFR2) was assessed. First, the representative structures for each kinase target were chosen by structural clustering of available crystal structures. Next, the performance of molecular docking to distinguish inhibitors from noninhibitors for each individual kinase target was evaluated. The results show that molecular docking-based virtual screening illustrates good capability to find known inhibitors for individual targets, but the prediction accuracy is structurally dependent. Finally, the performance of molecular docking to identify the dual-target kinase inhibitors for four kinase pairs was evaluated. The analyses show that molecular docking successfully filters out most noninhibitors and achieves promising performance for identifying dual-kinase inhibitors for CDK2-GSK3B and Lck-VEGFR2. But a high false-positive rate leads to low enrichment of true dual-target inhibitors in the final list. This study suggests that molecular docking serves as a useful tool in searching inhibitors against dual or even multiple kinase targets, but integration with other virtual screening tools is necessary for achieving better predictions.

  15. Loss of Cytoplasmic CDK1 Predicts Poor Survival in Human Lung Cancer and Confers Chemotherapeutic Resistance

    PubMed Central

    Zhang, Chunyu; Elkahloun, Abdel G.; Robertson, Matthew; Gills, Joell J.; Tsurutani, Junji; Shih, Joanna H.; Fukuoka, Junya; Hollander, M. Christine; Harris, Curtis C.; Travis, William D.; Jen, Jin; Dennis, Phillip A.

    2011-01-01

    The dismal lethality of lung cancer is due to late stage at diagnosis and inherent therapeutic resistance. The incorporation of targeted therapies has modestly improved clinical outcomes, but the identification of new targets could further improve clinical outcomes by guiding stratification of poor-risk early stage patients and individualizing therapeutic choices. We hypothesized that a sequential, combined microarray approach would be valuable to identify and validate new targets in lung cancer. We profiled gene expression signatures during lung epithelial cell immortalization and transformation, and showed that genes involved in mitosis were progressively enhanced in carcinogenesis. 28 genes were validated by immunoblotting and 4 genes were further evaluated in non-small cell lung cancer tissue microarrays. Although CDK1 was highly expressed in tumor tissues, its loss from the cytoplasm unexpectedly predicted poor survival and conferred resistance to chemotherapy in multiple cell lines, especially microtubule-directed agents. An analysis of expression of CDK1 and CDK1-associated genes in the NCI60 cell line database confirmed the broad association of these genes with chemotherapeutic responsiveness. These results have implications for personalizing lung cancer therapy and highlight the potential of combined approaches for biomarker discovery. PMID:21887332

  16. CDK1 Prevents Unscheduled PLK4-STIL Complex Assembly in Centriole Biogenesis.

    PubMed

    Zitouni, Sihem; Francia, Maria E; Leal, Filipe; Montenegro Gouveia, Susana; Nabais, Catarina; Duarte, Paulo; Gilberto, Samuel; Brito, Daniela; Moyer, Tyler; Kandels-Lewis, Steffi; Ohta, Midori; Kitagawa, Daiju; Holland, Andrew J; Karsenti, Eric; Lorca, Thierry; Lince-Faria, Mariana; Bettencourt-Dias, Mónica

    2016-05-09

    Centrioles are essential for the assembly of both centrosomes and cilia. Centriole biogenesis occurs once and only once per cell cycle and is temporally coordinated with cell-cycle progression, ensuring the formation of the right number of centrioles at the right time. The formation of new daughter centrioles is guided by a pre-existing, mother centriole. The proximity between mother and daughter centrioles was proposed to restrict new centriole formation until they separate beyond a critical distance. Paradoxically, mother and daughter centrioles overcome this distance in early mitosis, at a time when triggers for centriole biogenesis Polo-like kinase 4 (PLK4) and its substrate STIL are abundant. Here we show that in mitosis, the mitotic kinase CDK1-CyclinB binds STIL and prevents formation of the PLK4-STIL complex and STIL phosphorylation by PLK4, thus inhibiting untimely onset of centriole biogenesis. After CDK1-CyclinB inactivation upon mitotic exit, PLK4 can bind and phosphorylate STIL in G1, allowing pro-centriole assembly in the subsequent S phase. Our work shows that complementary mechanisms, such as mother-daughter centriole proximity and CDK1-CyclinB interaction with centriolar components, ensure that centriole biogenesis occurs once and only once per cell cycle, raising parallels to the cell-cycle regulation of DNA replication and centromere formation.

  17. Astrocyte NMDA receptors' activity sustains neuronal survival through a Cdk5–Nrf2 pathway

    PubMed Central

    Jimenez-Blasco, D; Santofimia-Castaño, P; Gonzalez, A; Almeida, A; Bolaños, J P

    2015-01-01

    Neurotransmission unavoidably increases mitochondrial reactive oxygen species. However, the intrinsic antioxidant defense of neurons is weak and hence the mechanism whereby these cells are physiologically protected against oxidative damage is unknown. Here we found that the antioxidant defense of neurons is repressed owing to the continuous protein destabilization of the master antioxidant transcriptional activator, nuclear factor-erythroid 2-related factor-2 (Nrf2). By contrast, Nrf2 is highly stable in neighbor astrocytes explaining their robust antioxidant defense and resistance against oxidative stress. We also show that subtle and persistent stimulation of N-methyl-d-aspartate receptors (NMDAR) in astrocytes, through a mechanism not requiring extracellular Ca2+ influx, upregulates a signal transduction pathway involving phospholipase C-mediated endoplasmic reticulum release of Ca2+ and protein kinase Cδ activation. Active protein kinase Cδ promotes, by phosphorylation, the stabilization of p35, a cyclin-dependent kinase-5 (Cdk5) cofactor. Active p35/Cdk5 complex in the cytosol phosphorylates Nrf2 at Thr395, Ser433 and Thr439 that is sufficient to promote Nrf2 translocation to the nucleus and induce the expression of antioxidant genes. Furthermore, this Cdk5–Nrf2 transduction pathway boosts glutathione metabolism in astrocytes efficiently protecting closely spaced neurons against oxidative damage. Thus, intercellular communication through NMDAR couples neurotransmission with neuronal survival. PMID:25909891

  18. Memory Enhancement by Targeting Cdk5 Regulation of NR2B

    PubMed Central

    Plattner, Florian; Hernandéz, Adan; Kistler, Tara M.; Pozo, Karine; Zhong, Ping; Yuen, Eunice Y.; Tan, Chunfeng; Hawasli, Ammar H.; Cooke, Sam F.; Nishi, Akinori; Guo, Ailan; Wiederhold, Thorsten; Yan, Zhen; Bibb, James A.

    2014-01-01

    SUMMARY Many psychiatric and neurological disorders are characterized by learning and memory deficits, for which cognitive enhancement is considered a valid treatment strategy. The N-methyl-D-aspartate receptor (NMDAR) is a prime target for the development of cognitive enhancers due to its fundamental role in learning and memory. In particular, the NMDAR subunit NR2B improves synaptic plasticity and memory when over-expressed in neurons. However, NR2B regulation is not well understood and no therapies potentiating NMDAR function have been developed. Here, we show that serine 1116 of NR2B is phosphorylated by cyclin-dependent kinase 5 (Cdk5). Cdk5-dependent NR2B phosphorylation is regulated by neuronal activity and controls the receptor’s cell surface expression. Disrupting NR2B-Cdk5 interaction using a small interfering peptide (siP) increases NR2B surface levels, facilitates synaptic transmission, and improves memory formation in vivo. Our results reveal a novel regulatory mechanism critical to NR2B function that can be targeted for the development of cognitive enhancers. PMID:24607229

  19. SUMOylation of Rb enhances its binding with CDK2 and phosphorylation at early G1 phase.

    PubMed

    Meng, Fengxi; Qian, Jiang; Yue, Han; Li, Xiaofeng; Xue, Kang

    2016-07-02

    Retinoblastoma protein (Rb) is a prototypical tumor suppressor that is vital to the negative regulation of the cell cycle and tumor progression. Hypo-phosphorylated Rb is associated with G0/G1 arrest by suppressing E2F transcription factor activity, whereas Rb hyper-phosphorylation allows E2F release and cell cycle progression from G0/G1 to S phase. However, the factors that regulate cyclin-dependent protein kinase (CDK)-dependent hyper-phosphorylation of Rb during the cell cycle remain obscure. In this study, we show that throughout the cell cycle, Rb is specifically small ubiquitin-like modifier (SUMO)ylated at early G1 phase. SUMOylation of Rb stimulates its phosphorylation level by recruiting a SUMO-interaction motif (SIM)-containing kinase CDK2, leading to Rb hyper-phosphorylation and E2F-1 release. In contrast, a SUMO-deficient Rb mutant results in reduced SUMOylation and phosphorylation, weakened CDK2 binding, and attenuated E2F-1 sequestration. Furthermore, we reveal that Rb SUMOylation is required for cell proliferation. Therefore, our study describes a novel mechanism that regulates Rb phosphorylation during cell cycle progression.

  20. S-nitrosylation-dependent proteasomal degradation restrains Cdk5 activity to regulate hippocampal synaptic strength

    PubMed Central

    Zhang, Peng; Fu, Wing-Yu; Fu, Amy K. Y.; Ip, Nancy Y.

    2015-01-01

    Precise regulation of synaptic strength requires coordinated activity and functions of synaptic proteins, which is controlled by a variety of post-translational modification. Here we report that S-nitrosylation of p35, the activator of cyclin-dependent kinase 5 (Cdk5), by nitric oxide (NO) is important for the regulation of excitatory synaptic strength. While blockade of NO signalling results in structural and functional synaptic deficits as indicated by reduced mature dendritic spine density and surface expression of glutamate receptor subunits, phosphorylation of numerous synaptic substrates of Cdk5 and its activity are aberrantly upregulated following reduced NO production. The results show that the NO-induced reduction in Cdk5 activity is mediated by S-nitrosylation of p35, resulting in its ubiquitination and degradation by the E3 ligase PJA2. Silencing p35 protein in hippocampal neurons partially rescues the NO blockade-induced synaptic deficits. These findings collectively demonstrate that p35 S-nitrosylation by NO signalling is critical for regulating hippocampal synaptic strength. PMID:26503494

  1. Replication factory activation can be decoupled from the replication timing program by modulating Cdk levels

    PubMed Central

    Thomson, Alexander M.; Gillespie, Peter J.

    2010-01-01

    In the metazoan replication timing program, clusters of replication origins located in different subchromosomal domains fire at different times during S phase. We have used Xenopus laevis egg extracts to drive an accelerated replication timing program in mammalian nuclei. Although replicative stress caused checkpoint-induced slowing of the timing program, inhibition of checkpoint kinases in an unperturbed S phase did not accelerate it. Lowering cyclin-dependent kinase (Cdk) activity slowed both replication rate and progression through the timing program, whereas raising Cdk activity increased them. Surprisingly, modest alteration of Cdk activity changed the amount of DNA synthesized during different stages of the timing program. This was associated with a change in the number of active replication factories, whereas the distribution of origins within active factories remained relatively normal. The ability of Cdks to differentially effect replication initiation, factory activation, and progression through the timing program provides new insights into the way that chromosomal DNA replication is organized during S phase. PMID:20083602

  2. A computational model of binding thermodynamics: the design of cyclin-dependent kinase 2 inhibitors.

    PubMed

    Sims, Peter A; Wong, Chung F; McCammon, J Andrew

    2003-07-17

    The cyclin-dependent protein kinases are important targets in drug discovery because of their role in cell cycle regulation. In this computational study, we have applied a continuum solvent model to study the interactions between cyclin-dependent kinase 2 (CDK2) and analogues of the clinically tested anticancer agent flavopiridol. The continuum solvent model uses Coulomb's law to account for direct electrostatic interactions, solves the Poisson equation to obtain the electrostatic contributions to solvation energy, and calculates scaled solvent-accessible surface area to account for hydrophobic interactions. The computed free energy of binding gauges the strength of protein-ligand interactions. Our model was first validated through a study on the binding of a number of flavopiridol derivatives to CDK2, and its ability to identify potent inhibitors was observed. The model was then used to aid in the design of novel CDK2 inhibitors with the aid of a computational sensitivity analysis. Some of these hypothetical structures could be significantly more potent than the lead compound flavopiridol. We applied two approaches to gain insights into designing selective inhibitors. One relied on the comparative analysis of the binding pocket for several hundred protein kinases to identify the parts of a lead compound whose modifications might lead to selective compounds. The other was based on building and using homology models for energy calculations. The homology models appear to be able to classify ligand potency into groups but cannot yet give reliable quantitative results.

  3. Structure-based lead discovery for protein kinase C zeta inhibitor design by exploiting kinase-inhibitor complex crystal structure data and potential therapeutics for preterm labour.

    PubMed

    Shao, Qing-Chun; Zhang, Cui-Juan; Li, Jie

    2014-10-14

    The protein kinase C (PKC) is a family of serine/threonine kinases with a broad range of cellular targets. Members of the PKC family participate at the diverse biological events involved in cellular proliferation, differentiation and survival. The PKC isoform zeta (PKCζ) is an atypical member that has recently been found to play an essential role in promoting human uterine contractility and thus been raised as a new target for treating preterm labour and other tocolytic diseases. In this study, an integrative protocol was described to graft hundreds of inhibitor ligands from their complex crystal structures with cognate kinases into the active pocket of PKCζ and, based on the modeled structures, to evaluate the binding strength of these inhibitors to the non-cognate PKCζ receptor by using a consensus scoring strategy. A total of 32 inhibitors with top score were compiled, and eight out of them were tested for inhibitory potency against PKCζ. Consequently, five compounds, i.e. CDK6 inhibitor fisetin, PIM1 inhibitor myricetin, CDK9 inhibitor flavopiridol and PknB inhibitor mitoxantrone as well as the promiscuous kinase inhibitor staurosporine showed high or moderate inhibitory activity on PKCζ, with IC50 values of 58 ± 9, 1.7 ± 0.4, 108 ± 17, 280 ± 47 and 0.019 ± 0.004 μM, respectively, while other three compounds, including two marketed drugs dasatinib and sunitinib as well as the Rho inhibitor fasudil, have not been detected to possess observable activity. Next, based on the modeled structure data we modified three flavonoid kinase inhibitors, i.e. fisetin, myricetin and flavopiridol, to generate a number of more potential molecular entities, two of which were found to have a moderately improved activity as compared to their parent compounds.

  4. Nuclear removal during terminal lens fiber cell differentiation requires CDK1 activity: appropriating mitosis-related nuclear disassembly

    PubMed Central

    Chaffee, Blake R.; Shang, Fu; Chang, Min-Lee; Clement, Tracy M.; Eddy, Edward M.; Wagner, Brad D.; Nakahara, Masaki; Nagata, Shigekazu; Robinson, Michael L.; Taylor, Allen

    2014-01-01

    Lens epithelial cells and early lens fiber cells contain the typical complement of intracellular organelles. However, as lens fiber cells mature they must destroy their organelles, including nuclei, in a process that has remained enigmatic for over a century, but which is crucial for the formation of the organelle-free zone in the center of the lens that assures clarity and function to transmit light. Nuclear degradation in lens fiber cells requires the nuclease DNase IIβ (DLAD) but the mechanism by which DLAD gains access to nuclear DNA remains unknown. In eukaryotic cells, cyclin-dependent kinase 1 (CDK1), in combination with either activator cyclins A or B, stimulates mitotic entry, in part, by phosphorylating the nuclear lamin proteins leading to the disassembly of the nuclear lamina and subsequent nuclear envelope breakdown. Although most post-mitotic cells lack CDK1 and cyclins, lens fiber cells maintain these proteins. Here, we show that loss of CDK1 from the lens inhibited the phosphorylation of nuclear lamins A and C, prevented the entry of DLAD into the nucleus, and resulted in abnormal retention of nuclei. In the presence of CDK1, a single focus of the phosphonuclear mitotic apparatus is observed, but it is not focused in CDK1-deficient lenses. CDK1 deficiency inhibited mitosis, but did not prevent DNA replication, resulting in an overall reduction of lens epithelial cells, with the remaining cells possessing an abnormally large nucleus. These observations suggest that CDK1-dependent phosphorylations required for the initiation of nuclear membrane disassembly during mitosis are adapted for removal of nuclei during fiber cell differentiation. PMID:25139855

  5. p53 is phosphorylated by CDK7-cyclin H in a p36MAT1-dependent manner.

    PubMed Central

    Ko, L J; Shieh, S Y; Chen, X; Jayaraman, L; Tamai, K; Taya, Y; Prives, C; Pan, Z Q

    1997-01-01

    The tumor suppressor protein p53 acts as a transcriptional activator that can mediate cellular responses to DNA damage by inducing apoptosis and cell cycle arrest. p53 is a nuclear phosphoprotein, and phosphorylation has been proposed to be a means by which the activity of p53 is regulated. The cyclin-dependent kinase (CDK)-activating kinase (CAK) was originally identified as a cellular kinase required for the activation of a CDK-cyclin complex, and CAK is comprised of three subunits: CDK7, cyclin H, and p36MAT1. CAK is part of the transcription factor IIH multiprotein complex, which is required for RNA polymerase II transcription and nucleotide excision repair. Because of the similarities between p53 and CAK in their involvement in the cell cycle, transcription, and repair, we investigated whether p53 could act as a substrate for phosphorylation by CAK. While CDK7-cyclin H is sufficient for phosphorylation of CDK2, we show that p36MAT1 is required for efficient phosphorylation of p53 by CDK7-cyclin H, suggesting that p36MAT1 can act as a substrate specificity-determining factor for CDK7-cyclin H. We have mapped a major site of phosphorylation by CAK to Ser-33 of p53 and have demonstrated as well that p53 is phosphorylated at this site in vivo. Both wild-type and tumor-derived mutant p53 proteins are efficiently phosphorylated by CAK. Furthermore, we show that p36 and p53 can interact both in vitro and in vivo. These studies reveal a potential mechanism for coupling the regulation of p53 with DNA repair and the basal transcriptional machinery. PMID:9372954

  6. S-nitrosylation of cyclin-dependent kinase 5 (cdk5) regulates its kinase activity and dendrite growth during neuronal development.

    PubMed

    Zhang, Peng; Yu, Pei-Chun; Tsang, Anthony H K; Chen, Yu; Fu, Amy K Y; Fu, Wing-Yu; Chung, Kenny K; Ip, Nancy Y

    2010-10-27

    Precise regulation of cyclin-dependent kinase 5 (Cdk5), a member of the cyclin-dependent kinase family, is critical for proper neuronal development and functions. Cdk5 is activated through its association with the neuron-specific activator p35 or p39. Nonetheless, how its kinase activity is regulated in neurons is not well understood. In this study, we found that Cdk5 activity is regulated by S-nitrosylation, a post-translational modification of protein that affects a plethora of neuronal functions. S-nitrosylation of Cdk5 occurs at Cys83, which is one of the critical amino acids within the ATP-binding pocket of the kinase. Upon S-nitrosylation, Cdk5 exhibits reduced kinase activity, whereas mutation of Cys83 to Ala on Cdk5 renders the kinase refractory to such inhibition. Importantly, S-nitrosylated Cdk5 can be detected in the mouse brain, and blocking the S-nitrosylation of Cdk5 in cultured hippocampal neurons enhances dendritic growth and branching. Together, our findings reveal an important role of S-nitrosylation in regulating Cdk5 kinase activity and dendrite growth in neurons during development.

  7. P-TEFb regulation of transcription termination factor Xrn2 revealed by a chemical genetic screen for Cdk9 substrates

    PubMed Central

    Sansó, Miriam; Levin, Rebecca S.; Lipp, Jesse J.; Wang, Vivien Ya-Fan; Greifenberg, Ann Katrin; Quezada, Elizabeth M.; Ali, Akbar; Ghosh, Animesh; Larochelle, Stéphane; Rana, Tariq M.; Geyer, Matthias; Tong, Liang; Shokat, Kevan M.; Fisher, Robert P.

    2016-01-01

    The transcription cycle of RNA polymerase II (Pol II) is regulated at discrete transition points by cyclin-dependent kinases (CDKs). Positive transcription elongation factor b (P-TEFb), a complex of Cdk9 and cyclin T1, promotes release of paused Pol II into elongation, but the precise mechanisms and targets of Cdk9 action remain largely unknown. Here, by a chemical genetic strategy, we identified ∼100 putative substrates of human P-TEFb, which were enriched for proteins implicated in transcription and RNA catabolism. Among the RNA processing factors phosphorylated by Cdk9 was the 5′-to-3′ “torpedo” exoribonuclease Xrn2, required in transcription termination by Pol II, which we validated as a bona fide P-TEFb substrate in vivo and in vitro. Phosphorylation by Cdk9 or phosphomimetic substitution of its target residue, Thr439, enhanced enzymatic activity of Xrn2 on synthetic substrates in vitro. Conversely, inhibition or depletion of Cdk9 or mutation of Xrn2-Thr439 to a nonphosphorylatable Ala residue caused phenotypes consistent with inefficient termination in human cells: impaired Xrn2 chromatin localization and increased readthrough transcription of endogenous genes. Therefore, in addition to its role in elongation, P-TEFb regulates termination by promoting chromatin recruitment and activation of a cotranscriptional RNA processing enzyme, Xrn2. PMID:26728557

  8. A truncated peptide from p35, a Cdk5 activator, prevents Alzheimer's disease phenotypes in model mice

    PubMed Central

    Shukla, Varsha; Zheng, Ya-Li; Mishra, Santosh K.; Amin, Niranjana D.; Steiner, Joseph; Grant, Philip; Kesavapany, Sashi; Pant, Harish C.

    2013-01-01

    Alzheimer's disease (AD), one of the leading neurodegenerative disorders of older adults, which causes major socioeconomic burdens globally, lacks effective therapeutics without significant side effects. Besides the hallmark pathology of amyloid plaques and neurofibrillary tangles (NFTs), it has been reported that cyclin-dependent kinase 5 (Cdk5), a critical neuronal kinase, is hyperactivated in AD brains and is, in part, responsible for the above pathology. Here we show that a modified truncated 24-aa peptide (TFP5), derived from the Cdk5 activator p35, penetrates the blood-brain barrier after intraperitoneal injections, inhibits abnormal Cdk5 hyperactivity, and significantly rescues AD pathology (up to 70–80%) in 5XFAD AD model mice. The mutant mice, injected with TFP5 exhibit behavioral rescue, whereas no rescue was observed in mutant mice injected with either saline or scrambled peptide. However, TFP5 does not inhibit cell cycle Cdks or normal Cdk5/p35 activity, and thereby has no toxic side effects (even at 200 mg/kg), a common problem in most current therapeutics for AD. In addition, treated mice displayed decreased inflammation, amyloid plaques, NFTs, cell death, and an extended life by 2 mo. These results suggest TFP5 as a potential therapeutic, toxicity-free candidate for AD.—Shukla, V., Zheng, Y.-L., Mishra, S. K., Amin, N. D., Steiner, J., Grant, P., Kesavapany, S., Pant, H. C. A truncated peptide from p35, a Cdk5 activator, prevents Alzheimer's disease phenotypes in model mice. PMID:23038754

  9. Phosphorylation-mediated stabilization of Bora in mitosis coordinates Plx1/Plk1 and Cdk1 oscillations

    PubMed Central

    Feine, Oren; Hukasova, Elvira; Bruinsma, Wytse; Freire, Raimundo; Fainsod, Abraham; Gannon, Julian; Mahbubani, Hiro M; Lindqvist, Arne; Brandeis, Michael

    2014-01-01

    Cdk1 and Plk1/Plx1 activation leads to their inactivation through negative feedback loops. Cdk1 deactivates itself by activating the APC/C, consequently generating embryonic cell cycle oscillations. APC/C inhibition by the mitotic checkpoint in somatic cells and the cytostatic factor (CSF) in oocytes sustain the mitotic state. Plk1/Plx1 targets its co-activator Bora for degradation, but it remains unclear how embryonic oscillations in Plx1 activity are generated, and how Plk1/Plx1 activity is sustained during mitosis. We show that Plx1-mediated degradation of Bora in interphase generates oscillations in Plx1 activity and is essential for development. In CSF extracts, phosphorylation of Bora on the Cdk consensus site T52 blocks Bora degradation. Upon fertilization, Calcineurin dephosphorylates T52, triggering Plx1 oscillations. Similarly, we find that GFP-Bora is degraded when Plk1 activity spreads to somatic cell cytoplasm before mitosis. Interestingly, GFP–Bora degradation stops upon mitotic entry when Cdk1 activity is high. We hypothesize that Cdk1 controls Bora through an incoherent feedforward loop synchronizing the activities of mitotic kinases. PMID:24675888

  10. Drosophila cyclin D/Cdk4 regulates mitochondrial biogenesis and aging and sensitizes animals to hypoxic stress

    PubMed Central

    Icreverzi, Amalia; Flor de la Cruz, Aida; Van Voorhies, Wayne A

    2012-01-01

    Drosophila cyclin D (CycD) is the single fly ortholog of the mammalian cyclin D1 and promotes both cell cycle progression and cellular growth. However, little is known about how CycD promotes cell growth. We show here that CycD/Cdk4 hyperactivity leads to increased mitochondrial biogenesis (mitobiogenesis), mitochondrial mass, NRF-1 activity (Tfam transcript levels) and metabolic activity in Drosophila, whereas loss of CycD/Cdk4 activity has the opposite effects. Surprisingly, both CycD/Cdk4 addition and loss of function increase mitochondrial superoxide production and decrease lifespan, indicating that an imbalance in mitobiogenesis may lead to oxidative stress and aging. In addition, we provide multiple lines of evidence indicating that CycD/Cdk4 activity affects the hypoxic status of cells and sensitizes animals to hypoxia. Both mitochondrial and hypoxia-related effects can be detected at global transcriptional level. We propose that mitobiogenesis and the hypoxic stress response have an antagonistic relationship, and that CycD/Cdk4 levels regulate mitobiogenesis contemporaneous to the cell cycle, such that only when cells are sufficiently oxygenated can they proliferate. PMID:22293404

  11. Fission yeast Cdk7 controls gene expression through both its CAK and C-terminal domain kinase activities.

    PubMed

    Devos, Maxime; Mommaerts, Elise; Migeot, Valerie; van Bakel, Harm; Hermand, Damien

    2015-05-01

    Cyclin-dependent kinase (Cdk) activation and RNA polymerase II transcription are linked by the Cdk7 kinase, which phosphorylates Cdks as a trimeric Cdk-activating kinase (CAK) complex, and serine 5 within the polymerase II (Pol II) C-terminal domain (CTD) as transcription factor TFIIH-bound CAK. However, the physiological importance of integrating these processes is not understood. Besides the Cdk7 ortholog Mcs6, fission yeast possesses a second CAK, Csk1. The two enzymes have been proposed to act redundantly to activate Cdc2. Using an improved analogue-sensitive Mcs6-as kinase, we show that Csk1 is not a relevant CAK for Cdc2. Further analyses revealed that Csk1 lacks a 20-amino-acid sequence required for its budding yeast counterpart, Cak1, to bind Cdc2. Transcriptome profiling of the Mcs6-as mutant in the presence or absence of the budding yeast Cak1 kinase, in order to uncouple the CTD kinase and CAK activities of Mcs6, revealed an unanticipated role of the CAK branch in the transcriptional control of the cluster of genes implicated in ribosome biogenesis and cell growth. The analysis of a Cdc2 CAK site mutant confirmed these data. Our data show that the Cdk7 kinase modulates transcription through its well-described RNA Pol II CTD kinase activity and also through the Cdc2-activating kinase activity.

  12. Fission Yeast Cdk7 Controls Gene Expression through both Its CAK and C-Terminal Domain Kinase Activities

    PubMed Central

    Devos, Maxime; Mommaerts, Elise; Migeot, Valerie; van Bakel, Harm

    2015-01-01

    Cyclin-dependent kinase (Cdk) activation and RNA polymerase II transcription are linked by the Cdk7 kinase, which phosphorylates Cdks as a trimeric Cdk-activating kinase (CAK) complex, and serine 5 within the polymerase II (Pol II) C-terminal domain (CTD) as transcription factor TFIIH-bound CAK. However, the physiological importance of integrating these processes is not understood. Besides the Cdk7 ortholog Mcs6, fission yeast possesses a second CAK, Csk1. The two enzymes have been proposed to act redundantly to activate Cdc2. Using an improved analogue-sensitive Mcs6-as kinase, we show that Csk1 is not a relevant CAK for Cdc2. Further analyses revealed that Csk1 lacks a 20-amino-acid sequence required for its budding yeast counterpart, Cak1, to bind Cdc2. Transcriptome profiling of the Mcs6-as mutant in the presence or absence of the budding yeast Cak1 kinase, in order to uncouple the CTD kinase and CAK activities of Mcs6, revealed an unanticipated role of the CAK branch in the transcriptional control of the cluster of genes implicated in ribosome biogenesis and cell growth. The analysis of a Cdc2 CAK site mutant confirmed these data. Our data show that the Cdk7 kinase modulates transcription through its well-described RNA Pol II CTD kinase activity and also through the Cdc2-activating kinase activity. PMID:25691663

  13. Cdk5/p35 phosphorylates lemur tyrosine kinase-2 to regulate protein phosphatase-1C phosphorylation and activity.

    PubMed

    Manser, Catherine; Vagnoni, Alessio; Guillot, Florence; Davies, Jennifer; Miller, Christopher C J

    2012-05-01

    Cyclin-dependent kinase-5 (cdk5)/p35 and protein phosphatase-1 (PP1) are two major enzymes that control a variety of physiological processes within the nervous system including neuronal differentiation, synaptic plasticity and axonal transport. Defective cdk5/p35 and PP1 function are also implicated in several major human neurodegenerative diseases. Cdk5/p35 and the catalytic subunit of PP1 (PP1C) both bind to the brain-enriched, serine-threonine kinase lemur tyrosine kinase-2 (LMTK2). Moreover, LMTK2 phosphorylates PP1C on threonine-320 (PP1Cthr³²⁰) to inhibit its activity. Here, we demonstrate that LMTK2 is phosphorylated on serine-1418 (LMTK2ser¹⁴¹⁸) by cdk5/p35 and present evidence that this regulates its ability to phosphorylate PP1Cthr³²⁰. We thus describe a new signalling pathway within the nervous system that links cdk5/p35 with PP1C and which has implications for a number of neuronal functions and neuronal dysfunction.

  14. Cytotoxicity of diacetoxyscirpenol is associated with apoptosis by activation of caspase-8 and interruption of cell cycle progression by down-regulation of cdk4 and cyclin B1 in human Jurkat T cells

    SciTech Connect

    Jun, Do Youn; Kim, Jun Seok; Park, Hae Sun; Song, Woo Sun; Bae, Young Seuk; Kim, Young Ho . E-mail: ykim@knu.ac.kr

    2007-07-15

    To understand the mechanism underlying T-cell toxicity of diacetoxyscirpenol (DAS) from Fusarium sambucinum, its apoptogenic as well as growth retardation activity was investigated in human Jurkat T cells. Exposure to DAS (0.01-0.15 {mu}M) caused apoptotic DNA fragmentation along with caspase-8 activation, Bid cleavage, mitochondrial cytochrome c release, activation of caspase-9 and caspase-3, and PARP degradation, without any alteration in the levels of Fas or FasL. Under these conditions, necrosis was not accompanied. The cytotoxicity of DAS was not blocked by the anti-Fas neutralizing antibody ZB-4. Although the DAS-induced apoptotic events were completely prevented by overexpression of Bcl-xL, the cells overexpressing Bcl-xL were unable to divide in the presence of DAS, resulting from the failure of cell cycle progression possibly due to down-regulation in the protein levels of cdk4 and cyclin B1. The DAS-mediated apoptosis and activation of caspase-8, -9, and -3 were abrogated by either pan-caspase inhibitor (z-VAD-fmk) or caspase-8 inhibitor (z-IETD-fmk). While the DAS-mediated apoptosis and activation of caspase-9 and caspase-3 were slightly suppressed by the mitochondrial permeability transition pore inhibitor (CsA), both caspase-8 activation and Bid cleavage were not affected by CsA. The activated normal peripheral T cells possessed a similar susceptibility to the cytotoxicity of DAS. These results demonstrate that the T-cell toxicity of DAS is attributable to not only apoptosis initiated by caspase-8 activation and subsequent mitochondrion-dependent or -independent activation of caspase cascades, which can be regulated by Bcl-xL, but also interruption of cell cycle progression caused by down-regulation of cdk4 and cyclin B1 proteins.

  15. A kinase-independent function of Cks1 and Cdk1 in regulation of transcription.

    PubMed

    Yu, Veronica P C C; Baskerville, Chris; Grünenfelder, Björn; Reed, Steven I

    2005-01-07

    We describe a function in transcription for the Saccharomyces cerevisiae cell cycle regulatory cyclin-dependent kinase Cdc28 (Cdk1) and its interacting protein, Cks1. The Cks1/Cdc28 complex is recruited to multiple coding regions in the genome and is necessary for efficient expression of a significant subset of genes. This transcriptional role is mediated through a requirement of Cdc28/Cks1 for recruiting proteasomes to coding regions. However, it is independent of the protein kinase activity of Cdc28. In the absence of Cks1, neither Cdc28 nor the proteasome can be recruited. Consequently, there is a failure to maintain efficient transcription.

  16. Regulation of T-Type Cyclin/CDK9 Complexes in Breast Cancer Cells

    DTIC Science & Technology

    2005-07-01

    2004) Cellular control of gene expression by T-type cyclin/CDK9 complexes. Gene.337:15- 23. Garriga, J., Limon , A., Mayol, X., Rane, S.G...Breast cancer susceptibility gene 1 (BRCAI) is a coactivator of the androgen receptor. Cancer Res 60, 5946-9. Parreño, M., Garriga, J., Limon , A...complexes. Oncogene, In press. Parreño, M., Garriga, J., Limon , A., Mayol, X., Beck, G.R., Jr., Moran, E., and Graña, X. (2000). E1A blocks

  17. c-Jun induces apoptosis of starved BM2 monoblasts by activating cyclin A-CDK2

    SciTech Connect

    Vanhara, Petr; Bryja, Vitezslav; Horvath, Viktor; Kozubik, Alois; Hampl, Ales; Smarda, Jan . E-mail: smarda@sci.muni.cz

    2007-02-02

    c-Jun is one of the major components of the activating protein-1 (AP-1), the transcription factor that participates in regulation of proliferation, differentiation, and apoptosis. In this study, we explored functional interactions of the c-Jun protein with several regulators of the G1/S transition in serum-deprived v-myb-transformed chicken monoblasts BM2. We show that the c-Jun protein induces expression of cyclin A, thus up-regulating activity of cyclin A-associated cyclin-dependent kinase 2 (CDK2), and causing massive programmed cell death of starved BM2cJUN cells. Specific inhibition of CDK2 suppresses frequency of apoptosis of BM2cJUN cells. We conclude that up-regulation of cyclin A expression and CDK2 activity can represent important link between the c-Jun protein, cell cycle machinery, and programmed cell death pathway in leukemic cells.

  18. Two Degradation Pathways of the p35 Cdk5 (Cyclin-dependent Kinase) Activation Subunit, Dependent and Independent of Ubiquitination.

    PubMed

    Takasugi, Toshiyuki; Minegishi, Seiji; Asada, Akiko; Saito, Taro; Kawahara, Hiroyuki; Hisanaga, Shin-ichi

    2016-02-26

    Cdk5 is a versatile protein kinase that is involved in various neuronal activities, such as the migration of newborn neurons, neurite outgrowth, synaptic regulation, and neurodegenerative diseases. Cdk5 requires the p35 regulatory subunit for activation. Because Cdk5 is more abundantly expressed in neurons compared with p35, the p35 protein levels determine the kinase activity of Cdk5. p35 is a protein with a short half-life that is degraded by proteasomes. Although ubiquitination of p35 has been previously reported, the degradation mechanism of p35 is not yet known. Here, we intended to identify the ubiquitination site(s) in p35. Because p35 is myristoylated at the N-terminal glycine, the possible ubiquitination sites are the lysine residues in p35. We mutated all 23 Lys residues to Arg (p35 23R), but p35 23R was still rapidly degraded by proteasomes at a rate similar to wild-type p35. The degradation of p35 23R in primary neurons and the Cdk5 activation ability of p35 23R suggested the occurrence of ubiquitin-independent degradation of p35 in physiological conditions. We found that p35 has the amino acid sequence similar to the ubiquitin-independent degron in the NKX3.1 homeodomain transcription factor. An Ala mutation at Pro-247 in the degron-like sequence made p35 stable. These results suggest that p35 can be degraded by two degradation pathways: ubiquitin-dependent and ubiquitin-independent. The rapid degradation of p35 by two different methods would be a mechanism to suppress the production of p25, which overactivates Cdk5 to induce neuronal cell death.

  19. Easy Identification of Residues Involved on Structural Differences Between Nonphosphorylated and Phosphorylated CDK2Cyclin A Complexes Using Two-Dimensional Networks.

    PubMed

    Riadi, Gonzalo; Caballero, Julio

    2014-02-01

    The structures of proteins in Protein Data Bank (PDB) contain a lot of information that can be revealed through the use of tools to facilitate their organization and analysis. The increase in available structural data of nonphosphorylated and phosphorylated CDK2cyclin A (npCDK2cycA and pCDK2cycA) complexes has enabled a more realistic description of the fine structural details of the interface residues of these proteins. This work reports the application of two-dimensional network representations (TDNRs) to the structures deposited in PDB to distinguish the differences in the surface between both complexes due to phosphorylation. As a result, a detailed map of the hydrogen bonds (HBs) and hydrophobic interactions between the T-loop residues of CDK2 and the residues of cycA that are different among nonphosphorylated and phosphorylated complexes were described. In addition, we found some interesting subtle differences in the CDK2cycA interface between nonphosphorylated and phosphorylated complexes due to residues that are not located at the T-loop of CDK2. We noted that some HB interactions in CDK2cycA complex are reinforced when the CDK2 is phosphorylated.

  20. A Whi7-anchored loop controls the G1 Cdk-cyclin complex at start.

    PubMed

    Yahya, Galal; Parisi, Eva; Flores, Alba; Gallego, Carme; Aldea, Martí

    2014-01-09

    Cells commit to a new cell cycle at Start by activation of the G1 Cdk-cyclin complex which, in turn, triggers a genome-wide transcriptional wave that executes the G1/S transition. In budding yeast, the Cdc28-Cln3 complex is regulated by an ER-retention mechanism that is important for proper cell size control. We have isolated small-cell-size CDC28 mutants showing impaired retention at the ER and premature accumulation of the Cln3 cyclin in the nucleus. The differential interactome of a quintuple Cdc28(wee) mutant pinpointed Whi7, a Whi5 paralog targeted by Cdc28 that associates to the ER in a phosphorylation-dependent manner. Our results demonstrate that the Cln3 cyclin and Whi7 act in a positive feedback loop to release the G1 Cdk-cyclin complex and trigger Start once a critical size has been reached, thus uncovering a key nonlinear mechanism at the earliest known events of cell-cycle entry.

  1. Xenopus Mcm10 is a CDK-substrate required for replication fork stability

    PubMed Central

    Chadha, Gaganmeet Singh; Gambus, Agnieszka; Gillespie, Peter J.; Blow, J. Julian

    2016-01-01

    ABSTRACT During S phase, following activation of the S phase CDKs and the DBF4-dependent kinases (DDK), double hexamers of Mcm2-7 at licensed replication origins are activated to form the core replicative helicase. Mcm10 is one of several proteins that have been implicated from work in yeasts to play a role in forming a mature replisome during the initiation process. Mcm10 has also been proposed to play a role in promoting replisome stability after initiation has taken place. The role of Mcm10 is particularly unclear in metazoans, where conflicting data has been presented. Here, we investigate the role and regulation of Mcm10 in Xenopus egg extracts. We show that Xenopus Mcm10 is recruited to chromatin late in the process of replication initiation and this requires prior action of DDKs and CDKs. We also provide evidence that Mcm10 is a CDK substrate but does not need to be phosphorylated in order to associate with chromatin. We show that in extracts depleted of more than 99% of Mcm10, the bulk of DNA replication still occurs, suggesting that Mcm10 is not required for the process of replication initiation. However, in extracts depleted of Mcm10, the replication fork elongation rate is reduced. Furthermore, the absence of Mcm10 or its phosphorylation by CDK results in instability of replisome proteins on DNA, which is particularly important under conditions of replication stress. PMID:27327991

  2. Evolution of the Cdk-activator Speedy/RINGO in vertebrates.

    PubMed

    Chauhan, Sangeeta; Zheng, Xinde; Tan, Yue Ying; Tay, Boon-Hui; Lim, Shuhui; Venkatesh, Byrappa; Kaldis, Philipp

    2012-11-01

    Successful completion of the cell cycle relies on the precise activation and inactivation of cyclin-dependent kinases (Cdks) whose activity is mainly regulated by binding to cyclins. Recently, a new family of Cdk regulators termed Speedy/RINGO has been discovered, which can bind and activate Cdks but shares no apparent amino acid sequence homology with cyclins. All Speedy proteins share a conserved domain of approximately 140 amino acids called "Speedy Box", which is essential for Cdk binding. Speedy/RINGO proteins display an important role in oocyte maturation in Xenopus. Interestingly, a common feature of all Speedy genes is their predominant expression in testis suggesting that meiotic functions may be the most important physiological feature of Speedy genes. Speedy homologs have been reported in mammals and can be traced back to the most primitive clade of chordates (Ciona intestinalis). Here, we investigated the evolution of the Speedy genes and have identified a number of new Speedy/RINGO proteins. Through extensive analysis of numerous species, we discovered diverse evolutionary histories: the number of Speedy genes varies considerably among species, with evidence of substantial gains and losses. Despite the interspecies variation, Speedy is conserved among most species examined. Our results provide a complete picture of the Speedy gene family and its evolution.

  3. PKCδ regulates cortical radial migration by stabilizing the Cdk5 activator p35

    PubMed Central

    Zhao, Chun-tao; Li, Kun; Li, Jun-tao; Zheng, Wang; Liang, Xu-jun; Geng, An-qi; Li, Ning; Yuan, Xiao-bing

    2009-01-01

    Cyclin-dependent kinase 5 (Cdk5) and its activator p35 are critical for radial migration and lamination of cortical neurons. However, how this kinase is regulated by extracellular and intracellular signals during cortical morphogenesis remains unclear. Here, we show that PKCδ, a member of novel PKC expressing in cortical neurons, could stabilize p35 by direct phosphorylation. PKCδ attenuated the degradation of p35 but not its mutant derivative, which could not be phosphorylated by PKCδ. Down-regulation of PKCδ by in utero electroporation of specific small interference RNA (siRNA) severely impaired the radial migration of cortical neurons. This migration defect was similar to that caused by down-regulation of p35 and could be prevented by cotransfection with the wild-type but not the mutant p35. Furthermore, PKCδ could be activated by the promigratory factor brain-derived neurotrophic factor (BDNF) and was required for the activation of Cdk5 by BDNF. Both PKCδ and p35 were required for the promigratory effect of BDNF on cultured newborn neurons. Thus, PKCδ may promote cortical radial migration through maintaining the proper level of p35 in newborn neurons. PMID:19965374

  4. p12CDK2-AP1 interacts with CD82 to regulate the proliferation and survival of human oral squamous cell carcinoma cells.

    PubMed

    Chai, Juan; Ju, Jun; Zhang, Shao-Wu; Shen, Zhi-Yuan; Liang, Liang; Yang, Xiang-Ming; Ma, Chao; Ni, Qian-Wei; Sun, Mo-Yi

    2016-08-01

    p12 cyclin-dependent kinase 2 (CDK2)-associating protein 1 (p12CDK2-AP1) has been demonstrated to negatively regulate the activity of CDK2. However, the underlying molecular mechanism remains largely unknown. We aimed to determine the potential binding proteins of p12CDK2-AP1 and to elucidate the role of p12CDK2-AP1 in the regulation of the proliferation, invasion, apoptosis, and in vivo growth of human oral squamous cell carcinoma cells. The protein-protein interaction was predicted using computational decision templates. The predicted p12CDK2‑AP1 interacting proteins were overexpressed in human oral squamous cell carcinoma OSCC-15 cells, and the protein binding was examined using co-precipitation (Co-IP). Cell proliferation and invasion were determined via MTT assay and Transwell system, respectively. Cell apoptosis was evaluated using Annexin V-FITC/PI double staining followed by flow cytometric analysis. The in vivo growth of OSCC-15 cells was examined in nude mouse tumor xenografts. We found that overexpression of either p12CDK2-AP1 or CD82 significantly suppressed the proliferation and invasion but promoted the apoptosis of OSCC-15 cells (P<0.05). Importantly, combined overexpression of p12CDK2-AP1 and CD82 showed synergistic antitumor activity compared with the overexpression of a single protein alone (P<0.05). Additionally, the simultaneous overexpression of p12CDK2-AP1 and CD82 significantly suppressed the in vivo tumor growth of OSCC-15 cells in nude mice compared with the negative control (P<0.05). Our findings indicate that p12CDK2-AP1 interacts with CD82 to play a functional role in suppressing the in vitro and in vivo growth of OSCC-15 cells.

  5. Butyrate, an HDAC inhibitor, stimulates interplay between different posttranslational modifications of histone H3 and differently alters G1-specific cell cycle proteins in vascular smooth muscle cells.

    PubMed

    Mathew, Omana P; Ranganna, Kasturi; Yatsu, Frank M

    2010-12-01

    HDACs and HATs regulate histone acetylation, an epigenetic modification that controls chromatin structure and through it, gene expression. Butyrate, a dietary HDAC inhibitor, inhibits VSMC proliferation, a crucial factor in atherogenesis, and the principle mechanism in arterial and in-stent restenosis. Here, the link between antiproliferation action of butyrate and the portraits of global covalent modifications of histone H3 that it induces are characterized to understand the mechanics of butyrate-arrested VSMC proliferation. Analysis of histone H3 modifications specific to butyrate arrested VSMC proliferation display induction of histone H3-Lysine9 acetylation, inhibition of histone H3-Serine10 phosphorylation, reduction of histone H3-Lysine9 dimethylation and stimulation of histone H3-Lysine4 di-methylation, which is linked to transcriptional activation, cell cycle/mitosis, transcriptional suppression and activation, respectively. Conversely, untreated VSMCs exhibit inhibition of H3-Lysine9 acetylation, induction of H3-Serine10 phosphorylation, stimulation of H3-Lysine9 di-methylation and reduction in H3-Lysine4 di-methylation. Butyrate's cooperative effects on H3-Lysine9 acetylation and H3-Serine10 phosphorylation, and contrasting effects on di-methylation of H3-Lysine9 and H3-Lysine4 suggests that the interplay between these site-specific modifications cause distinct chromatin alterations that allow cyclin D1 and D3 induction, G1-specific cdk4, cdk6 and cdk2 downregulation, and upregulation of cdk inhibitors, p15INK4b and p21Cip1. Regardless of butyrate's effect on D-type cyclins, downregulation of G1-specific cdks and upregulation of cdk inhibitors by butyrate prevents cell cycle progression by failing to inactivate Rb. Overall, through chromatin remodeling, butyrate appears to differentially alter G1-specific cell cycle proteins to ensure proliferation arrest of VSMCs, a crucial cellular component of blood vessel wall.

  6. Dephosphorylation of threonine-821 of the retinoblastoma tumor suppressor protein (Rb) is required for apoptosis induced by UV and Cdk inhibition.

    PubMed

    Lentine, Brandon; Antonucci, Lisa; Hunce, Ray; Edwards, Justina; Marallano, Valerie; Krucher, Nancy A

    2012-09-01

    The Retinoblastoma protein (Rb) is important in the control of cell proliferation and apoptosis. Its activity is controlled by reversible phosphorylation on several serine and threonine residues. When Rb is hypophosphorylated, it inhibits proliferation by preventing passage through the G 1- S phase transition. Hyperphosphorylated Rb promotes cell cycle progression. The role of Rb phosphorylation in the control of apoptosis is largely unknown, although several apoptotic stimuli result in dephosphorylation of Rb. It may be that dephosphorylation of specific amino acids signals apoptosis vs. cell cycle arrest. Using glutamic acid mutagenesis, we have generated 15 single phosphorylation site mutants of Rb to alter serine/threonine to glutamic acid to mimic the phosphorylated state. By calcium phosphate transfection, mutant plasmids were introduced into C33A Rb-null cells, and apoptosis was induced using UV. Apoptosis was measured by ELISA detection of degraded DNA and by immunoblotting to assess proteolytic cleavage of PARP. Our results show that only mutation of threonine-821 to glutamic acid (T821E) blocked apoptosis by 50%, whereas other sites tested had little effect. In Rb-null Saos-2 and SKUT-1 cells, the T821E mutation also blocked apoptosis induced by the cdk inhibitor, Roscovitine, by 50%. In addition, we show that endogenous Rb is dephosphorylated on threonine-821 when cells are undergoing apoptosis. Thus, our data indicates that dephosphorylation of threonine-821 of Rb is required for cells to undergo apoptosis.

  7. Nephrotoxicity of epigenetic inhibitors used for the treatment of cancer.

    PubMed

    Scholpa, N E; Kolli, R T; Moore, M; Arnold, R D; Glenn, T C; Cummings, B S

    2016-10-25

    This study determined the anti-neoplastic activity and nephrotoxicity of epigenetic inhibitors in vitro. The therapeutic efficacy of epigenetic inhibitors was determined in human prostate cancer cells (PC-3 and LNCaP) using the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-Aza) and the histone deacetylase inhibitor trichostatin A (TSA). Cells were also treated with carbamazepine (CBZ), an anti-convulsant with histone deacetylase inhibitor-like properties. 5-Aza, TSA or CBZ alone did not decrease MTT staining in PC-3 or LNCaP cells after 48 h. In contrast, docetaxel, a frontline chemotherapeutic induced concentration-dependent decreases in MTT staining. Pretreatment with 5-Aza or TSA increased docetaxel-induced cytotoxicity in LNCaP cells, but not PC-3 cells. TSA pretreatment also increased cisplatin-induced toxicity in LNCaP cells. Carfilzomib (CFZ), a protease inhibitor approved for the treatment of multiple myeloma had minimal effect on LNCaP cell viability, but reduced MTT staining 50% in PC-3 cells compared to control, and pretreatment with 5-Aza further enhanced toxicity. Treatment of normal rat kidney (NRK) and human embryonic kidney 293 (HEK293) cells with the same concentrations of epigenetic inhibitors used in prostate cancer cells significantly decreased MTT staining in all cell lines after 48 h. Interestingly, we found that the toxicity of epigenetic inhibitors to kidney cells was dependent on both the compound and the stage of cell growth. The effect of 5-Aza and TSA on DNA methyltransferase and histone deacetylase activity, respectively, was confirmed by assessing the methylation and acetylation of the CDK inhibitor p21. Collectively, these data show that combinatorial treatment with epigenetic inhibitors alters the efficacy of chemotherapeutics in cancer cells in a compound- and cell-specific manner; however, this treatment also has the potential to induce nephrotoxic cell injury.

  8. Cdk5-mediated inhibition of APC/C-Cdh1 switches on the cyclin D1-Cdk4-pRb pathway causing aberrant S-phase entry of postmitotic neurons.

    PubMed

    Veas-Pérez de Tudela, Miguel; Maestre, Carolina; Delgado-Esteban, María; Bolaños, Juan P; Almeida, Angeles

    2015-12-10

    The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase that regulates cell cycle progression in proliferating cells. To enter the S-phase, APC/C must be inactivated by phosphorylation of its cofactor, Cdh1. In post-mitotic cells such as neurons APC/C-Cdh1 complex is highly active and responsible for the continuous degradation of mitotic cyclins. However, the specific molecular pathway that determines neuronal cell cycle blockade in post-mitotic neurons is unknown. Here, we show that activation of glutamatergic receptors in rat cortical primary neurons endogenously triggers cyclin-dependent kinase-5 (Cdk5)-mediated phosphorylation of Cdh1 leading to its cytoplasmic accumulation and disassembly from the APC3 core protein, causing APC/C inactivation. Conversely, pharmacological or genetic inhibition of Cdk5 promotes Cdh1 ubiquitination and proteasomal degradation. Furthermore, we show that Cdk5-mediated phosphorylation and inactivation of Cdh1 leads to p27 depletion, which switches on the cyclin D1-cyclin-dependent kinase-4 (Cdk4)-retinoblastoma protein (pRb) pathway to allow the S-phase entry of neurons. However, neurons do not proceed through the cell cycle and die by apoptosis. These results indicate that APC/C-Cdh1 actively suppresses an aberrant cell cycle entry and death of neurons, highlighting its critical function in neuroprotection.

  9. Angiogenesis Inhibitors

    MedlinePlus

    ... inhibitors: current strategies and future prospects. CA: A Cancer Journal for Clinicians 2010; 60(4):222–243. [PubMed Abstract] Chen HX, Cleck JN. Adverse effects of anticancer agents that target the VEGF pathway. Nature Reviews Clinical Oncology 2009; 6(8):465– ...

  10. Carboxylesterase inhibitors

    PubMed Central

    Hatfield, M. Jason; Potter, Philip M.

    2011-01-01

    Introduction Carboxylesterases play major roles in the hydrolysis of numerous therapeutically active compounds. This is, in part, due to the prevalence of the ester moiety in these small molecules. However, the impact these enzymes may play on drug stability and pharmacokinetics is rarely considered prior to molecule development. Therefore, the application of selective inhibitors of this class of proteins may have utility in modulating the metabolism, distribution and toxicity of agents that are subjected to enzyme hydrolysis. Areas covered This review details the development of all such compounds dating back to 1986, but principally focuses on the very recent identification of selective human carboxylesterases inhibitors. Expert opinion The implementation of carboxylesterase inhibitors may significantly revolutionize drug discovery. Such molecules may allow for improved efficacy of compounds inactivated by this class of enzymes and/or reduce the toxicity of agents that are activated by these proteins. Furthermore, since lack of carboxylesterase activity appears to have no obvious biological consequence, these compounds could be applied in combination with virtually any esterified drug. Therefore, inhibitors of these proteins may have utility in altering drug hydrolysis and distribution in vivo. The characteristics, chemical and biological properties, and potential uses of such agents, are discussed here. PMID:21609191

  11. Polycomb protein SCML2 regulates the cell cycle by binding and modulating CDK/CYCLIN/p21 complexes.

    PubMed

    Lecona, Emilio; Rojas, Luis Alejandro; Bonasio, Roberto; Johnston, Andrew; Fernández-Capetillo, Oscar; Reinberg, Danny

    2013-12-01

    Polycomb group (PcG) proteins are transcriptional repressors of genes involved in development and differentiation, and also maintain repression of key genes involved in the cell cycle, indirectly regulating cell proliferation. The human SCML2 gene, a mammalian homologue of the Drosophila PcG protein SCM, encodes two protein isoforms: SCML2A that is bound to chromatin and SCML2B that is predominantly nucleoplasmic. Here, we purified SCML2B and found that it forms a stable complex with CDK/CYCLIN/p21 and p27, enhancing the inhibitory effect of p21/p27. SCML2B participates in the G1/S checkpoint by stabilizing p21 and favoring its interaction with CDK2/CYCE, resulting in decreased kinase activity and inhibited progression through G1. In turn, CDK/CYCLIN complexes phosphorylate SCML2, and the interaction of SCML2B with CDK2 is regulated through the cell cycle. These findings highlight a direct crosstalk between the Polycomb system of cellular memory and the cell-cycle machinery in mammals.

  12. Enhancement of BACE1 Activity by p25/Cdk5-Mediated Phosphorylation in Alzheimer’s Disease

    PubMed Central

    Lee, Hye-Won; Seo, Hyemyung; Kim, Jeong Hee; Chung, Sul-Hee

    2015-01-01

    The activity of beta-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) is elevated during aging and in sporadic Alzheimer’s disease (AD), but the underlying mechanisms of this change are not well understood. p25/Cyclin-dependent kinase 5 (Cdk5) has been implicated in the pathogenesis of several neurodegenerative diseases, including AD. Here, we describe a potential mechanism by which BACE activity is increased in AD brains. First, we show that BACE1 is phosphorylated by the p25/Cdk5 complex at Thr252 and that this phosphorylation increases BACE1 activity. Then, we demonstrate that the level of phospho-BACE1 is increased in the brains of AD patients and in mammalian cells and transgenic mice that overexpress p25. Furthermore, the fraction of p25 prepared from iodixanol gradient centrifugation was unexpectedly protected by protease digestion, suggesting that p25/Cdk5-mediated BACE1 phosphorylation may occur in the lumen. These results reveal a link between p25 and BACE1 in AD brains and suggest that upregulated Cdk5 activation by p25 accelerates AD pathogenesis by enhancing BACE1 activity via phosphorylation. PMID:26317805

  13. Cdk1 and okadaic acid-sensitive phosphatases control assembly of nuclear pore complexes in Drosophila embryos.

    PubMed

    Onischenko, Evgeny A; Gubanova, Natalia V; Kiseleva, Elena V; Hallberg, Einar

    2005-11-01

    Disassembly and reassembly of the nuclear pore complexes (NPCs) is one of the major events during open mitosis in higher eukaryotes. However, how this process is controlled by the mitotic machinery is not clear. To investigate this we developed a novel in vivo model system based on syncytial Drosophila embryos. We microinjected different mitotic effectors into the embryonic cytoplasm and monitored the dynamics of disassembly/reassembly of NPCs in live embryos using fluorescently labeled wheat germ agglutinin (WGA) or in fixed embryos using electron microscopy and immunostaining techniques. We found that in live embryos Cdk1 activity was necessary and sufficient to induce disassembly of NPCs as well as their cytoplasmic mimics: annulate lamellae pore complexes (ALPCs). Cdk1 activity was also required for keeping NPCs and ALPCs disassembled during mitosis. In agreement recombinant Cdk1/cyclin B was able to induce phosphorylation and dissociation of nucleoporins from the NPCs in vitro. Conversely, reassembly of NPCs and ALPCs was dependent on the activity of protein phosphatases, sensitive to okadaic acid (OA). Our findings suggest a model where mitotic disassembly/reassembly of the NPCs is regulated by a dynamic equilibrium of Cdk1 and OA-sensitive phosphatase activities and provide evidence that mitotic phosphorylation mediates disassembly of the NPC.

  14. TARANIS Functions with Cyclin A and Cdk1 in a Novel Arousal Center to Control Sleep in Drosophila.

    PubMed

    Afonso, Dinis J S; Liu, Die; Machado, Daniel R; Pan, Huihui; Jepson, James E C; Rogulja, Dragana; Koh, Kyunghee

    2015-06-29

    Sleep is an essential and conserved behavior whose regulation at the molecular and anatomical level remains to be elucidated. Here, we identify TARANIS (TARA), a Drosophila homolog of the Trip-Br (SERTAD) family of transcriptional coregulators, as a molecule that is required for normal sleep patterns. Through a forward-genetic screen, we isolated tara as a novel sleep gene associated with a marked reduction in sleep amount. Targeted knockdown of tara suggests that it functions in cholinergic neurons to promote sleep. tara encodes a conserved cell-cycle protein that contains a Cyclin A (CycA)-binding homology domain. TARA regulates CycA protein levels and genetically and physically interacts with CycA to promote sleep. Furthermore, decreased levels of Cyclin-dependent kinase 1 (Cdk1), a kinase partner of CycA, rescue the short-sleeping phenotype of tara and CycA mutants, while increased Cdk1 activity mimics the tara and CycA phenotypes, suggesting that Cdk1 mediates the role of TARA and CycA in sleep regulation. Finally, we describe a novel wake-promoting role for a cluster of ∼14 CycA-expressing neurons in the pars lateralis (PL), previously proposed to be analogous to the mammalian hypothalamus. We propose that TARANIS controls sleep amount by regulating CycA protein levels and inhibiting Cdk1 activity in a novel arousal center.

  15. MicroRNA-206 induces G1 arrest in melanoma by inhibition of CDK4 and Cyclin D.

    PubMed

    Georgantas, Robert W; Streicher, Katie; Luo, Xiaobing; Greenlees, Lydia; Zhu, Wei; Liu, Zheng; Brohawn, Philip; Morehouse, Christopher; Higgs, Brandon W; Richman, Laura; Jallal, Bahija; Yao, Yihong; Ranade, Koustubh

    2014-03-01

    Expression profiling of microRNAs in melanoma lesional skin biopsies compared with normal donor skin biopsies, as well as melanoma cell lines compared with normal melanocytes, revealed that hsa-miR-206 was down-regulated in melanoma (-75.4-fold, P = 1.7 × 10(-4)). MiR-206 has been implicated in a large number of cancers, including breast, lung, colorectal, ovarian, and prostate cancers; however, its role in tumor development remains largely unknown, its biologic function is poorly characterized, and its targets affecting cancer cells are largely unknown. MiR-206 reduced growth and migration/invasion of multiple melanoma cell lines. Bioinformatics identified cell cycle genes CDK2, CDK4, Cyclin C, and Cyclin D1 as strong candidate targets. Western blots and 3'UTR reporter gene assays revealed that miR-206 inhibited translation of CDK4, Cyclin D1, and Cyclin C. Additionally, hsa-miR-206 transfection induced G1 arrest in multiple melanoma cell lines. These observations support hsa-miR-206 as a tumor suppressor in melanoma and identify Cyclin C, Cyclin D1, and CDK4 as miR-206 targets.

  16. Pyrrolopyridine inhibitors of mitogen-activated protein kinase-activated protein kinase 2 (MK-2).

    PubMed

    Anderson, David R; Meyers, Marvin J; Vernier, William F; Mahoney, Matthew W; Kurumbail, Ravi G; Caspers, Nicole; Poda, Gennadiy I; Schindler, John F; Reitz, David B; Mourey, Robert J

    2007-05-31

    A new class of potent kinase inhibitors selective for mitogen-activated protein kinase-activated protein kinase 2 (MAPKAP-K2 or MK-2) for the treatment of rheumatoid arthritis has been prepared and evaluated. These inhibitors have IC50 values as low as 10 nM against the target and have good selectivity profiles against a number of kinases including CDK2, ERK, JNK, and p38. These MK-2 inhibitors have been shown to suppress TNFalpha production in U397 cells and to be efficacious in an acute inflammation model. The structure-activity relationships of this series, the selectivity for MK-2 and their activity in both in vitro and in vivo models are discussed. The observed selectivity is discussed with the aid of an MK-2/inhibitor crystal structure.

  17. Interplay between cdk9 and NF-kappaB factors determines the level of HIV-1 gene transcription in astrocytic cells.

    PubMed

    Amini, Shohreh; Clavo, Anaira; Nadraga, Yuri; Giordano, Antonio; Khalili, Kamel; Sawaya, Bassel E

    2002-08-22

    Basal transcription of the HIV-1 genome is controlled by a variety of ubiquitous and inducible regulatory factors, some with the ability to associate with the viral DNA sequences within the promoter spanning the long terminal repeat (LTR). In this report we demonstrate that activation of the HIV-1 promoter through the inducible DNA binding NF-kappaB transcription factors can be affected by cdk9 in human astrocytic cells. Our results show that ectopic expression of cdk9, but not its mutant variant which lacks the domain responsible for its kinase activity, augments transcription of the LTR. Moreover, we demonstrate that induction of the NF-kappaB pathway by PMA, or overexpression of its subunits including p50/p65 have a negative effect on the ability of cdk9 to stimulate viral gene transcription in these cells. Results from band-shift experiments demonstrated significant suppression of p50/p65 association to its DNA target motif by cdk9. Further, data from GST pull-down and combined immunoprecipitation/Western blot analysis of the protein extracts from cells expressing cdk9, p50 and p65 have revealed the interaction of cdk9 with both p50 and p65 in the absence of DNA containing the kappaB motif. All of these observations led us to conclude that the interaction of cdk9 with the NF-kappaB factors can determine the ability of NF-kappaB to modulate HIV-1 gene transcription.

  18. Cyclin I-like (CCNI2) is a cyclin-dependent kinase 5 (CDK5) activator and is involved in cell cycle regulation

    PubMed Central

    Liu, Chengcheng; Zhai, Xiaoyan; Zhao, Bin; Wang, Yanfei; Xu, Zhigang

    2017-01-01

    In contrast to conventional cyclin-dependent kinases that are important for mitotic cell division, cyclin-dependent kinase 5 (CDK5) is predominantly activated in post-mitotic cells and is involved in various cellular events. The kinase activity of CDK5 is tightly regulated by specific activators including p35, p39, and cyclin I (CCNI). Here we show that cyclin I-like (CCNI2), a homolog of CCNI, interacts with CDK5 and activates the kinase activity of CDK5. Different from CCNI, which colocalizes with CDK5 in the nuclei in transfected cells, CCNI2 mainly retains CDK5 in the cytoplasm as well as on the cell membrane. Furthermore, although the expression level of CCNI2 mRNA and CCNI2 protein do not change significantly during cell cycle, depletion of CCNI2 with siRNA affects cell cycle progression as well as cell proliferation. In conclusion, our data strongly suggest that CCNI2 is a novel CDK5 activator and is involved in cell cycle regulation. PMID:28112194

  19. Lack of cyclin D-Cdk complexes in Rb-negative cells correlates with high levels of p16INK4/MTS1 tumour suppressor gene product.

    PubMed Central

    Parry, D; Bates, S; Mann, D J; Peters, G

    1995-01-01

    D-type cyclins, in association with the cyclin-dependent kinases Cdk4 or Cdk6, regulate events in the G1 phase of the cell cycle and may contribute to the phosphorylation of the retinoblastoma gene product (Rb). However, in cells in which the function of Rb has been compromised, either by naturally arising mutations or through binding to proteins encoded by DNA tumour viruses, Cdk4 and Cdk6 are not associated with D cyclins. Instead, both kinases form binary complexes with a stable 16 kDa protein (p16) encoded by the putative tumour suppressor gene INK4/MTS1 on human chromosome 9p21. Here we show an inverse correlation between Rb status and the expression of p16. Since Rb-negative cells express high levels of p16, we suggest that in these cells p16 competes with D cyclins for binding to Cdk4 and Cdk6 and prevents formation of active complexes. In line with these predictions, DNA tumour virus oncoproteins do not disrupt cyclin D1-Cdk4 complexes in cells lacking p16. Images PMID:7859739

  20. The Design and Synthesis of Potent and Selective Inhibitors of Trypanosoma brucei Glycogen Synthase Kinase 3 for the Treatment of Human African Trypanosomiasis

    PubMed Central

    2014-01-01

    Glycogen synthase kinase 3 (GSK3) is a genetically validated drug target for human African trypanosomiasis (HAT), also called African sleeping sickness. We report the synthesis and biological evaluation of aminopyrazole derivatives as Trypanosoma brucei GSK3 short inhibitors. Low nanomolar inhibitors, which had high selectivity over the off-target human CDK2 and good selectivity over human GSK3β enzyme, have been prepared. These potent kinase inhibitors demonstrated low micromolar levels of inhibition of the Trypanosoma brucei brucei parasite grown in culture. PMID:25198388

  1. CDKN2A and CDK4 variants in Latvian melanoma patients: analysis of a clinic-based population.

    PubMed

    Pjanova, Dace; Engele, Ludmila; Randerson-Moor, Juliette A; Harland, Mark; Bishop, D Timothy; Newton Bishop, Julia A; Taylor, Claire; Debniak, Tadeusz; Lubinski, Jan; Kleina, Regina; Heisele, Olita

    2007-06-01

    Germline mutations of the CDKN2A and CDK4 genes explain a significant proportion of familial melanoma. To date, there have been few published estimations of the prevalence of such mutations in sporadic melanoma patients. In this study, we investigated CDKN2A and CDK4 exon 2 for germline mutations in 125 consecutive cutaneous malignant melanoma patients recruited through the Latvian Oncological Center, using amplicon melting analysis and sequencing. No disease-related CDKN2A germline mutations were identified in any of the melanoma patients analysed but the previously described CDK4 mutation, Arg24His, was found in one patient with a family history of melanoma. CDKN2A polymorphisms were studied as putative low penetrance susceptibility genes. The proportion of cases with polymorphisms in this Latvian melanoma population was Ala148Thr (c.442G>A) (6%), 500 C/G (c.*29C>G) (18%), and 540 C/T (c.*69C>T) (20%); however, only the frequency of the Ala148Thr polymorphism was higher in melanoma patients than in 203 controls (6 versus 1%, P=0.03). Ala148Thr has also been reported in association with melanoma in a Polish series but not in an English series. We therefore examined the Ala148Thr carrier's haplotype in 10 Latvian and 39 Polish samples. No significant difference was seen between these populations and the predominant haplotype observed in English samples, giving no indication that the discrepancy could be explained by population differences in linkage disequilibrium. In summary, our results show that germline mutations at the CDKN2A locus are rare in sporadic melanoma in Latvia. The study does, however, provide some additional evidence for a role for the CDKN2A polymorphism Ala148Thr as a low penetrance susceptibility gene. The detected CDK4 exon 2 mutation was found in only the seventh family identified worldwide with a germline CDK4 mutation.

  2. Analysis of Candida albicans Mutants Defective in the Cdk8 Module of Mediator Reveal Links between Metabolism and Biofilm Formation

    PubMed Central

    Lindsay, Allia K.; Morales, Diana K.; Liu, Zhongle; Grahl, Nora; Zhang, Anda; Willger, Sven D.; Myers, Lawrence C.; Hogan, Deborah A.

    2014-01-01

    Candida albicans biofilm formation is a key virulence trait that involves hyphal growth and adhesin expression. Pyocyanin (PYO), a phenazine secreted by Pseudomonas aeruginosa, inhibits both C. albicans biofilm formation and development of wrinkled colonies. Using a genetic screen, we identified two mutants, ssn3Δ/Δ and ssn8Δ/Δ, which continued to wrinkle in the presence of PYO. Ssn8 is a cyclin-like protein and Ssn3 is similar to cyclin-dependent kinases; both proteins are part of the heterotetrameric Cdk8 module that forms a complex with the transcriptional co-regulator, Mediator. Ssn3 kinase activity was also required for PYO sensitivity as a kinase dead mutant maintained a wrinkled colony morphology in the presence of PYO. Furthermore, similar phenotypes were observed in mutants lacking the other two components of the Cdk8 module—Srb8 and Srb9. Through metabolomics analyses and biochemical assays, we showed that a compromised Cdk8 module led to increases in glucose consumption, glycolysis-related transcripts, oxidative metabolism and ATP levels even in the presence of PYO. In the mutant, inhibition of respiration to levels comparable to the PYO-treated wild type inhibited wrinkled colony development. Several lines of evidence suggest that PYO does not act through Cdk8. Lastly, the ssn3 mutant was a hyperbiofilm former, and maintained higher biofilm formation in the presence of PYO than the wild type. Together these data provide novel insights into the role of the Cdk8 module of Mediator in regulation of C. albicans physiology and the links between respiratory activity and both wrinkled colony and biofilm development. PMID:25275466

  3. Cloning and characterization of human IC53-2, a novel CDK5 activator binding protein.

    PubMed

    Xie, Yi Hu; He, Xiang Huo; Tang, Yun Tian; Li, Jin Jun; Pan, Zhi Mei; Qin, Wen Xin; Wan, Da Fang; Gu, Jian Ren

    2003-04-01

    We have identified IC53-2, a human homologue of the rat C53 gene from a human placenta cDNA library (GeneBank Accession No.AF217982). IC53-2 can bind to the CDK5 activator p35 by in vitro association assay. IC53-2 is mapped to human chromosome 17q21.31. The IC53-2 transcript is highly expressed in kidney, liver, skeletal muscle and placenta. It is abundantly expressed in SMMC-7721, C-33A, 3AO, A431 and MCF-7 cancer cell lines by RT-PCR assay. Stable transfection of IC53-2 cDNA into the hepatocellular carcinoma SMMC-7721 cell remarkably stimulates its growth in vitro. The above results indicate that IC53-2 is a novel human gene, which may be involved in the regulation of cell proliferation.

  4. Phosphorylation of Lte1 by Cdk prevents polarized growth during mitotic arrest in S. cerevisiae

    PubMed Central

    Spanos, Adonis; Jensen, Sanne; Sedgwick, Steven G.

    2010-01-01

    Lte1 is known as a regulator of mitotic progression in budding yeast. Here we demonstrate phosphorylation-dependent inhibition of polarized bud growth during G2/M by Lte1. Cla4 activity first localizes Lte1 to the polarity cap and thus specifically to the bud. This localization is a prerequisite for subsequent Clb–Cdk-dependent phosphorylation of Lte1 and its relocalization to the entire bud cortex. There, Lte1 interferes with activation of the small GTPases, Ras and Bud1. The inhibition of Bud1 prevents untimely polarization until mitosis is completed and Cdc14 phosphatase is released. Inhibition of Bud1 and Ras depends on Lte1’s GEF-like domain, which unexpectedly inhibits these small G proteins. Thus, Lte1 has dual functions for regulation of mitotic progression: it both induces mitotic exit and prevents polarized growth during mitotic arrest, thereby coupling cell cycle progression and morphological development. PMID:21149565

  5. DRG2 Regulates G2/M Progression via the Cyclin B1-Cdk1 Complex

    PubMed Central

    Jang, Soo Hwa; Kim, Ah-Ram; Park, Neung-Hwa; Park, Jeong Woo; Han, In-Seob

    2016-01-01

    Developmentally regulated GTP-binding protein 2 (DRG2) plays an important role in cell growth. Here we explored the linkage between DRG2 and G2/M phase checkpoint function in cell cycle progression. We observed that knockdown of DRG2 in HeLa cells affected growth in a wound-healing assay, and tumorigenicity in nude mice xenografts. Flow cytometry assays and [3H] incorporation assays indicated that G2/M phase arrest was responsible for the decreased proliferation of these cells. Knockdown of DRG2 elicited down-regulation of the major mitotic promoting factor, the cyclin B1/Cdk1 complex, but up-regulation of the cell cycle arresting proteins, Wee1, Myt1, and p21. These findings identify a novel role of DRG2 in G2/M progression. PMID:27669826

  6. Roles of CDK and DDK in Genome Duplication and Maintenance: Meiotic Singularities

    PubMed Central

    Gómez-Escoda, Blanca; Wu, Pei-Yun Jenny

    2017-01-01

    Cells reproduce using two types of divisions: mitosis, which generates two daughter cells each with the same genomic content as the mother cell, and meiosis, which reduces the number of chromosomes of the parent cell by half and gives rise to four gametes. The mechanisms that promote the proper progression of the mitotic and meiotic cycles are highly conserved and controlled. They require the activities of two types of serine-threonine kinases, the cyclin-dependent kinases (CDKs) and the Dbf4-dependent kinase (DDK). CDK and DDK are essential for genome duplication and maintenance in both mitotic and meiotic divisions. In this review, we aim to highlight how these kinases cooperate to orchestrate diverse processes during cellular reproduction, focusing on meiosis-specific adaptions of their regulation and functions in DNA metabolism. PMID:28335524

  7. Mitochondrial Ribosomal Protein L10 Associates with Cyclin B1/Cdk1 Activity and Mitochondrial Function

    PubMed Central

    Li, Hai-Bo; Wang, Ruo-Xi; Jiang, Hai-Bo; Zhang, En-dong; Tan, Jie-Qiong; Xu, Hui-Zhuo

    2016-01-01

    Mitochondrial ribosomal proteins are important for mitochondrial-encoded protein synthesis and mitochondrial function. In addition to their roles in mitoribosome assembly, several mitochondrial ribosome proteins are also implicated in cellular processes like cell cycle regulation, apoptosis, and mitochondrial homeostasis regulation. Here, we demonstrate that MRPL10 regulates cyclin B1/Cdk1 (cyclin-dependent kinase 1) activity and mitochondrial protein synthesis in mammalian cells. In Drosophila, inactivation of mRpL10 (the Drosophila ortholog of mammalian MRPL10) in eyes results in abnormal eye development. Furthermore, expression of human cyclin B1 suppresses eye phenotypes and mitochondrial abnormality of mRpL10 knockdown Drosophila. This study identified that the physiological regulatory pathway of MRPL10 and providing new insights into the role of MRPL10 in growth control and mitochondrial function. PMID:27726420

  8. CDK1-mediated SIRT3 Activation Enhances Mitochondrial Function and Tumor Radioresistance

    PubMed Central

    Liu, Rui; Fan, Ming; Candas, Demet; Qin, Lili; Zhang, Xiaodi; Eldridge, Angela; Zou, June X.; Zhang, Tieqiao; Juma, Shuaib; Jin, Cuihong; Li, Robert F.; Perks, Julian; Sun, Lun-Quan; Vaughan, Andrew T.M.; Hai, Chun-Xu; Gius, David R.; Li, Jian Jian

    2015-01-01

    The tumor adaptive resistance to therapeutic radiation remains to be a barrier for further improvement of local cancer control. SIRT3, a member of the sirtuin family of NAD+-dependent protein deacetylases in mitochondria, promotes metabolic homeostasis through regulation of mitochondrial protein deacetylation and plays a key role in prevention of cell aging. Here, we demonstrate that SIRT3 expression is induced in an array of radiation-treated human tumor cells and their corresponding xenograft tumors including colon cancer HCT-116, glioblastoma U87 and breast cancer MDA-MB231 cells. The SIRT3 transcriptional activation is due to SIRT3 promoter activation controlled by the stress transcription factor NF-κB. Post-transcriptionally, the SIRT3 enzymatic activity is further enhanced via Thr150/Ser159 phosphorylation by Cyclin B1/CDK1, which is also induced by radiation and relocated to mitochondria together with SIRT3. Cells expressing the Thr150Ala/Ser159Ala mutant SIRT3 show a reduction in the mitochondrial protein lysine deacetylation, ΔΨm, MnSOD activity and mitochondrial ATP generation. The clonogenicity of Thr150Ala/Ser159Ala mutant transfectants is lower and significantly decreased under radiation. Tumors harboring the Thr150Ala/Ser159Ala mutant SIRT3 show inhibited growth and sensitivity to in vivo local irradiation. These results demonstrate that enhanced SIRT3 transcription and post-translational modifications in mitochondria contribute to the adaptive radioresistance in tumor cells. The CDK1-mediated SIRT3 phosphorylation is a potential effective target to sensitize tumor cells to radiotherapy. PMID:26141949

  9. Systematic Determination of Human Cyclin Dependent Kinase (CDK)-9 Interactome Identifies Novel Functions in RNA Splicing Mediated by the DEAD Box (DDX)-5/17 RNA Helicases.

    PubMed

    Yang, Jun; Zhao, Yingxin; Kalita, Mridul; Li, Xueling; Jamaluddin, Mohammad; Tian, Bing; Edeh, Chukwudi B; Wiktorowicz, John E; Kudlicki, Andrzej; Brasier, Allan R

    2015-10-01

    Inducible transcriptional elongation is a rapid, stereotypic mechanism for activating immediate early immune defense genes by the epithelium in response to viral pathogens. Here, the recruitment of a multifunctional complex containing the cyclin dependent kinase 9 (CDK9) triggers the process of transcriptional elongation activating resting RNA polymerase engaged with innate immune response (IIR) genes. To identify additional functional activity of the CDK9 complex, we conducted immunoprecipitation (IP) enrichment-stable isotope labeling LC-MS/MS of the CDK9 complex in unstimulated cells and from cells activated by a synthetic dsRNA, polyinosinic/polycytidylic acid [poly (I:C)]. 245 CDK9 interacting proteins were identified with high confidence in the basal state and 20 proteins in four functional classes were validated by IP-SRM-MS. These data identified that CDK9 interacts with DDX 5/17, a family of ATP-dependent RNA helicases, important in alternative RNA splicing of NFAT5, and mH2A1 mRNA two proteins controlling redox signaling. A direct comparison of the basal versus activated state was performed using stable isotope labeling and validated by IP-SRM-MS. Recruited into the CDK9 interactome in response to poly(I:C) stimulation are HSPB1, DNA dependent kinases, and cytoskeletal myosin proteins that exchange with 60S ribosomal structural proteins. An integrated human CDK9 interactome map was developed containing all known human CDK9- interacting proteins. These data were used to develop a probabilistic global map of CDK9-dependent target genes that predicted two functional states controlling distinct cellular functions, one important in immune and stress responses. The CDK9-DDX5/17 complex was shown to be functionally important by shRNA-mediated knockdown, where differential accumulation of alternatively spliced NFAT5 and mH2A1 transcripts and alterations in downstream redox signaling were seen. The requirement of CDK9 for DDX5 recruitment to NFAT5 and mH2A1

  10. The interaction of Munc 18 (p67) with the p10 domain of p35 protects in vivo Cdk5/p35 activity from inhibition by TFP5, a peptide derived from p35

    PubMed Central

    Amin, Niranjana D.; Zheng, Yali; BK, Binukumar; Shukla, Varsha; Skuntz, Susan; Grant, Philip; Steiner, Joseph; Bhaskar, Manju; Pant, Harish C.

    2016-01-01

    In a series of studies, we have identified TFP5, a truncated fragment of p35, the Cdk5 kinase regulatory protein, which inhibits Cdk5/p35 and the hyperactive Cdk5/p25 activities in test tube experiments. In cortical neurons, however, and in vivo in Alzheimer’s disease (AD) model mice, the peptide specifically inhibits the Cdk5/p25 complex and not the endogenous Cdk5/p35. To account for the selective inhibition of Cdk5/p25 activity, we propose that the “p10” N-terminal domain of p35, absent in p25, spares Cdk5/p35 because p10 binds to macromolecules (e.g., tubulin and actin) as a membrane-bound multimeric complex that favors p35 binding to Cdk5 and catalysis. To test this hypothesis, we focused on Munc 18, a key synapse-associated neuronal protein, one of many proteins copurifying with Cdk5/p35 in membrane-bound multimeric complexes. Here we show that, in vitro, the addition of p67 protects Cdk5/p35 and has no effect on Cdk5/p25 activity in the presence of TFP5. In cortical neurons transfected with p67siRNA, we also show that TFP5 inhibits Cdk5/p35 activity, whereas in the presence of p67 the activity is protected. It does so without affecting any other kinases of the Cdk family of cyclin kinases. This difference may be of significant therapeutic value because the accumulation of the deregulated, hyperactive Cdk5/p25 complex in human brains has been implicated in pathology of AD and other neurodegenerative disorders. PMID:27630261

  11. Functional Analysis of the Cyclin-Dependent Kinase Inhibitor Pho81 Identifies a Novel Inhibitory Domain

    PubMed Central

    Huang, Sidong; Jeffery, Douglas A.; Anthony, Malcolm D.; O'Shea, Erin K.

    2001-01-01

    In response to phosphate limitation, Saccharomyces cerevisiae induces transcription of a set of genes important for survival. A phosphate-responsive signal transduction pathway mediates this response by controlling the activity of the transcription factor Pho4. Three components of this signal transduction pathway resemble those used to regulate the eukaryotic cell cycle: a cyclin-dependent kinase (CDK), Pho85; a cyclin, Pho80; and a CDK inhibitor (CKI), Pho81. Pho81 forms a stable complex with Pho80-Pho85 under both high- and low-phosphate conditions, but it only inhibits the kinase when cells are starved for phosphate. Pho81 contains six tandem repeats of the ankyrin consensus domain homologous to the INK4 family of mammalian CKIs. INK4 proteins inhibit kinase activity through an interaction of the ankyrin repeats and the CDK subunits. Surprisingly, we find that a region of Pho81 containing 80 amino acids C terminal to the ankyrin repeats is necessary and sufficient for Pho81's CKI function. The ankyrin repeats of Pho81 appear to have no significant role in Pho81 inhibition. Our results suggest that Pho81 inhibits Pho80-Pho85 with a novel motif. PMID:11533256

  12. Prevalence of p16 and CDK4 germline mutations in 48 melanoma-prone families in France. The French Familial Melanoma Study Group.

    PubMed

    Soufir, N; Avril, M F; Chompret, A; Demenais, F; Bombled, J; Spatz, A; Stoppa-Lyonnet, D; Bénard, J; Bressac-de Paillerets, B

    1998-02-01

    Germline mutations in the p16 and CDK4 genes have been reported in a subset of melanoma pedigrees, but their prevalence is not well known. We searched for such germline mutations in 48 French melanoma-prone families selected according to two major criteria: families with at least three affected members (n = 20) or families with two affected members, one of them affected before the age of 50 (n = 28), and one additional minor criterion. Sixteen different p16 germline mutations were found in 21 families, while one germline mutation, Arg24His, was detected in the CDK4 gene. The frequency of p16 gene mutation in our sample (44%) is among the highest rates yet reported and the CDK4 mutation is the second mutation detected in this gene worldwide. In summary, our results show frequent involvement of the p16 gene in familial melanoma and confirm the role of the CDK4 gene as a melanoma-predisposing gene.

  13. CDC-25.1 controls the rate of germline mitotic cell cycle by counteracting WEE-1.3 and by positively regulating CDK-1 in Caenorhabditis elegans.

    PubMed

    Yoon, Sunghee; Kawasaki, Ichiro; Shim, Yhong-Hee

    2012-04-01

    In Caenorhabditis elegans, cdc-25.1 loss-of-function mutants display a lack of germline proliferation. We found that the proliferation defect of cdc-25.1 mutants was suppressed by wee-1.3 RNAi. Further, among the seven cdk and seven cyclin homologs examined, cdk-1 and cyb-3 RNAi treatment caused the most severe germline proliferation defects in an rrf-1 mutant background, which were similar to those of the cdc-25.1 mutants. In addition, while RNAi of cyd-1 and cye-1 caused significant germline proliferation defects, RNAi of cdk-2 and cdk-4 did not. Compared with the number of germ nuclei in wee-1.3(RNAi) worms, the number in wee-1.3(RNAi);cdk-1(RNAi) and wee-1.3(RNAi);cyb-3(RNAi) worms further decreased to the level of cdk-1(RNAi) and cyb-3(RNAi) worms, respectively, indicating that cdk-1 and cyb-3 are epistatic and function downstream of cdc-25.1 and wee-1.3 in the control of the cell cycle. BrdU labeling of adult worms showed that, while 100% of the wild-type germ nuclei in the mitotic region incorporated BrdU when labeled for more than 12 h at 20°C, a small fraction of the cdc-25.1 mutant germ nuclei failed to incorporate BrdU even when labeled for 68 h. These results indicate that CDC-25.1 is required for maintaining proper rate of germline mitotic cell cycle. We propose that CDC-25.1 regulates the rate of germline mitotic cell cycle by counteracting WEE-1.3 and by positively controlling CDK-1, which forms a complex primarily with CYB-3, but also possibly with CYD-1 and CYE-1.

  14. Cdk1 Phosphorylates Drosophila Sas-4 to Recruit Polo to Daughter Centrioles and Convert Them to Centrosomes.

    PubMed

    Novak, Zsofia A; Wainman, Alan; Gartenmann, Lisa; Raff, Jordan W

    2016-06-20

    Centrosomes and cilia are organized by a centriole pair comprising an older mother and a younger daughter. Centriole numbers are tightly regulated, and daughter centrioles (which assemble in S phase) cannot themselves duplicate or organize centrosomes until they have passed through mitosis. It is unclear how this mitotic "centriole conversion" is regulated, but it requires Plk1/Polo kinase. Here we show that in flies, Cdk1 phosphorylates the conserved centriole protein Sas-4 during mitosis. This creates a Polo-docking site that helps recruit Polo to daughter centrioles and is required for the subsequent recruitment of Asterless (Asl), a protein essential for centriole duplication and mitotic centrosome assembly. Point mutations in Sas-4 that prevent Cdk1 phosphorylation or Polo docking do not block centriole disengagement during mitosis, but block efficient centriole conversion and lead to embryonic lethality. These observations can explain why daughter centrioles have to pass through mitosis before they can duplicate and organize a centrosome.

  15. Mastl is required for timely activation of APC/C in meiosis I and Cdk1 reactivation in meiosis II.

    PubMed

    Adhikari, Deepak; Diril, M Kasim; Busayavalasa, Kiran; Risal, Sanjiv; Nakagawa, Shoma; Lindkvist, Rebecca; Shen, Yan; Coppola, Vincenzo; Tessarollo, Lino; Kudo, Nobuaki R; Kaldis, Philipp; Liu, Kui

    2014-09-29

    In mitosis, the Greatwall kinase (called microtubule-associated serine/threonine kinase like [Mastl] in mammals) is essential for prometaphase entry or progression by suppressing protein phosphatase 2A (PP2A) activity. PP2A suppression in turn leads to high levels of Cdk1 substrate phosphorylation. We have used a mouse model with an oocyte-specific deletion of Mastl to show that Mastl-null oocytes resume meiosis I and reach metaphase I normally but that the onset and completion of anaphase I are delayed. Moreover, after the completion of meiosis I, Mastl-null oocytes failed to enter meiosis II (MII) because they reassembled a nuclear structure containing decondensed chromatin. Our results show that Mastl is required for the timely activation of anaphase-promoting complex/cyclosome to allow meiosis I exit and for the rapid rise of Cdk1 activity that is needed for the entry into MII in mouse oocytes.

  16. Evaluation of germline CDKN2A, ARF, CDK4, PTEN, and BRAF alterations in atypical mole syndrome.

    PubMed

    Celebi, J T; Ward, K M; Wanner, M; Polsky, D; Kopf, A W

    2005-01-01

    Atypical mole syndrome is a sporadic or an inherited condition with an increased risk of melanoma. Germline mutations in the CDKN2A, ARF, CDK4 and somatic mutations in the PTEN and BRAF genes have been associated with melanoma. In this study, we evaluated genes associated with familial and sporadic melanoma for mutations in 28 probands with the atypical mole syndrome. No sequence alterations in the coding regions or in the splice junctions of CDKN2A, ARF, CDK4, PTEN or BRAF were identified. These data suggest that genes evaluated in this study are unlikely to be candidate genes for atypical mole syndrome and support the notion that unknown susceptibility gene/s for this disease exist.

  17. Mice lacking p35, a neuronal specific activator of Cdk5, display cortical lamination defects, seizures, and adult lethality.

    PubMed

    Chae, T; Kwon, Y T; Bronson, R; Dikkes, P; Li, E; Tsai, L H

    1997-01-01

    The adult mammalian cortex is characterized by a distinct laminar structure generated through a well-defined pattern of neuronal migration. Successively generated neurons are layered in an "inside-out" manner to produce six cortical laminae. We demonstrate here that p35, the neuronal-specific activator of cyclin-dependent kinase 5, plays a key role in proper neuronal migration. Mice lacking p35, and thus p35/cdk5 kinase activity, display severe cortical lamination defects and suffer from sporadic adult lethality and seizures. Histological examination reveals that the mutant mice lack the characteristic laminated structure of the cortex. Neuronal birth-dating experiments indicate a reversed packing order of cortical neurons such that earlier born neurons reside in superficial layers and later generated neurons occupy deep layers. The phenotype of p35 mutant mice thus demonstrates that the formation of cortical laminar structure depends on the action of the p35/cdk5 kinase.

  18. Computational study of the inhibitory mechanism of the kinase CDK5 hyperactivity by peptide p5 and derivation of a pharmacophore

    NASA Astrophysics Data System (ADS)

    Cardone, A.; Brady, M.; Sriram, R.; Pant, H. C.; Hassan, S. A.

    2016-06-01

    The hyperactivity of the cyclic dependent kinase 5 (CDK5) induced by the activator protein p25 has been linked to a number of pathologies of the brain. The CDK5-p25 complex has thus emerged as a major therapeutic target for Alzheimer's disease (AD) and other neurodegenerative conditions. Experiments have shown that the peptide p5 reduces the CDK5-p25 activity without affecting the endogenous CDK5-p35 activity, whereas the peptide TFP5, obtained from p5, elicits similar inhibition, crosses the blood-brain barrier, and exhibits behavioral rescue of AD mice models with no toxic side effects. The molecular basis of the kinase inhibition is not currently known, and is here investigated by computer simulations. It is shown that p5 binds the kinase at the same CDK5/p25 and CDK5/p35 interfaces, and is thus a non-selective competitor of both activators, in agreement with available experimental data in vitro. Binding of p5 is enthalpically driven with an affinity estimated in the low µM range. A quantitative description of the binding site and pharmacophore is presented, and options are discussed to increase the binding affinity and selectivity in the design of drug-like compounds against AD.

  19. Cyclin A/Cdk2 regulates Cdh1 and claspin during late S/G2 phase of the cell cycle.

    PubMed

    Oakes, Vanessa; Wang, Weili; Harrington, Brittney; Lee, Won Jae; Beamish, Heather; Chia, Kee Ming; Pinder, Alex; Goto, Hidemasa; Inagaki, Masaki; Pavey, Sandra; Gabrielli, Brian

    2014-01-01

    Whereas many components regulating the progression from S phase through G2 phase into mitosis have been identified, the mechanism by which these components control this critical cell cycle progression is still not fully elucidated. Cyclin A/Cdk2 has been shown to regulate the timing of Cyclin B/Cdk1 activation and progression into mitosis although the mechanism by which this occurs is only poorly understood. Here we show that depletion of Cyclin A or inhibition of Cdk2 during late S/early G2 phase maintains the G2 phase arrest by reducing Cdh1 transcript and protein levels, thereby stabilizing Claspin and maintaining elevated levels of activated Chk1 which contributes to the G2 phase observed. Interestingly, the Cyclin A/Cdk2 regulated APC/C(Cdh1) activity is selective for only a subset of Cdh1 targets including Claspin. Thus, a normal role for Cyclin A/Cdk2 during early G2 phase is to increase the level of Cdh1 which destabilises Claspin which in turn down regulates Chk1 activation to allow progression into mitosis. This mechanism links S phase exit with G2 phase transit into mitosis, provides a novel insight into the roles of Cyclin A/Cdk2 in G2 phase progression, and identifies a novel role for APC/C(Cdh1) in late S/G2 phase cell cycle progression.

  20. Autophagy inhibitors.

    PubMed

    Pasquier, Benoit

    2016-03-01

    Autophagy is a lysosome-dependent mechanism of intracellular degradation. The cellular and molecular mechanisms underlying this process are highly complex and involve multiple proteins, including the kinases ULK1 and Vps34. The main function of autophagy is the maintenance of cell survival when modifications occur in the cellular environment. During the past decade, extensive studies have greatly improved our knowledge and autophagy has exploded as a research field. This process is now widely implicated in pathophysiological processes such as cancer, metabolic, and neurodegenerative disorders, making it an attractive target for drug discovery. In this review, we will summarize the different types of inhibitors that affect the autophagy machinery and provide some potential therapeutic perspectives.

  1. Targeting Cyclin-Dependent Kinases in Human Cancers: From Small Molecules to Peptide Inhibitors

    PubMed Central

    Peyressatre, Marion; Prével, Camille; Pellerano, Morgan; Morris, May C.

    2015-01-01

    Cyclin-dependent kinases (CDK/Cyclins) form a family of heterodimeric kinases that play central roles in regulation of cell cycle progression, transcription and other major biological processes including neuronal differentiation and metabolism. Constitutive or deregulated hyperactivity of these kinases due to amplification, overexpression or mutation of cyclins or CDK, contributes to proliferation of cancer cells, and aberrant activity of these kinases has been reported in a wide variety of human cancers. These kinases therefore constitute biomarkers of proliferation and attractive pharmacological targets for development of anticancer therapeutics. The structural features of several of these kinases have been elucidated and their molecular mechanisms of regulation characterized in depth, providing clues for development of drugs and inhibitors to disrupt their function. However, like most other kinases, they constitute a challenging class of therapeutic targets due to their highly conserved structural features and ATP-binding pocket. Notwithstanding, several classes of inhibitors have been discovered from natural sources, and small molecule derivatives have been synthesized through rational, structure-guided approaches or identified in high throughput screens. The larger part of these inhibitors target ATP pockets, but a growing number of peptides targeting protein/protein interfaces are being proposed, and a small number of compounds targeting allosteric sites have been reported. PMID:25625291

  2. Synthesis and biological evaluation of novel coumarin-based inhibitors of Cdc25 phosphatases.

    PubMed

    Valente, Sergio; Bana, Emilie; Viry, Elodie; Bagrel, Denyse; Kirsch, Gilbert

    2010-10-01

    The cell division cycle 25 (Cdc25) family of proteins are dual specificity phosphatases that activate cyclin-dependent kinase (CDK) complexes, which in turn regulate progression through the cell division cycle. Overexpression of Cdc25 proteins has been reported in a wide variety of cancers; their inhibition may thus represent a novel approach for the development of anticancer therapeutics. Herein we report new coumarin-based scaffolds endowed with a selective inhibition against Cdc25A and Cdc25C, being 6a and 6d the most efficient inhibitors and worthy of further investigation as anticancer agents.

  3. Discovery of azabenzimidazole derivatives as potent, selective inhibitors of TBK1/IKKε kinases.

    PubMed

    Wang, Tao; Block, Michael A; Cowen, Scott; Davies, Audrey M; Devereaux, Erik; Gingipalli, Lakshmaiah; Johannes, Jeffrey; Larsen, Nicholas A; Su, Qibin; Tucker, Julie A; Whitston, David; Wu, Jiaquan; Zhang, Hai-Jun; Zinda, Michael; Chuaqui, Claudio

    2012-03-01

    The design, synthesis and biological evaluation of a series of azabenzimidazole derivatives as TBK1/IKKε kinase inhibitors are described. Starting from a lead compound 1a, iterative design and SAR exploitation of the scaffold led to analogues with nM enzyme potencies against TBK1/IKKε. These compounds also exhibited excellent cellular activity against TBK1. Further structure-based design to improve selectivity over CDK2 and Aurora B resulted in compounds such as 5b-e. These probe compounds will facilitate study of the complex cancer biology of TBK1 and IKKε.

  4. Draft Genome Sequence of Halolamina pelagica CDK2 Isolated from Natural Salterns from Rann of Kutch, Gujarat, India

    PubMed Central

    Gaba, Sonam; Abrol, Shrutica; Yadav, Ajar Nath; Saxena, Anil Kumar

    2017-01-01

    ABSTRACT Halolamina pelagica strain CDK2, a halophilic archaeon (growth range 1.36 to 5.12 M NaCl), was isolated from rhizosphere of wild grasses of hypersaline soil of the Rann of Kutch, Gujarat, India. Its draft genome contains 2,972,542 bp and 3,485 coding sequences, depicting genes for halophilic serine proteases and trehalose synthesis. PMID:28183764

  5. Inhibition of Cdk2 activity decreases Aurora-A kinase centrosomal localization and prevents centrosome amplification in breast cancer cells.

    PubMed

    Leontovich, Alexey A; Salisbury, Jeffrey L; Veroux, Massimiliano; Tallarita, Tiziano; Billadeau, Daniel; McCubrey, James; Ingle, James; Galanis, Evanthia; D'Assoro, Antonino B

    2013-05-01

    Centrosome amplification plays a key role in the origin of chromosomal instability (CIN) during cancer development and progression. In this study, MCF-7 breast cancer cell lines harboring abrogated p53 function (vMCF-7DNp53) were employed to investigate the relationship between induction of genotoxic stress, activation of cyclin-A/Cdk2 and Aurora-A oncogenic signalings and development of centrosome amplification. Introduction of genotoxic stress in the vMCF-7DNp53 cell line by treatment with hydroxyurea (HU) induced centrosome amplification that was mechanistically linked to Aurora-A kinase activity. In cells carrying defective p53, the development of centrosome amplification also occurred following treatment with another DNA damaging agent, methotrexate. Importantly, we demonstrated that Aurora-A kinase-induced centrosome amplification was mediated by Cdk2 kinase since molecular inhibition of Cdk2 activity by SU9516 suppressed Aurora-A centrosomal localization and consequent centrosome amplification. In addition, we employed vMCF-7DRaf-1 cells that display high levels of endogenous cyclin-A and demonstrated that molecular targeting of Aurora-A by Alisertib reduces cyclin-A expression. Taken together, these findings demonstrate a novel positive feed-back loop between cyclin-A/Cdk2 and Aurora-A pathways in the development of centrosome amplification in breast cancer cells. They also provide the translational rationale for targeting 'druggable cell cycle regulators' as an innovative therapeutic strategy to inhibit centrosome amplification and CIN in breast tumors resistant to conventional chemotherapeutic drugs.

  6. Altered expression of cell cycle regulators Cyclin D1, p14, p16, CDK4 and Rb in nodular melanomas.

    PubMed

    Bachmann, Ingeborg M; Straume, Oddbjørn; Akslen, Lars A

    2004-12-01

    Cell cycle regulating proteins are important in tumour development. To investigate whether alterations in Cyclin D1, p14, CDK4 and Rb are associated with tumour cell proliferation, tumour progression and patient survival in malignant melanoma, we examined 202 vertical growth phase tumours and 68 corresponding metastases for expression of Cyclin D1, p14, CDK4 and Rb, and compared the results with Ki-67 expression, p16 and p53 expression, clinico-pathological variables, and survival data. Nuclear staining of Cyclin D1 was strong in 35% of cases, and correlated with high levels of Rb (p=0.05), but not with survival or other markers tested. Strong staining of p14 was found in 63% of nodular melanomas and was associated with strong p53 expression (p=0.014), and with high levels of CDK4 (p<0.0001). Low p14 expression was associated with increased tumour thickness (p=0.008) and increasing level of invasion (p=0.020). Strong nuclear staining for CDK4 was found in 81% of cases and was associated with tumour thickness below the median value of 3.7 mm and improved survival (log-rank test, p=0.024). Further, 56% of the tumours showed strong nuclear staining for Rb, and these cases were significantly associated with absent/low levels of p16 staining (p=0.030), high levels of p14 (p=0.010), as well as high Ki-67 expression (p=0.005). Our results seem to confirm that the p16-Rb pathway plays an important role in tumour progression and prognosis in vertical growth phase melanomas, whereas alterations in the p14-p53 pathway might be less important.

  7. CDK5RAP2 interaction with components of the Hippo signaling pathway may play a role in primary microcephaly.

    PubMed

    Sukumaran, Salil K; Stumpf, Maria; Salamon, Sarah; Ahmad, Ilyas; Bhattacharya, Kurchi; Fischer, Sarah; Müller, Rolf; Altmüller, Janine; Budde, Birgit; Thiele, Holger; Tariq, Muhammad; Malik, Naveed Altaf; Nürnberg, Peter; Baig, Shahid Mahmood; Hussain, Muhammad Sajid; Noegel, Angelika A

    2017-04-01

    Autosomal recessive primary microcephaly (MCPH) is characterized by a substantial reduction in brain size but with normal architecture. It is often linked to mutations in genes coding for centrosomal proteins; however, their role in brain size regulation is not completely understood. By combining homozygosity mapping and whole-exome sequencing in an MCPH family from Pakistan, we identified a novel mutation (XM_011518861.1; c.4114C > T) in CDK5RAP2, the gene associated with primary microcephaly-3 (MCPH3), leading to a premature stop codon (p.Arg1372*). CDK5RAP2 is a component of the pericentriolar material important for the microtubule-organizing function of the centrosome. Patient-derived primary fibroblasts had strongly decreased CDK5RAP2 amounts, showed centrosomal and nuclear abnormalities and exhibited changes in cell size and migration. We further identified an interaction of CDK5RAP2 with the Hippo pathway components MST1 kinase and the transcriptional regulator TAZ. This finding potentially provides a mechanism through which the Hippo pathway with its roles in the regulation of centrosome number is linked to the centrosome. In the patient fibroblasts, we observed higher levels of TAZ and YAP. However, common target genes of the Hippo pathway were downregulated as compared to the control with the exception of BIRC5 (Survivin), which was significantly upregulated. We propose that the centrosomal deficiencies and the altered cellular properties in the patient fibroblasts can also result from the observed changes in the Hippo pathway components which could thus be relevant for MCPH and play a role in brain size regulation and development.

  8. TRPV1 function is modulated by Cdk5-mediated phosphorylation: insights into the molecular mechanism of nociception.

    PubMed

    Jendryke, Thomas; Prochazkova, Michaela; Hall, Bradford E; Nordmann, Grégory C; Schladt, Moritz; Milenkovic, Vladimir M; Kulkarni, Ashok B; Wetzel, Christian H

    2016-02-23

    TRPV1 is a polymodally activated cation channel acting as key receptor in nociceptive neurons. Its function is strongly affected by kinase-mediated phosphorylation leading to hyperalgesia and allodynia. We present behavioral and molecular data indicating that TRPV1 is strongly modulated by Cdk5-mediated phosphorylation at position threonine-407(mouse)/T406(rat). Increasing or decreasing Cdk5 activity in genetically engineered mice has severe consequences on TRPV1-mediated pain perception leading to altered capsaicin consumption and sensitivity to heat. To understand the molecular and structural/functional consequences of TRPV1 phosphorylation, we generated various rTRPV1T406 receptor variants to mimic phosphorylated or dephosphorylated receptor protein. We performed detailed functional characterization by means of electrophysiological whole-cell and single-channel recordings as well as Ca(2+)-imaging and challenged recombinant rTRPV1 receptors with capsaicin, low pH, or heat. We found that position T406 is critical for the function of TRPV1 by modulating ligand-sensitivity, activation, and desensitization kinetics as well as voltage-dependence. Based on high resolution structures of TRPV1, we discuss T406 being involved in the molecular transition pathway, its phosphorylation leading to a conformational change and influencing the gating of the receptor. Cdk5-mediated phosphorylation of T406 can be regarded as an important molecular switch modulating TRPV1-related behavior and pain sensitivity.

  9. Usefulness of CDK5RAP3, CCNB2, and RAGE genes for the diagnosis of lung adenocarcinoma.

    PubMed

    Stav, D; Bar, I; Sandbank, J

    2007-01-01

    We used oligonucleotide microarrays with probe sets to 22,283 genes to analyze the gene expression profile of lung adenocarcinoma. Cancerous and noncancerous tissue samples were obtained from 23 patients with stage I or II lung cancer; 18 tissue pairs and 5 cancerous tissues. A list of 2065 genes that differentiate between cancerous and noncancerous tissues was generated using Winsorized paired t-tests. We analyzed CDK5RAP3 and CCNB2, which are involved in cell cycle progression, and RAGE. The first 2 of these 3 genes proved to be overexpressed in tumor tissue, whereas the RAGE gene was suppressed in tumor tissue. When CDK5RAP3 and CCNB2 were examined in individual patients we found that in cases where one of these genes was only slightly overexpressed the other was highly overexpressed. The combined expression of the 2 cell cycle genes was found to be statistically significant for differentiating between cancerous and noncancerous tissues. Inclusion of the data for the RAGE gene made the differentiation more powerful. The gene expression ratio gave a clear result: when CDK5RAP3 was expressed more than RAGE, the tissue was carcinomatous, and vice versa. We therefore conclude that these 3 genes may be used as a very reliable biomarker of lung adenocarcinoma.

  10. Cks1, Cdk1, and the 19S proteasome collaborate to regulate gene induction-dependent nucleosome eviction in yeast.

    PubMed

    Chaves, Susana; Baskerville, Chris; Yu, Veronica; Reed, Steven I

    2010-11-01

    Cks1, Cdk1 (Cdc28), and the proteasome are required for efficient transcriptional induction of GAL1 and other genes in Saccharomyces cerevisiae. We show here that one function of these proteins is to reduce nucleosome density on chromatin in a gene induction-specific manner. The transcriptional requirement for Cks1 can be bypassed if nucleosome density is reduced by an alternative pathway, indicating that this is the primary function of Cks1 in the context of gene induction. We further show that Cks1, Cdk1, and the 19S subunit of the proteasome are recruited to chromatin by binding directly to the histone H4 amino-terminal tail. However, this activity of the proteasome does not require the protease activity associated with the 20S subunit. These data suggest a model where binding of a complex consisting of Cks1, Cdk1, and the 19S proteasome to histone H4 leads to removal of nucleosomes via a nonproteolytic activity of the proteasome.

  11. Phosphorylation of p62 by cdk1 Controls the Timely Transit of Cells through Mitosis and Tumor Cell Proliferation ▿

    PubMed Central

    Linares, Juan F.; Amanchy, Ramars; Diaz-Meco, Maria T.; Moscat, Jorge

    2011-01-01

    The protein scaffold and signaling regulator p62 is important in critical cellular functions, including bone homeostasis, obesity, and cancer, because of its interactions with various signaling intermediaries. p62 is overexpressed in human cancers and is induced during cell transformation. Its genetic ablation inhibits lung tumorigenesis in vivo and cell proliferation in culture by regulating the TRAF6/NF-κB signaling cascade to control reactive oxygen species (ROS) production and apoptosis. Here we show that cdk1 phosphorylates p62 in vitro and in vivo at T269 and S272, which is necessary for the maintenance of appropriate cyclin B1 levels and the levels of cdk1 activity necessary to allow cells to properly enter and exit mitosis. The lack of cdk1-mediated phosphorylation of p62 leads to a faster exit from mitosis, which translates into enhanced cell proliferation and tumorigenesis in response to Ras-induced transformation. Therefore, p62 emerges as a node for the control of not only cell survival but also cell transit through mitosis. PMID:20974803

  12. PCAF acts as a gastric cancer suppressor through a novel PCAF-p16-CDK4 axis

    PubMed Central

    Fei, Hong-Jun; Zu, Li-Dong; Wu, Jun; Jiang, Xiao-Shu; Wang, Jing-Long; Chin, Y Eugene; Fu, Guo-Hui

    2016-01-01

    Gastric cancer (GC) is a leading cause of cancer-related death worldwide and the pathogenesis of GC remains largely unknown. Here, we demonstrate a novel mechanism by which P300/CBP associating factor (PCAF) acts as a tumor suppressor in GC cells. We showed that both PCAF mRNA and protein were downregulated in GC cells, and that this downregulation correlated with poor survival. Meanwhile, the interaction between human anion exchanger 1 (AE1) and p16 is a key event in GC development. We found that PCAF inhibited GC growth by interacting with AE1 and p16 to promote ubiquitin-mediated degradation of AE1 and p16 upregulation and translocation into the nucleus. Binding of nuclear p16 to CDK4 prevented the CDK4-Cyclin D1 interaction to inhibit GC proliferation. Furthermore, reduced PCAF levels in GC cells were associated with intracellular alkalinization and decreased immunity. Together these results suggest that PCAF acts as a GC suppressor through a novel PCAF-p16-CDK4 axis. The downregulation of PCAF expression in GC cells that follows intracellular alkalinization and decreased immune response, indicates that GC therapies should focus on restoring PCAF levels. PMID:28042499

  13. Stress-induced nuclear translocation of CDK5 suppresses neuronal death by downregulating ERK activation via VRK3 phosphorylation

    PubMed Central

    Song, Haengjin; Kim, Wanil; Choi, Jung-Hyun; Kim, Sung-Hoon; Lee, Dohyun; Park, Choon-Ho; Kim, Sangjune; Kim, Do-Yeon; Kim, Kyong-Tai

    2016-01-01

    Although extracellular signal-related kinase 1/2 (ERK 1/2) activity is generally associated with cell survival, prolonged ERK activation induced by oxidative stress also mediates neuronal cell death. Here we report that oxidative stress-induced cyclin-dependent kinase 5 (CDK5) activation stimulates neuroprotective signaling via phosphorylation of vaccinia-related kinase 3 (VRK3) at Ser 108. The binding of vaccinia H1-related (VHR) phosphatase to phosphorylated VRK3 increased its affinity for phospho-ERK and subsequently downregulated ERK activation. Overexpression of VRK3 protected human neuroblastoma SH-SY5Y cells against hydrogen peroxide (H2O2)-induced apoptosis. However the CDK5 was unable to phosphorylate mutant VRK3, and thus the mutant forms of VRK3 could not attenuate apoptotic process. Suppression of CDK5 activity results in increase of ERK activation and elevation of proapoptotic protein Bak expression in mouse cortical neurons. Results from VRK3-deficient neurons were further confirmed the role of VRK3 phosphorylation in H2O2-evoked ERK regulation. Importantly, we showed an association between phospho-VRK3 levels and the progression of human Alzheimer’s disease (AD) and Parkinson’s disease (PD). Together our work reveals endogenous protective mechanism against oxidative stress-induced neuronal cell death and suggest VRK3 as a potential therapeutic target in neurodegenerative diseases. PMID:27346674

  14. CCND1-CDK4-mediated cell cycle progression provides a competitive advantage for human hematopoietic stem cells in vivo.

    PubMed

    Mende, Nicole; Kuchen, Erika E; Lesche, Mathias; Grinenko, Tatyana; Kokkaliaris, Konstantinos D; Hanenberg, Helmut; Lindemann, Dirk; Dahl, Andreas; Platz, Alexander; Höfer, Thomas; Calegari, Federico; Waskow, Claudia

    2015-07-27

    Maintenance of stem cell properties is associated with reduced proliferation. However, in mouse hematopoietic stem cells (HSCs), loss of quiescence results in a wide range of phenotypes, ranging from functional failure to extensive self-renewal. It remains unknown whether the function of human HSCs is controlled by the kinetics of cell cycle progression. Using human HSCs and human progenitor cells (HSPCs), we report here that elevated levels of CCND1-CDK4 complexes promoted the transit from G0 to G1 and shortened the G1 cell cycle phase, resulting in protection from differentiation-inducing signals in vitro and increasing human leukocyte engraftment in vivo. Further, CCND1-CDK4 overexpression conferred a competitive advantage without impacting HSPC numbers. In contrast, accelerated cell cycle progression mediated by elevated levels of CCNE1-CDK2 led to the loss of functional HSPCs in vivo. Collectively, these data suggest that the transition kinetics through the early cell cycle phases are key regulators of human HSPC function and important for lifelong hematopoiesis.

  15. The Saccharomyces cerevisiae Cdk8 Mediator Represses AQY1 Transcription by Inhibiting Set1p-Dependent Histone Methylation

    PubMed Central

    Law, Michael J.; Finger, Michael A.

    2017-01-01

    In the budding yeast Saccharomyces cerevisiae, nutrient depletion induces massive transcriptional reprogramming that relies upon communication between transcription factors, post-translational histone modifications, and the RNA polymerase II holoenzyme complex. Histone H3Lys4 methylation (H3Lys4 me), regulated by the Set1p-containing COMPASS methyltransferase complex and Jhd2p demethylase, is one of the most well-studied histone modifications. We previously demonstrated that the RNA polymerase II mediator components cyclin C-Cdk8p inhibit locus-specific H3Lys4 3me independently of Jhd2p. Here, we identify loci subject to cyclin C- and Jhd2p-dependent histone H3Lys4 3me inhibition using chromatin immunoprecipitation (ChIP)-seq. We further characterized the independent and combined roles of cyclin C and Jhd2p in controlling H3Lys4 3me and transcription in response to fermentable and nonfermentable carbon at multiple loci. These experiments suggest that H3Lys4 3me alone is insufficient to induce transcription. Interestingly, we identified an unexpected role for cyclin C-Cdk8p in repressing AQY1 transcription, an aquaporin whose expression is normally induced during nutrient deprivation. These experiments, combined with previous work in other labs, support a two-step model in which cyclin C-Cdk8p mediate AQY1 transcriptional repression by stimulating transcription factor proteolysis and preventing Set1p recruitment to the AQY1 locus. PMID:28143948

  16. Cdk1 and Plk1 mediate a CLASP2 phospho-switch that stabilizes kinetochore–microtubule attachments

    PubMed Central

    Garcia, Zaira; Kabeche, Lilian; Barisic, Marin; Maffini, Stefano; Macedo-Ribeiro, Sandra; Cheeseman, Iain M.; Compton, Duane A.; Kaverina, Irina

    2012-01-01

    Accurate chromosome segregation during mitosis relies on a dynamic kinetochore (KT)–microtubule (MT) interface that switches from a labile to a stable condition in response to correct MT attachments. This transition is essential to satisfy the spindle-assembly checkpoint (SAC) and couple MT-generated force with chromosome movements, but the underlying regulatory mechanism remains unclear. In this study, we show that during mitosis the MT- and KT-associated protein CLASP2 is progressively and distinctively phosphorylated by Cdk1 and Plk1 kinases, concomitant with the establishment of KT–MT attachments. CLASP2 S1234 was phosphorylated by Cdk1, which primed CLASP2 for association with Plk1. Plk1 recruitment to KTs was enhanced by CLASP2 phosphorylation on S1234. This was specifically required to stabilize KT–MT attachments important for chromosome alignment and to coordinate KT and non-KT MT dynamics necessary to maintain spindle bipolarity. CLASP2 C-terminal phosphorylation by Plk1 was also required for chromosome alignment and timely satisfaction of the SAC. We propose that Cdk1 and Plk1 mediate a fine CLASP2 “phospho-switch” that temporally regulates KT–MT attachment stability. PMID:23045552

  17. TRPV1 function is modulated by Cdk5-mediated phosphorylation: insights into the molecular mechanism of nociception

    PubMed Central

    Jendryke, Thomas; Prochazkova, Michaela; Hall, Bradford E.; Nordmann, Grégory C.; Schladt, Moritz; Milenkovic, Vladimir M.; Kulkarni, Ashok B.; Wetzel, Christian H.

    2016-01-01

    TRPV1 is a polymodally activated cation channel acting as key receptor in nociceptive neurons. Its function is strongly affected by kinase-mediated phosphorylation leading to hyperalgesia and allodynia. We present behavioral and molecular data indicating that TRPV1 is strongly modulated by Cdk5-mediated phosphorylation at position threonine-407(mouse)/T406(rat). Increasing or decreasing Cdk5 activity in genetically engineered mice has severe consequences on TRPV1-mediated pain perception leading to altered capsaicin consumption and sensitivity to heat. To understand the molecular and structural/functional consequences of TRPV1 phosphorylation, we generated various rTRPV1T406 receptor variants to mimic phosphorylated or dephosphorylated receptor protein. We performed detailed functional characterization by means of electrophysiological whole-cell and single-channel recordings as well as Ca2+-imaging and challenged recombinant rTRPV1 receptors with capsaicin, low pH, or heat. We found that position T406 is critical for the function of TRPV1 by modulating ligand-sensitivity, activation, and desensitization kinetics as well as voltage-dependence. Based on high resolution structures of TRPV1, we discuss T406 being involved in the molecular transition pathway, its phosphorylation leading to a conformational change and influencing the gating of the receptor. Cdk5-mediated phosphorylation of T406 can be regarded as an important molecular switch modulating TRPV1-related behavior and pain sensitivity. PMID:26902776

  18. Cdk2-dependent phosphorylation of Id2 modulates activity of E2A-related transcription factors.

    PubMed Central

    Hara, E; Hall, M; Peters, G

    1997-01-01

    The helix-loop-helix (HLH) protein Id2 is thought to affect the balance between cell growth and differentiation by negatively regulating the function of basic-helix-loop-helix (bHLH) transcription factors. Id2 acts by forming heterodimers that are unable to bind to specific (E-box) DNA sequences. Here we show that this activity can be overcome by phosphorylation of a serine residue within a consensus target site for cyclin-dependent kinases (Cdks). In vitro, Id2 can be phosphorylated by either cyclin E-Cdk2 or cyclin A-Cdk2 but not by cyclin D-dependent kinases. Analogous phosphorylation occurs in serum-stimulated human diploid fibroblasts at a time in late G1 consistent with the appearance of active cyclin E-Cdk2. The phosphorylation of Id2 in these cells correlates with the restoration of a distinct E-box-dependent DNA-binding complex, suggesting that the levels of this complex are modulated by both the abundance and phosphorylation status of Id2. These data provide a link between cyclin-dependent kinases and bHLH transcription factors that may be critical for the regulation of cell proliferation and differentiation. PMID:9029153

  19. The ω-3 epoxide of eicosapentaenoic acid inhibits endothelial cell proliferation by p38 MAP kinase activation and cyclin D1/CDK4 down-regulation

    PubMed Central

    Cui, Pei H; Petrovic, Nenad; Murray, Michael

    2011-01-01

    BACKGROUND AND PURPOSE Dietary intake of ω-3 polyunsaturated fatty acids (ω-3 PUFAs) like eicosapentaenoic acid (EPA) decreases cancer risk, while arachidonic acid and other ω-6 PUFAs increase risk, but the underlying mechanisms are unclear. Cytochrome P450 (CYP)-derived epoxides contribute to enhanced tumourigenesis due to ω-6 PUFA intake. Thus, ω-6 arachidonic acid epoxides (EETs) inhibit apoptosis and stimulate proliferation by up-regulating cyclin D1 expression in cells. The present study evaluated the corresponding ω-3 PUFA epoxides and assessed their role in the regulation of cell proliferation. EXPERIMENTAL APPROACH Four chemically stable EPA epoxides (formed at the 8,9-, 11,12-, 14,15- and 17,18-olefinic bonds) were synthesized and tested against growth-related signalling pathways in brain microvascular endothelial bEND.3 cells. Cell cycle distribution was determined by flow cytometry and cyclin gene expression by immunoblotting and real-time PCR. The role of the p38 mitogen-activated protein (MAP) kinase in cyclin D1 dysregulation was assessed using specific inhibitors and dominant-negative expression plasmids. KEY RESULTS The ω-3 17,18-epoxide of EPA decreased cell proliferation, interrupted the cell cycle in S-phase and down-regulated the cyclin D1/cyclin-dependent kinase (CDK)-4 complex, whereas the 8,9-, 11,12- and 14,15-epoxides were either inactive or enhanced proliferation. Cyclin D1 down-regulation by 17,18-epoxy-EPA was mediated by activation of the growth-suppressing p38 MAP kinase, but the alternate EPA-epoxides were inactive. CONCLUSIONS AND IMPLICATIONS The present findings suggest that the epoxide formed by CYP enzymes at the ω-3 olefinic bond may contribute to the beneficial effects of ω-3 PUFA by down-regulating cyclin D1 and suppressing cell proliferation. PMID:21077851

  20. Virtual screening of selective multitarget kinase inhibitors by combinatorial support vector machines.

    PubMed

    Ma, X H; Wang, R; Tan, C Y; Jiang, Y Y; Lu, T; Rao, H B; Li, X Y; Go, M L; Low, B C; Chen, Y Z

    2010-10-04

    Multitarget agents have been increasingly explored for enhancing efficacy and reducing countertarget activities and toxicities. Efficient virtual screening (VS) tools for searching selective multitarget agents are desired. Combinatorial support vector machines (C-SVM) were tested as VS tools for searching dual-inhibitors of 11 combinations of 9 anticancer kinase targets (EGFR, VEGFR, PDGFR, Src, FGFR, Lck, CDK1, CDK2, GSK3). C-SVM trained on 233-1,316 non-dual-inhibitors correctly identified 26.8%-57.3% (majority >36%) of the 56-230 intra-kinase-group dual-inhibitors (equivalent to the 50-70% yields of two independent individual target VS tools), and 12.2% of the 41 inter-kinase-group dual-inhibitors. C-SVM were fairly selective in misidentifying as dual-inhibitors 3.7%-48.1% (majority <20%) of the 233-1,316 non-dual-inhibitors of the same kinase pairs and 0.98%-4.77% of the 3,971-5,180 inhibitors of other kinases. C-SVM produced low false-hit rates in misidentifying as dual-inhibitors 1,746-4,817 (0.013%-0.036%) of the 13.56 M PubChem compounds, 12-175 (0.007%-0.104%) of the 168 K MDDR compounds, and 0-84 (0.0%-2.9%) of the 19,495-38,483 MDDR compounds similar to the known dual-inhibitors. C-SVM was compared to other VS methods Surflex-Dock, DOCK Blaster, kNN and PNN against the same sets of kinase inhibitors and the full set or subset of the 1.02 M Zinc clean-leads data set. C-SVM produced comparable dual-inhibitor yields, slightly better false-hit rates for kinase inhibitors, and significantly lower false-hit rates for the Zinc clean-leads data set. Combinatorial SVM showed promising potential for searching selective multitarget agents against intra-kinase-group kinases without explicit knowledge of multitarget agents.

  1. Redundancy or specificity? The role of the CDK Pho85 in cell cycle control

    PubMed Central

    Jiménez, Javier; Ricco, Natalia; Grijota-Martínez, Carmen; Fadó, Rut; Clotet, Josep

    2013-01-01

    It is generally accepted that progression through the eukaryotic cell cycle is driven by cyclin-dependent kinases (CDKs), which are regulated by interaction with oscillatory expressed proteins called cyclins. CDKs may be separated into 2 categories: essential and non-essential. Understandably, more attention has been focused on essential CDKs because they are shown to control cell cycle progression to a greater degree. After clearly determining the basic and “core” mechanisms of essential CDKs, several questions arise. What role do non-essential CDKs play? Are these CDKs functionally redundant and do they serve as a mere backup? Or might they be responsible for some accessory tasks in cell cycle progression or control? In the present review we will try to answer these questions based on recent findings on the involvement of non-essential CDKs in cell cycle progression. We will analyse the most recent information with regard to these questions in the yeast Saccharomyces cerevisiae, a well-established eukaryotic model, and in its unique non-essential CDK involved in the cell cycle, Pho85. We will also briefly extend our discussion to higher eukaryotic systems. PMID:24049669

  2. Nucleophosmin phosphorylation by v-cyclin-CDK6 controls KSHV latency.

    PubMed

    Sarek, Grzegorz; Järviluoma, Annika; Moore, Henna M; Tojkander, Sari; Vartia, Salla; Biberfeld, Peter; Laiho, Marikki; Ojala, Päivi M

    2010-03-19

    Nucleophosmin (NPM) is a multifunctional nuclear phosphoprotein and a histone chaperone implicated in chromatin organization and transcription control. Oncogenic Kaposi's sarcoma herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman disease (MCD). In the infected host cell KSHV displays two modes of infection, the latency and productive viral replication phases, involving extensive viral DNA replication and gene expression. A sustained balance between latency and reactivation to the productive infection state is essential for viral persistence and KSHV pathogenesis. Our study demonstrates that the KSHV v-cyclin and cellular CDK6 kinase phosphorylate NPM on threonine 199 (Thr199) in de novo and naturally KSHV-infected cells and that NPM is phosphorylated to the same site in primary KS tumors. Furthermore, v-cyclin-mediated phosphorylation of NPM engages the interaction between NPM and the latency-associated nuclear antigen LANA, a KSHV-encoded repressor of viral lytic replication. Strikingly, depletion of NPM in PEL cells leads to viral reactivation, and production of new infectious virus particles. Moreover, the phosphorylation of NPM negatively correlates with the level of spontaneous viral reactivation in PEL cells. This work demonstrates that NPM is a critical regulator of KSHV latency via functional interactions with v-cyclin and LANA.

  3. In silico 3D structure modeling and inhibitor binding studies of human male germ cell-associated kinase.

    PubMed

    Tanneeru, Karunakar; Balla, Ashok Raja; Guruprasad, Lalitha

    2015-01-01

    Human male germ cell-associated kinase (hMAK) is an androgen-inducible gene in prostate epithelial cells, and it acts as a coactivator of androgen receptor signaling in prostate cancer. The 3D structure of the hMAK kinase was modeled based on the crystal structure of CDK2 kinase using comparative modeling methods, and the ATP-binding site was characterized. We have collected five inhibitors of hMAK from the literature and docked into the ATP-binding site of the kinase domain. Solvated interaction energies (SIE) of inhibitor binding are calculated from the molecular dynamics simulations trajectories of protein-inhibitor complexes. The contribution from each active site residue in hMAK toward inhibitor binding revealed the nature and extent of interactions between inhibitors and individual residues. The main chain atoms of Met79 invariably form hydrogen bonds with all five inhibitors. The amino acids Leu7, Val15, and Leu129 stabilize the inhibitors via CH-pi interactions. The Asp140 in the active site and Glu77 in hinge region show characteristic hydrogen bonding interactions with inhibitors. From SIE, the residue-wise interactions revealed the nature of non-bonding contacts and modifications required to increase the inhibitor activity. Our work provides 3D model structure of hMAK and molecular basis for the mechanisms of hMAK inhibition at atomic level that aid in designing new potent inhibitors.

  4. Cell death in leukemia: passenger protein regulation by topoisomerase inhibitors.

    PubMed

    Jahnke, Ulrike; Higginbottom, Karen; Newland, Adrian C; Cotter, Finbarr E; Allen, Paul D

    2007-10-05

    Etoposide is a potent inducer of mitotic catastrophe; a type of cell death resulting from aberrant mitosis. It is important in p53 negative cells where p53 dependent apoptosis and events at the G1 and G2 cell cycle checkpoints are compromised. Passenger proteins regulate many aspects of mitosis and siRNA interference or direct inhibition of Aurora B kinase results in mitotic catastrophe. However, there is little available data of clinical relevance in leukaemia models. Here, in p53 negative K562 myeloid leukemia cells, etoposide-induced mitotic catastrophe is shown to be time and/or concentration dependent. Survivin and Aurora remained bound to chromosomes. Survivin and Aurora were also associated with Cdk1 and were shown to form complexes, which in pull down experiments, included INCENP. There was no evidence of Aurora B kinase suppression. These data suggests etoposide will complement Aurora B kinase inhibitors currently in clinical trials for cancer.

  5. SKLB70326, a novel small-molecule inhibitor of cell-cycle progression, induces G{sub 0}/G{sub 1} phase arrest and apoptosis in human hepatic carcinoma cells

    SciTech Connect

    Han, Yuanyuan; He, Haiyun; Peng, Feng; Liu, Jiyan; Dai, Xiaoyun; Lin, Hongjun; Xu, Youzhi; Zhou, Tian; Mao, Yongqiu; Xie, Gang; Yang, Shengyong; Yu, Luoting; Yang, Li; Zhao, Yinglan

    2012-05-18

    Highlights: Black-Right-Pointing-Pointer SKLB70326 is a novel compound and has activity of anti-HCC. Black-Right-Pointing-Pointer SKLB70326 induces cell cycle arrest and apoptosis in HepG2 cells. Black-Right-Pointing-Pointer SKLB70326 induces G{sub 0}/G{sub 1} phase arrest via inhibiting the activity of CDK2, CDK4 and CDK6. Black-Right-Pointing-Pointer SKLB70326 induces apoptosis through the intrinsic pathway. -- Abstract: We previously reported the potential of a novel small molecule 3-amino-6-(3-methoxyphenyl)thieno[2.3-b]pyridine-2-carboxamide (SKLB70326) as an anticancer agent. In the present study, we investigated the anticancer effects and possible mechanisms of SKLB70326 in vitro. We found that SKLB70326 treatment significantly inhibited human hepatic carcinoma cell proliferation in vitro, and the HepG2 cell line was the most sensitive to its treatment. The inhibition of cell proliferation correlated with G{sub 0}/G{sub 1} phase arrest, which was followed by apoptotic cell death. The SKLB70326-mediated cell-cycle arrest was associated with the downregulation of cyclin-dependent kinase (CDK) 2, CDK4 and CDK6 but not cyclin D1 or cyclin E. The phosphorylation of the retinoblastoma protein (Rb) was also observed. SKLB70326 treatment induced apoptotic cell death via the activation of PARP, caspase-3, caspase-9 and Bax as well as the downregulation of Bcl-2. The expression levels of p53 and p21 were also induced by SKLB70326 treatment. Moreover, SKLB70326 treatment was well tolerated. In conclusion, SKLB70326, a novel cell-cycle inhibitor, notably inhibits HepG2 cell proliferation through the induction of G{sub 0}/G{sub 1} phase arrest and subsequent apoptosis. Its potential as a candidate anticancer agent warrants further investigation.

  6. FGF inhibits the activity of the cyclin B1/CDK1 kinase to induce a transient G₂arrest in RCS chondrocytes.

    PubMed

    Tran, Tri; Kolupaeva, Victoria; Basilico, Claudio

    2010-11-01

    Fibroblast growth factors (FGFs) negatively regulate long bone development by inhibiting the proliferation of chondrocytes that accumulate in the G₁ phase of the cycle following FGF treatment. Here we report that FGF also causes a striking but transient delay in mitotic entry in RCS chondrocytes by inactivating the cyclin B1-associated CDK1(CDC2) kinase. As a consequence of this inactivation, cells accumulate in the G₂ phase of the cycle for the first 4-6 hours of the treatment. Cyclin B1/CDK1 activity is then restored and cells reach a G₁ arrest. The reduced cyclin B1/CDK1 activity was accompanied by increased CDK1 inhibitory phosphorylation, likely caused by increased activity and expression of the Myt1 kinase. FGF1 also caused dephosphorylation of the CDC25C phosphatase, that however appears due the inactivation of cyclin B1/CDK1 complex in the CDK1 feedback loop, and not the activation of specific phosphatases. the inactivation of the cyclin B1/CDK1 complex is a direct effect of FGF signaling, and not a consequence of the G₂ arrest as it can be observed also in cells blocked at mitosis by Nocodazole. The Chk1 and AtM/ATR kinase are known to play essential roles in the G₂ checkpoint induced by DNA damage/genotoxic stress, but inhibition of Chk1 or ATM/ATR not only did not prevent, but rather potentiated the FGF-induced G₂ arrest. Additionally our results indicate that the transient G₂ arrest is induced by FGF in RCS cell through mechanisms that are independent of the G₁ arrest, and that the G₂ block is not strictly required for the sustained G₁ arrest but may provide a pausing mechanism that allows the FGF response to be fully established.

  7. Cdk5 activator-binding protein C53 regulates apoptosis induced by genotoxic stress via modulating the G2/M DNA damage checkpoint.

    PubMed

    Jiang, Hai; Luo, Shouqing; Li, Honglin

    2005-05-27

    In response to DNA damage, the cellular decision of life versus death involves an intricate network of multiple factors that play critical roles in regulation of DNA repair, cell cycle, and cell death. DNA damage checkpoint proteins are crucial for maintaining DNA integrity and normal cellular functions, but they may also reduce the effectiveness of cancer treatment. Here we report the involvement of Cdk5 activator p35-binding protein C53 in regulation of apoptosis induced by genotoxic stress through modulating Cdk1-cyclin B1 function. C53 was originally identified as a Cdk5 activator p35-binding protein and a caspase substrate. Importantly, our results demonstrated that C53 deficiency conferred partial resistance to genotoxic agents such as etoposide and x-ray irradiation, whereas ectopic expression of C53 rendered cells susceptible to multiple genotoxins that usually trigger G(2)/M arrest. Furthermore, we found that Cdk1 activity was required for etoposide-induced apoptosis of HeLa cells. Overexpression of C53 promoted Cdk1 activity and nuclear accumulation of cyclin B1, whereas C53 deficiency led to more cytoplasmic retention of cyclin B1, suggesting that C53 acts as a pivotal player in modulating the G(2)/M DNA damage checkpoint. Finally, C53 and cyclin B1 co-localize and associate in vivo, indicating a direct role of C53 in regulating the Cdk1-cyclin B1 complex. Taken together, our results strongly indicate that in response to genotoxic stress, C53 serves as an important regulatory component of the G(2)/M DNA damage checkpoint. By overriding the G(2)/M checkpoint-mediated inhibition of Cdk1-cyclin B1 function, ectopic expression of C53 may represent a novel approach for chemo- and radio-sensitization of cancer cells.

  8. Inhibitory phosphorylation of Cdk1 mediates prolonged prophase I arrest in female germ cells and is essential for female reproductive lifespan

    PubMed Central

    Adhikari, Deepak; Busayavalasa, Kiran; Zhang, Jingjing; Hu, Mengwen; Risal, Sanjiv; Bayazit, Mustafa Bilal; Singh, Meenakshi; Diril, M Kasim; Kaldis, Philipp; Liu, Kui

    2016-01-01

    A unique feature of female germ cell development in mammals is their remarkably long arrest at the prophase of meiosis I, which lasts up to 50 years in humans. Both dormant and growing oocytes are arrested at prophase I and completely lack the ability to resume meiosis. Here, we show that the prolonged meiotic arrest of female germ cells is largely achieved via the inhibitory phosphorylation of Cdk1 (cyclin-dependent kinase 1). In two mouse models where we have introduced mutant Cdk1T14AY15F which cannot be inhibited by phosphorylation (Cdk1AF) in small meiotically incompetent oocytes, the prophase I arrest is interrupted, leading to a premature loss of female germ cells. We show that in growing oocytes, Cdk1AF leads to premature resumption of meiosis with condensed chromosomes and germinal vesicle breakdown followed by oocyte death, whereas in dormant oocytes, Cdk1AF leads to oocyte death directly, and both situations damage the ovarian reserve that maintains the female reproductive lifespan, which should be around 1 year in mice. Furthermore, interruption of the inhibitory phosphorylation of Cdk1 results in DNA damage, which is accompanied by induction of the Chk2 (checkpoint kinase 2)-p53/p63-dependent cell death pathway, which eventually causes global oocyte death. Together, our data demonstrate that the phosphorylation-mediated suppression of Cdk1 activity is one of the crucial factors that maintain the lengthy prophase arrest in mammalian female germ cells, which is essential for preserving the germ cell pool and reproductive lifespan in female mammals. PMID:27767095

  9. Cdk5 is required for multipolar-to-bipolar transition during radial neuronal migration and proper dendrite development of pyramidal neurons in the cerebral cortex.

    PubMed

    Ohshima, Toshio; Hirasawa, Motoyuki; Tabata, Hidenori; Mutoh, Tetsuji; Adachi, Tomoko; Suzuki, Hiromi; Saruta, Keiko; Iwasato, Takuji; Itohara, Shigeyoshi; Hashimoto, Mistuhiro; Nakajima, Kazunori; Ogawa, Masaharu; Kulkarni, Ashok B; Mikoshiba, Katsuhiko

    2007-06-01

    The mammalian cerebral cortex consists of six layers that are generated via coordinated neuronal migration during the embryonic period. Recent studies identified specific phases of radial migration of cortical neurons. After the final division, neurons transform from a multipolar to a bipolar shape within the subventricular zone-intermediate zone (SVZ-IZ) and then migrate along radial glial fibres. Mice lacking Cdk5 exhibit abnormal corticogenesis owing to neuronal migration defects. When we introduced GFP into migrating neurons at E14.5 by in utero electroporation, we observed migrating neurons in wild-type but not in Cdk5(-/-) embryos after 3-4 days. Introduction of the dominant-negative form of Cdk5 into the wild-type migrating neurons confirmed specific impairment of the multipolar-to-bipolar transition within the SVZ-IZ in a cell-autonomous manner. Cortex-specific Cdk5 conditional knockout mice showed inverted layering of the cerebral cortex and the layer V and callosal neurons, but not layer VI neurons, had severely impaired dendritic morphology. The amount of the dendritic protein Map2 was decreased in the cerebral cortex of Cdk5-deficient mice, and the axonal trajectory of cortical neurons within the cortex was also abnormal. These results indicate that Cdk5 is required for proper multipolar-to-bipolar transition, and a deficiency of Cdk5 results in abnormal morphology of pyramidal neurons. In addition, proper radial neuronal migration generates an inside-out pattern of cerebral cortex formation and normal axonal trajectories of cortical pyramidal neurons.

  10. GADD45α and γ interaction with CDK11p58 regulates SPDEF protein stability and SPDEF-mediated effects on cancer cell migration.

    PubMed

    Tamura, Rodrigo E; Paccez, Juliano D; Duncan, Kristal C; Morale, Mirian G; Simabuco, Fernando M; Dillon, Simon; Correa, Ricardo G; Gu, Xuesong; Libermann, Towia A; Zerbini, Luiz F

    2016-03-22

    The epithelium-specific Ets transcription factor, SPDEF, plays a critical role in metastasis of prostate and breast cancer cells. While enhanced SPDEF expression blocks migration and invasion, knockdown of SPDEF expression enhances migration, invasion, and metastasis of cancer cells. SPDEF expression and activation is tightly regulated in cancer cells; however, the precise mechanism of SPDEF regulation has not been explored in detail. In this study we provide evidence that the cell cycle kinase CDK11p58, a protein involved in G2/M transition and degradation of several transcription factors, directly interacts with and phosphorylates SPDEF on serine residues, leading to subsequent ubiquitination and degradation of SPDEF through the proteasome pathway. As a consequence of CDK11p58 mediated degradation of SPDEF, this loss