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1
Matsuoka, M., J. Y. Kato, R. P. Fisher, D. O. Morgan, and C. J. Sherr. "Activation of cyclin-dependent kinase 4 (cdk4) by mouse MO15-associated kinase." Molecular and Cellular Biology 14, no. 11 (November 1994): 7265–75. http://dx.doi.org/10.1128/mcb.14.11.7265.
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The assembly of functional holoenzymes composed of regulatory D-type cyclins and cyclin-dependent kinases (cdks) is rate limiting for progression through the G1 phase of the mammalian somatic cell cycle. Complexes between D-type cyclins and their major catalytic subunit, cdk4, are catalytically inactive until cyclin-bound cdk4 undergoes phosphorylation on a single threonyl residue (Thr-172). This step is catalyzed by a cdk-activating kinase (CAK) functionally analogous to the enzyme which phosphorylates cdc2 and cdk2 at Thr-161/160. Here, we demonstrate that the catalytic subunit of mouse cdc2/cdk2 CAK (a 39-kDa protein designated p39MO15) can assemble with a regulatory protein present in either insect or mammalian cells to generate a CAK activity capable of phosphorylating and enzymatically activating both cdk2 and cdk4 in complexes with their respective cyclin partners. A newly identified 37-kDa cyclin-like protein (cyclin H [R. P. Fisher and D. O. Morgan, Cell 78:713-724, 1994]) can assemble with p39MO15 to activate both cyclin A-cdk2 and cyclin D-cdk4 in vitro, implying that CAK is structurally reminiscent of cyclin-cdk complexes themselves. Antisera produced to the p39MO15 subunit can completely deplete mammalian cell lysates of CAK activity for both cyclin A-cdk2 and cyclin D-cdk4, with recovery of activity in the resulting immune complexes. By using an immune complex CAK assay, CAK activity for cyclin A-cdk2 and cyclin D-cdk4 was detected both in quiescent cells and invariantly throughout the cell cycle. Therefore, although it is essential for the enzymatic activation of cyclin-cdk complexes, CAK appears to be neither rate limiting for the emergence of cells from quiescence nor subject to upstream regulatory control by stimulatory mitogens.
2
Matsuoka, M., J. Y. Kato, R. P. Fisher, D. O. Morgan, and C. J. Sherr. "Activation of cyclin-dependent kinase 4 (cdk4) by mouse MO15-associated kinase." Molecular and Cellular Biology 14, no. 11 (November 1994): 7265–75. http://dx.doi.org/10.1128/mcb.14.11.7265-7275.1994.
Full textAbstract:
The assembly of functional holoenzymes composed of regulatory D-type cyclins and cyclin-dependent kinases (cdks) is rate limiting for progression through the G1 phase of the mammalian somatic cell cycle. Complexes between D-type cyclins and their major catalytic subunit, cdk4, are catalytically inactive until cyclin-bound cdk4 undergoes phosphorylation on a single threonyl residue (Thr-172). This step is catalyzed by a cdk-activating kinase (CAK) functionally analogous to the enzyme which phosphorylates cdc2 and cdk2 at Thr-161/160. Here, we demonstrate that the catalytic subunit of mouse cdc2/cdk2 CAK (a 39-kDa protein designated p39MO15) can assemble with a regulatory protein present in either insect or mammalian cells to generate a CAK activity capable of phosphorylating and enzymatically activating both cdk2 and cdk4 in complexes with their respective cyclin partners. A newly identified 37-kDa cyclin-like protein (cyclin H [R. P. Fisher and D. O. Morgan, Cell 78:713-724, 1994]) can assemble with p39MO15 to activate both cyclin A-cdk2 and cyclin D-cdk4 in vitro, implying that CAK is structurally reminiscent of cyclin-cdk complexes themselves. Antisera produced to the p39MO15 subunit can completely deplete mammalian cell lysates of CAK activity for both cyclin A-cdk2 and cyclin D-cdk4, with recovery of activity in the resulting immune complexes. By using an immune complex CAK assay, CAK activity for cyclin A-cdk2 and cyclin D-cdk4 was detected both in quiescent cells and invariantly throughout the cell cycle. Therefore, although it is essential for the enzymatic activation of cyclin-cdk complexes, CAK appears to be neither rate limiting for the emergence of cells from quiescence nor subject to upstream regulatory control by stimulatory mitogens.
3
Harper, J. W., S. J. Elledge, K. Keyomarsi, B. Dynlacht, L. H. Tsai, P. Zhang, S. Dobrowolski, C. Bai, L. Connell-Crowley, and E. Swindell. "Inhibition of cyclin-dependent kinases by p21." Molecular Biology of the Cell 6, no. 4 (April 1995): 387–400. http://dx.doi.org/10.1091/mbc.6.4.387.
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p21Cip1 is a cyclin-dependent kinase (Cdk) inhibitor that is transcriptionally activated by p53 in response to DNA damage. We have explored the interaction of p21 with the currently known Cdks. p21 effectively inhibits Cdk2, Cdk3, Cdk4, and Cdk6 kinases (Ki 0.5-15 nM) but is much less effective toward Cdc2/cyclin B (Ki approximately 400 nM) and Cdk5/p35 (Ki > 2 microM), and does not associate with Cdk7/cyclin H. Overexpression of P21 arrests cells in G1. Thus, p21 is not a universal inhibitor of Cdks but displays selectivity for G1/S Cdk/cyclin complexes. Association of p21 with Cdks is greatly enhanced by cyclin binding. This property is shared by the structurally related inhibitor p27, suggesting a common biochemical mechanism for inhibition. With respect to Cdk2 and Cdk4 complexes, p27 shares the inhibitory potency of p21 but has slightly different kinase specificities. In normal diploid fibroblasts, the vast majority of active Cdk2 is associated with p21, but this active kinase can be fully inhibited by addition of exogenous p21. Reconstruction experiments using purified components indicate that multiple molecules of p21 can associate with Cdk/cyclin complexes and inactive complexes contain more than one molecule of p21. Together, these data suggest a model whereby p21 functions as an inhibitory buffer whose levels determine the threshold kinase activity required for cell cycle progression.
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Musgrove, Elizabeth A., Alexander Swarbrick, Christine S. L. Lee, Ann L. Cornish, and Robert L. Sutherland. "Mechanisms of Cyclin-Dependent Kinase Inactivation by Progestins." Molecular and Cellular Biology 18, no. 4 (April 1, 1998): 1812–25. http://dx.doi.org/10.1128/mcb.18.4.1812.
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ABSTRACT The steroid hormone progesterone regulates proliferation and differentiation in the mammary gland and uterus by cell cycle phase-specific actions. In breast cancer cells the predominant effect of synthetic progestins is long-term growth inhibition and arrest in G1 phase. Progestin-mediated growth arrest of T-47D breast cancer cells was preceded by inhibition of cyclin D1-Cdk4, cyclin D3-Cdk4, and cyclin E-Cdk2 kinase activities in vitro and reduced phosphorylation of pRB and p107. This was accompanied by decreases in the expression of cyclins D1, D3, and E, decreased abundance of cyclin D1- and cyclin D3-Cdk4 complexes, increased association of the cyclin-dependent kinase (CDK) inhibitor p27 with the remaining Cdk4 complexes, and changes in the molecular masses and compositions of cyclin E complexes. In control cells cyclin E eluted from Superdex 200 as two peaks of ∼120 and ∼200 kDa, with the 120-kDa peak displaying greater cyclin E-associated kinase activity. Following progestin treatment, almost all of the cyclin E was in the 200-kDa, low-activity form, which was associated with the CDK inhibitors p21 and p27; this change preceded the inhibition of cell cycle progression. These data suggest preferential formation of this higher-molecular-weight, CDK inhibitor-bound form and a reduced number of cyclin E-Cdk2 complexes as mechanisms for the decreased cyclin E-associated kinase activity following progestin treatment. Ectopic expression of cyclin D1 in progestin-inhibited cells led to the reappearance of the 120-kDa active form of cyclin E-Cdk2 preceding the resumption of cell cycle progression. Thus, decreased cyclin expression and consequent increased CDK inhibitor association are likely to mediate the decreases in CDK activity accompanying progestin-mediated growth inhibition.
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Endicott, Jane A., and Martin E. M. Noble. "Structural characterization of the cyclin-dependent protein kinase family." Biochemical Society Transactions 41, no. 4 (July 18, 2013): 1008–16. http://dx.doi.org/10.1042/bst20130097.
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Structural studies of members of the CDK (cyclin-dependent protein kinase) family have made a significant contribution to our understanding of the regulation of protein kinases. The structure of monomeric unphosphorylated CDK2 was the first of an inactive protein kinase to be determined and, since then, structures of other members of the CDK family, alone, in complex with regulatory proteins and in differing phosphorylation states, have enhanced our understanding of the molecular mechanisms regulating protein kinase activity. Recently, our knowledge of the structural biology of the CDK family has been extended by determination of structures for members of the transcriptional CDK and CDK-like kinase branches of the extended family. We include these recent structures in the present review and consider them in the light of current models for CDK activation and regulation.
6
DINARINA, Ana, Laurent H. PEREZ, Amparo DAVILA, Markus SCHWAB, Tim HUNT, and Angel R. NEBREDA. "Characterization of a new family of cyclin-dependent kinase activators." Biochemical Journal 386, no. 2 (February 22, 2005): 349–55. http://dx.doi.org/10.1042/bj20041779.
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Progression through the cell cycle is regulated by CDKs (cyclin-dependent kinases), which associate with activating partners, named cyclins, to efficiently phosphorylate substrates. We previously reported the identification of RINGO, a Xenopus protein that can activate CDK1 and CDK2 despite lack of sequence similarity to cyclins, which plays a role in the regulation of the meiotic cell cycle in oocytes. In the present study we report the characterization of four mammalian RINGO proteins, which are 53–68% identical with Xenopus RINGO in a central core of about 75 residues. We show that all RINGO family members can bind to and activate CDK1 and CDK2, albeit with different efficiencies, but they do not bind to CDK4 or CDK6. The core RINGO sequences are critical for CDK activation. We also identified key residues in CDK2 that are required for RINGO binding. All RINGO proteins can also bind the CDK inhibitor p27Kip1, but with an inverse efficiency of their ability to bind to CDK1. Our results identify a new family of mammalian proteins that can activate CDKs and therefore potentially function as cell cycle regulators. The ability of RINGO proteins to activate CDK1 and CDK2 suggest also cyclin-independent roles for these kinases.
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Shapiro, Geoffrey I. "Cyclin-Dependent Kinase Pathways As Targets for Cancer Treatment." Journal of Clinical Oncology 24, no. 11 (April 20, 2006): 1770–83. http://dx.doi.org/10.1200/jco.2005.03.7689.
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Cyclin-dependent kinases (cdks) are critical regulators of cell cycle progression and RNA transcription. A variety of genetic and epigenetic events cause universal overactivity of the cell cycle cdks in human cancer, and their inhibition can lead to both cell cycle arrest and apoptosis. However, built-in redundancy may limit the effects of highly selective cdk inhibition. Cdk4/6 inhibition has been shown to induce potent G1 arrest in vitro and tumor regression in vivo; cdk2/1 inhibition has the most potent effects during the S and G2 phases and induces E2F transcription factor–dependent cell death. Modulation of cdk2 and cdk1 activities also affects survival checkpoint responses after exposure to DNA-damaging and microtubule-stabilizing agents. The transcriptional cdks phosphorylate the carboxy-terminal domain of RNA polymerase II, facilitating efficient transcriptional initiation and elongation. Inhibition of these cdks primarily affects the accumulation of transcripts with short half-lives, including those encoding antiapoptosis family members, cell cycle regulators, as well as p53 and nuclear factor-kappa B–responsive gene targets. These effects may account for apoptosis induced by cdk9 inhibitors, especially in malignant hematopoietic cells, and may also potentiate cytotoxicity mediated by disruption of a variety of pathways in many transformed cell types. Current work is focusing on overcoming pharmacokinetic barriers that hindered development of flavopiridol, a pan-cdk inhibitor, as well as assessing novel classes of compounds potently targeting groups of cell cycle cdks (cdk4/6 or cdk2/1) with variable effects on the transcriptional cdks 7 and 9. These efforts will establish whether the strategy of cdk inhibition is able to produce therapeutic benefit in the majority of human tumors.
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Chen, J., P. Saha, S. Kornbluth, B. D. Dynlacht, and A. Dutta. "Cyclin-binding motifs are essential for the function of p21CIP1." Molecular and Cellular Biology 16, no. 9 (September 1996): 4673–82. http://dx.doi.org/10.1128/mcb.16.9.4673.
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The cyclin-dependent kinase (Cdk) inhibitor p21 is induced by the tumor suppressor p53 and is required for the G1-S block in cells with DNA damage. We report that there are two copies of a cyclin-binding motif in p21, Cy1 and Cy2, which interact with the cyclins independently of Cdk2. The cyclin-binding motifs of p21 are required for optimum inhibition of cyclin-Cdk kinases in vitro and for growth suppression in vivo. Peptides containing only the Cy1 or Cy2 motif partially inhibit cyclin-Cdk kinase activity in vitro and DNA replication in Xenopus egg extracts. A monoclonal antibody which recognizes the Cy1 site of p21 specifically disrupts the association of p21 with cyclin E-Cdk2 and with cyclin D1-Cdk4 in cell extracts. Taken together, these observations suggest that the cyclin-binding motif of p21 is important for kinase inhibition and for formation of p21-cyclin-Cdk complexes in the cell. Finally, we show that the cyclin-Cdk complex is partially active if associated with only the cyclin-binding motif of p21, providing an explanation for how p21 is found associated with active cyclin-Cdk complexes in vivo. The Cy sequences may be general motifs used by Cdk inhibitors or substrates to interact with the cyclin in a cyclin-Cdk complex.
9
Gitig, Diana M., and Andrew Koff. "Cdk Pathway: Cyclin-Dependent Kinases and Cyclin-Dependent Kinase Inhibitors." Molecular Biotechnology 19, no. 2 (2001): 179–88. http://dx.doi.org/10.1385/mb:19:2:179.
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Susanti, Ni Made Pitri, and Daryono Hadi Tjahjono. "Cyclin-Dependent Kinase 4 and 6 Inhibitors in Cell Cycle Dysregulation for Breast Cancer Treatment." Molecules 26, no. 15 (July 24, 2021): 4462. http://dx.doi.org/10.3390/molecules26154462.
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In cell development, the cell cycle is crucial, and the cycle progression’s main controllers are endogenous CDK inhibitors, cyclin-dependent kinases (CDKs), and cyclins. In response to the mitogenic signal, cyclin D is produced and retinoblastoma protein (Rb) is phosphorylated due to activated CDK4/CDK6. This causes various proteins required in the cell cycle progression to be generated. In addition, complexes of CDK1-cyclin A/B, CDK2-cyclin E/A, and CDK4/CDK6-cyclin D are required in each phase of this progression. Cell cycle dysregulation has the ability to lead to cancer. Based on its role in the cell cycle, CDK has become a natural target of anticancer therapy. Therefore, understanding the CDK structures and the complex formed with the drug, helps to foster the development of CDK inhibitors. This development starts from non-selective CDK inhibitors to selective CDK4/CDK6 inhibitors, and these have been applied in clinical cancer treatment. However, these inhibitors currently require further development for various hematologic malignancies and solid tumors, based on the results demonstrated. In drug development, the main strategy is primarily to prevent and asphyxiate drug resistance, thus a determination of specific biomarkers is required to increase the therapy’s effectiveness as well as patient selection suitability in order to avoid therapy failure. This review is expected to serve as a reference for early and advanced-stage researchers in designing new molecules or repurposing existing molecules as CDK4/CDK6 inhibitors to treat breast cancer.
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Latham, K. M., S. W. Eastman, A. Wong, and P. W. Hinds. "Inhibition of p53-mediated growth arrest by overexpression of cyclin-dependent kinases." Molecular and Cellular Biology 16, no. 8 (August 1996): 4445–55. http://dx.doi.org/10.1128/mcb.16.8.4445.
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Rat fibroblasts transformed by a temperature-sensitive mutant of murine p53 undergo a reversible growth arrest in G1 at 32.5 degrees C, the temperature at which p53 adopts a wild-type conformation. The arrested cells contain inactive cyclin-dependent kinase 2 (cdk2) despite the presence of high levels of cyclin E and cdk-activating kinase activity. This is due in part to p53-dependent expression of the p2l cdk inhibitor. Upon shift to 39 degrees C, wild-type p53 is lost and cdk2 activation and pRb phosphorylation occur concomitantly with loss of p2l. This p53-mediated growth arrest can be abrogated by overexpression of cdk4 and cdk6 but not cdk2 or cyclins, leading to continuous proliferation of transfected cells in the presence of wild-type p53 and p2l. Kinase-inactive counterparts of cdk4 and cdk6 also rescue these cells from growth arrest, implicating a noncatalytic role for cdk4 and cdk6 in this resistance to p53-mediated growth arrest. Aberrant expression of these cell cycle kinases may thus result in an oncogenic interference with inhibitors of cell cycle progression.
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Diehl, J. A., and C. J. Sherr. "A dominant-negative cyclin D1 mutant prevents nuclear import of cyclin-dependent kinase 4 (CDK4) and its phosphorylation by CDK-activating kinase." Molecular and Cellular Biology 17, no. 12 (December 1997): 7362–74. http://dx.doi.org/10.1128/mcb.17.12.7362.
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Cyclins contain two characteristic cyclin folds, each consisting of five alpha-helical bundles, which are connected to one another by a short linker peptide. The first repeat makes direct contact with cyclin-dependent kinase (CDK) subunits in assembled holoenzyme complexes, whereas the second does not contribute directly to the CDK interface. Although threonine 156 in mouse cyclin D1 is predicted to lie at the carboxyl terminus of the linker peptide that separates the two cyclin folds and is buried within the cyclin subunit, mutation of this residue to alanine has profound effects on the behavior of the derived cyclin D1-CDK4 complexes. CDK4 in complexes with mutant cyclin D1 (T156A or T156E but not T156S) is not phosphorylated by recombinant CDK-activating kinase (CAK) in vitro, fails to undergo activating T-loop phosphorylation in vivo, and remains catalytically inactive and unable to phosphorylate the retinoblastoma protein. Moreover, when it is ectopically overexpressed in mammalian cells, cyclin D1 (T156A) assembles with CDK4 in the cytoplasm but is not imported into the cell nucleus. CAK phosphorylation is not required for nuclear transport of cyclin D1-CDK4 complexes, because complexes containing wild-type cyclin D1 and a CDK4 (T172A) mutant lacking the CAK phosphorylation site are efficiently imported. In contrast, enforced overexpression of the CDK inhibitor p21Cip1 together with mutant cyclin D1 (T156A)-CDK4 complexes enhanced their nuclear localization. These results suggest that cyclin D1 (T156A or T156E) forms abortive complexes with CDK4 that prevent recognition by CAK and by other cellular factors that are required for their nuclear localization. These properties enable ectopically overexpressed cyclin D1 (T156A), or a more stable T156A/T286A double mutant that is resistant to ubiquitination, to compete with endogenous cyclin D1 in mammalian cells, thereby mobilizing CDK4 into cytoplasmic, catalytically inactive complexes and dominantly inhibiting the ability of transfected NIH 3T3 fibroblasts to enter S phase.
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Albrecht, Jeffrey H., Brenda M. Rieland, Christopher J. Nelsen, and Cory L. Ahonen. "Regulation of G1 cyclin-dependent kinases in the liver: role of nuclear localization and p27 sequestration." American Journal of Physiology-Gastrointestinal and Liver Physiology 277, no. 6 (December 1, 1999): G1207—G1216. http://dx.doi.org/10.1152/ajpgi.1999.277.6.g1207.
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Recent studies suggest that cyclin D1 mediates progression of hepatocytes through G1 phase of the cell cycle. The present study further examines the regulation of cyclin D1-dependent kinase activity and the interplay between cyclin D1 and other G1phase regulatory proteins during liver regeneration. After 70% partial hepatectomy in rats, there was upregulation of kinase activity associated with cyclins (A, D1, D3, and E), cyclin-dependent kinases (Cdk2 and Cdk4), and Cdk-inhibitory proteins (p27, p107, and p130). Although cyclin D1/Cdk4 complexes were more abundant in the cytoplasmic fraction after partial hepatectomy, kinase activity was detected primarily in the nuclear fraction. Cytoplasmic cyclin D1/Cdk4 complexes were activated by recombinant cyclin H/Cdk7. Because endogenous Cdk7 activity was found in the nucleus, this suggests that activation of cyclin D1/Cdk4 requires nuclear importation and subsequent phosphorylation by cyclin H/Cdk7. Recombinant cyclin E/Cdk2 was inhibited by extracts from quiescent liver, and cyclin D1 could titrate out this inhibitory activity. Induction of cyclin D1 was accompanied by increased abundance of cyclin D1/p27 complexes, and most p27 was sequestered by cyclin D1 after partial hepatectomy. Thus cyclin D1 appears to play two roles during G1 phase progression in the regenerating liver: it forms a nuclear kinase complex, and it promotes activation of Cdk2 by sequestering inhibitory proteins such as p27. These experiments underscore the complexity of cyclin/Cdk regulatory networks in the regenerating liver.
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Desai, D., H. C. Wessling, R. P. Fisher, and D. O. Morgan. "Effects of phosphorylation by CAK on cyclin binding by CDC2 and CDK2." Molecular and Cellular Biology 15, no. 1 (January 1995): 345–50. http://dx.doi.org/10.1128/mcb.15.1.345.
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The cyclin-dependent protein kinases (CDKs) are activated by association with cyclins and by phosphorylation at a conserved threonine residue by the CDK-activating kinase (CAK). We have studied the binding of various human CDK and cyclin subunits in vitro, using purified proteins derived from baculovirus-infected insect cells. We find that most CDK-cyclin complexes known to exist in human cells (CDC2-cyclin B, CDK2-cyclin A, and CDK2-cyclin E) form with high affinity in the absence of phosphorylation or other cellular components. One complex (CDC2-cyclin A) forms with high affinity only after CAK-mediated phosphorylation of CDC2 at the activating threonine residue. CDC2 does not bind with high affinity to cyclin E in vitro, even after phosphorylation of the CDC2 subunit. Thus, phosphorylation is of varying importance in the formation of high-affinity CDK-cyclin complexes.
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Adams, P. D., W. R. Sellers, S. K. Sharma, A. D. Wu, C. M. Nalin, and W. G. Kaelin. "Identification of a cyclin-cdk2 recognition motif present in substrates and p21-like cyclin-dependent kinase inhibitors." Molecular and Cellular Biology 16, no. 12 (December 1996): 6623–33. http://dx.doi.org/10.1128/mcb.16.12.6623.
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Understanding how cyclin-cdk complexes recognize their substrates is a central problem in cell cycle biology. We identified an E2F1-derived eight-residue peptide which blocked the binding of cyclin A and E-cdk2 complexes to E2F1 and p21. Short peptides spanning similar sequences in p107, p130, and p21-like cdk inhibitors likewise bound to cyclin A-cdk2 and cyclin E-cdk2. In addition, these peptides promoted formation of stable cyclin A-cdk2 complexes in vitro but inhibited the phosphorylation of the retinoblastoma protein by cyclin A- but not cyclin B-associated kinases. Mutation of the cyclin-cdk2 binding motifs in p107 and E2F1 likewise prevented their phosphorylation by cyclin A-associated kinases in vitro. The cdk inhibitor p21 was found to contain two functional copies of this recognition motif, as determined by in vitro kinase binding/inhibition assays and in vivo growth suppression assays. Thus, these studies have identified a cyclin A- and E-cdk2 substrate recognition motif. Furthermore, these data suggest that p21-like cdk inhibitors function, at least in part, by blocking the interaction of substrates with cyclin-cdk2 complexes.
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Feichtinger, Sabine, Thomas Stamminger, Regina Müller, Laura Graf, Bert Klebl, Jan Eickhoff, and Manfred Marschall. "Recruitment of cyclin-dependent kinase 9 to nuclear compartments during cytomegalovirus late replication: importance of an interaction between viral pUL69 and cyclin T1." Journal of General Virology 92, no. 7 (July 1, 2011): 1519–31. http://dx.doi.org/10.1099/vir.0.030494-0.
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Cyclin-dependent protein kinases (CDKs) are important regulators of cellular processes and are functionally integrated into the replication of human cytomegalovirus (HCMV). Recently, a regulatory impact of CDK activity on the viral mRNA export factor pUL69 was shown. Here, specific aspects of the mode of interaction between CDK9/cyclin T1 and pUL69 are described. Intracellular localization was studied in the presence of a novel selective CDK9 inhibitor, R22, which exerts anti-cytomegaloviral activity in vitro. A pronounced R22-induced formation of nuclear speckled aggregation of pUL69 was demonstrated. Multi-labelling confocal laser-scanning microscopy revealed that CDK9 and cyclin T1 co-localized perfectly with pUL69 in individual speckles. The effects were similar to those described recently for the broad CDK inhibitor roscovitine. Co-immunoprecipitation and yeast two-hybrid analyses showed that cyclin T1 interacted with both CDK9 and pUL69. The interaction region of pUL69 for cyclin T1 could be attributed to aa 269–487. Moreover, another component of CDK inhibitor-induced speckled aggregates was identified with RNA polymerase II, supporting earlier reports that strongly suggested an association of pUL69 with transcription complexes. Interestingly, when using a UL69-deleted recombinant HCMV, no speckled aggregates were formed by CDK inhibitor treatment. This indicated that pUL69 is the defining component of aggregates and generally may represent a crucial viral interactor of cyclin T1. In conclusion, these data emphasize that HCMV inter-regulation with CDK9/cyclin T1 is at least partly based on a pUL69–cylin T1 interaction, thus contributing to the importance of CDK9 for HCMV replication.
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Kranenburg, O., V. Scharnhorst, A. J. Van der Eb, and A. Zantema. "Inhibition of cyclin-dependent kinase activity triggers neuronal differentiation of mouse neuroblastoma cells." Journal of Cell Biology 131, no. 1 (October 1, 1995): 227–34. http://dx.doi.org/10.1083/jcb.131.1.227.
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Studies on the molecular mechanisms underlying neuronal differentiation are frequently performed using cell lines established from neuroblastomas. In this study we have used mouse N1E-115 neuroblastoma cells that undergo neuronal differentiation in response to DMSO. During differentiation, cyclin-dependent kinase (cdk) activities decline and phosphorylation of the retinoblastoma gene product (pRb) is lost, leading to the appearance of a pRb-containing E2F DNA-binding complex. The loss of cdk2 activity is due to a decrease in cdk2 abundance whereas loss of cdk4 activity is caused by strong association with the cdk inhibitor (CKI) p27KIP1 and concurrent loss of cdk4 phosphorylation. Moreover, neuronal differentiation can be induced by overexpression of p27KIP1 or pRb, suggesting that inhibition of cdk activity leading to loss of pRb phosphorylation, is the major determinant for neuronal differentiation.
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Eapen, Alex K., Matthew K. Henry, Dawn E. Quelle, and Frederick W. Quelle. "DNA Damage-Induced G1 Arrest in Hematopoietic Cells Is Overridden following Phosphatidylinositol 3-Kinase-Dependent Activation of Cyclin-Dependent Kinase 2." Molecular and Cellular Biology 21, no. 18 (September 15, 2001): 6113–21. http://dx.doi.org/10.1128/mcb.21.18.6113-6121.2001.
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ABSTRACT Exposure of hematopoietic cells to DNA-damaging agents induces p53-independent cell cycle arrest at a G1 checkpoint. Previously, we have shown that this growth arrest can be overridden by cytokine growth factors, such as erythropoietin or interleukin-3, through activation of a phosphatidylinositol 3-kinase (PI 3-kinase)/Akt-dependent signaling pathway. Here, we show that γ-irradiated murine myeloid 32D cells arrest in G1 with active cyclin D–cyclin-dependent kinase 4 (Cdk4) but with inactive cyclin E-Cdk2 kinases. The arrest was associated with elevated levels of the Cdk inhibitors p21Cip1 and p27Kip1, yet neither was associated with Cdk2. Instead, irradiation-induced inhibition of cyclin E-Cdk2 correlated with absence of the activating threonine-160 phosphorylation on Cdk2. Cytokine treatment of irradiated cells induced Cdk2 phosphorylation and activation, and cells entered into S phase despite sustained high-level expression of p21 and p27. Notably, the PI 3-kinase inhibitor, LY294002, completely blocked cytokine-induced Cdk2 activation and cell growth in irradiated 32D cells but not in nonirradiated cells. Together, these findings demonstrate a novel mechanism underlying the DNA damage-induced G1 arrest of hematopoietic cells, that is, inhibition of Cdk2 phosphorylation and activation. These observations link PI 3-kinase signaling pathways with the regulation of Cdk2 activity.
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Kato, J. Y., M. Matsuoka, D. K. Strom, and C. J. Sherr. "Regulation of cyclin D-dependent kinase 4 (cdk4) by cdk4-activating kinase." Molecular and Cellular Biology 14, no. 4 (April 1994): 2713–21. http://dx.doi.org/10.1128/mcb.14.4.2713.
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The accumulation of assembled holoenzymes composed of regulatory D-type cyclins and their catalytic partner, cyclin-dependent kinase 4 (cdk4), is rate limiting for progression through the G1 phase of the cell cycle in mammalian fibroblasts. Both the synthesis and assembly of D-type cyclins and cdk4 depend upon serum stimulation, but even when both subunits are ectopically overproduced, they do not assemble into complexes in serum-deprived cells. When coexpressed from baculoviral vectors in intact Sf9 insect cells, cdk4 assembles with D-type cyclins to form active protein kinases. In contrast, recombinant D-type cyclin and cdk4 subunits produced in insect cells or in bacteria do not assemble as efficiently into functional holoenzymes when combined in vitro but can be activated in the presence of lysates obtained from proliferating mammalian cells. Assembly of cyclin D-cdk4 complexes in coinfected Sf9 cells facilitates phosphorylation of cdk4 on threonine 172 by a cdk-activating kinase (CAK). Assembly can proceed in the absence of this modification, but cdk4 mutants which cannot be phosphorylated by CAK remain catalytically inactive. Therefore, formation of the cyclin D-cdk4 complex and phosphorylation of the bound catalytic subunit are independently regulated, and in addition to the requirement for CAK activity, serum stimulation is required to promote assembly of the complexes in mammalian cells.
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Kato, J. Y., M. Matsuoka, D. K. Strom, and C. J. Sherr. "Regulation of cyclin D-dependent kinase 4 (cdk4) by cdk4-activating kinase." Molecular and Cellular Biology 14, no. 4 (April 1994): 2713–21. http://dx.doi.org/10.1128/mcb.14.4.2713-2721.1994.
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The accumulation of assembled holoenzymes composed of regulatory D-type cyclins and their catalytic partner, cyclin-dependent kinase 4 (cdk4), is rate limiting for progression through the G1 phase of the cell cycle in mammalian fibroblasts. Both the synthesis and assembly of D-type cyclins and cdk4 depend upon serum stimulation, but even when both subunits are ectopically overproduced, they do not assemble into complexes in serum-deprived cells. When coexpressed from baculoviral vectors in intact Sf9 insect cells, cdk4 assembles with D-type cyclins to form active protein kinases. In contrast, recombinant D-type cyclin and cdk4 subunits produced in insect cells or in bacteria do not assemble as efficiently into functional holoenzymes when combined in vitro but can be activated in the presence of lysates obtained from proliferating mammalian cells. Assembly of cyclin D-cdk4 complexes in coinfected Sf9 cells facilitates phosphorylation of cdk4 on threonine 172 by a cdk-activating kinase (CAK). Assembly can proceed in the absence of this modification, but cdk4 mutants which cannot be phosphorylated by CAK remain catalytically inactive. Therefore, formation of the cyclin D-cdk4 complex and phosphorylation of the bound catalytic subunit are independently regulated, and in addition to the requirement for CAK activity, serum stimulation is required to promote assembly of the complexes in mammalian cells.
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Garrett, Sarah, William A. Barton, Ronald Knights, Pei Jin, David O. Morgan, and Robert P. Fisher. "Reciprocal Activation by Cyclin-Dependent Kinases 2 and 7 Is Directed by Substrate Specificity Determinants outside the T Loop." Molecular and Cellular Biology 21, no. 1 (January 1, 2001): 88–99. http://dx.doi.org/10.1128/mcb.21.1.88-99.2001.
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ABSTRACT Cyclin-dependent kinase 7 (CDK7) is the catalytic subunit of the metazoan CDK-activating kinase (CAK), which activates CDKs, such as CDC2 and CDK2, through phosphorylation of a conserved threonine residue in the T loop. Full activation of CDK7 requires association with a positive regulatory subunit, cyclin H, and phosphorylation of a conserved threonine residue at position 170 in its own T loop. We show that threonine-170 of CDK7 is phosphorylated in vitro by its targets, CDC2 and CDK2, which also phosphorylate serine-164 in the CDK7 T loop, a site that perfectly matches their consensus phosphorylation site. In contrast, neither CDK4 nor CDK7 itself can phosphorylate the CDK7 T loop in vitro. The ability of CDC2 or CDK2 and CDK7 to phosphorylate each other but not themselves implies that each kinase can discriminate among closely related sequences and can recognize a substrate site that diverges from its usual preferred site. To understand the basis for this paradoxical substrate specificity, we constructed a chimeric CDK with the T loop of CDK7 grafted onto the body of CDK2. Surprisingly, the hybrid enzyme, CDK2-7, was efficiently activated in cyclin A-dependent fashion by CDK7 but not at all by CDK2. CDK2-7, moreover, phosphorylated wild-type CDK7 but not CDK2. Our results suggest that the primary amino acid sequence of the T loop plays only a minor role, if any, in determining the specificity of cyclin-dependent CAKs for their CDK substrates and that protein-protein interactions involving sequences outside the T loop can influence substrate specificity both positively and negatively.
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Ezhevsky, Sergei A., Alan Ho, Michelle Becker-Hapak, Penny K. Davis, and Steven F. Dowdy. "Differential Regulation of Retinoblastoma Tumor Suppressor Protein by G1 Cyclin-Dependent Kinase Complexes In Vivo." Molecular and Cellular Biology 21, no. 14 (July 15, 2001): 4773–84. http://dx.doi.org/10.1128/mcb.21.14.4773-4784.2001.
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ABSTRACT The retinoblastoma tumor suppressor protein (pRB) negatively regulates early-G1 cell cycle progression, in part, by sequestering E2F transcription factors and repressing E2F-responsive genes. Although pRB is phosphorylated on up to 16 cyclin-dependent kinase (Cdk) sites by multiple G1 cyclin-Cdk complexes, the active form(s) of pRB in vivo remains unknown. pRB is present as an unphosphorylated protein in G0 quiescent cells and becomes hypophosphorylated (∼2 mol of PO4 to 1 mol of pRB) in early G1 and hyperphosphorylated (∼10 mol of PO4 to 1 mol of pRB) in late G1 phase. Here, we report that hypophosphorylated pRB, present in early G1, represents the biologically active form of pRB in vivo that is assembled with E2Fs and E1A but that both unphosphorylated pRB in G0 and hyperphosphorylated pRB in late G1 fail to become assembled with E2Fs and E1A. Furthermore, using transducible dominant-negative TAT fusion proteins that differentially target cyclin D-Cdk4 or cyclin D-Cdk6 (cyclin D-Cdk4/6) and cyclin E-Cdk2 complexes, namely, TAT-p16 and TAT–dominant-negative Cdk2, respectively, we found that, in vivo, cyclin D-Cdk4/6 complexes hypophosphorylate pRB in early G1 and that cyclin E-Cdk2 complexes inactivate pRB by hyperphosphorylation in late G1. Moreover, we found that cycling human tumor cells expressing deregulated cyclin D-Cdk4/6 complexes, due to deletion of the p16 INK4a gene, contained hypophosphorylated pRB that was bound to E2Fs in early G1and that E2F-responsive genes, including those for dihydrofolate reductase and cyclin E, were transcriptionally repressed. Thus, we conclude that, physiologically, pRB is differentially regulated by G1 cyclin-Cdk complexes.
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Egan, Elizabeth A., and Mark J. Solomon. "Cyclin-Stimulated Binding of Cks Proteins to Cyclin-Dependent Kinases." Molecular and Cellular Biology 18, no. 7 (July 1, 1998): 3659–67. http://dx.doi.org/10.1128/mcb.18.7.3659.
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ABSTRACT Although Cks proteins were the first identified binding partners of cyclin-dependent protein kinases (cdks), their cell cycle functions have remained unclear. To help elucidate the function of Cks proteins, we examined whether their binding to p34 cdc2 (the mitotic cdk) varies during the cell cycle in Xenopusegg extracts. We observed that binding of human CksHs2 to p34 cdc2 was stimulated by cyclin B. This stimulation was dependent on the activating phosphorylation of p34 cdc2 on Thr-161, which follows cyclin binding and is mediated by the cdk-activating kinase. Neither the inhibitory phosphorylations of p34 cdc2 nor the catalytic activity of p34 cdc2 was required for this stimulation. Stimulated binding of CksHs2 to another cdk, p33 cdk2 , required both cyclin A and activating phosphorylation. Our findings support recent models that suggest that Cks proteins target active forms of p34 cdc2 to substrates.
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Haller, Kerstin, Yalin Wu, Elisabeth Derow, Iris Schmitt, Kuan-Teh Jeang, and Ralph Grassmann. "Physical Interaction of Human T-Cell Leukemia Virus Type 1 Tax with Cyclin-Dependent Kinase 4 Stimulates the Phosphorylation of Retinoblastoma Protein." Molecular and Cellular Biology 22, no. 10 (May 15, 2002): 3327–38. http://dx.doi.org/10.1128/mcb.22.10.3327-3338.2002.
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ABSTRACT The Tax oncoprotein of human T-cell leukemia virus type 1 (HTLV-1) induces leukemia in transgenic mice and permanent T-cell growth in vitro. In transformed lymphocytes, it acts as an essential growth factor. Tax stimulates the cell cycle in the G1 phase by activating the cyclin-dependent kinase (CDK) CDK4 and CDK6 holoenzyme complexes. Here we show that Tax directly interacts with CDK4. This binding to CDK4 was specific, since Tax did not bind to either CDK2 or CDK1. The interaction with CDK4/cyclin D complexes was observed in vitro, in transfected fibroblasts, in HTLV-1-infected T cells, and in adult T-cell leukemia-derived cultures. Binding studies with several point and deletion mutants indicated that the N terminus of Tax mediates the interaction with CDK4. The Tax/CDK complex represented an active holoenzyme which capably phosphorylates the Rb protein in vitro and is resistant to repression by the inhibitor p21CIP. Binding-deficient Tax mutants failed to activate CDK4, indicating that direct association with Tax is required for enhanced kinase activity. Tax also increased the association of CDK4 with its positive cyclin regulatory subunit. Thus, protein-protein contact between Tax and the components of the cyclin D/CDK complexes provides a further mechanistic explanation for the mitogenic and immortalizing effects of this HTLV-1 oncoprotein.
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Kaldis, Philipp, Alicia A. Russo, Hubert S. Chou, Nikola P. Pavletich, and Mark J. Solomon. "Human and Yeast Cdk-activating Kinases (CAKs) Display Distinct Substrate Specificities." Molecular Biology of the Cell 9, no. 9 (September 1998): 2545–60. http://dx.doi.org/10.1091/mbc.9.9.2545.
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Cell cycle progression is controlled by the sequential functions of cyclin-dependent kinases (cdks). Cdk activation requires phosphorylation of a key residue (on sites equivalent to Thr-160 in human cdk2) carried out by the cdk-activating kinase (CAK). Human CAK has been identified as a p40MO15/cyclin H/MAT1 complex that also functions as part of transcription factor IIH (TFIIH) where it phosphorylates multiple transcriptional components including the C-terminal domain (CTD) of the large subunit of RNA polymerase II. In contrast, CAK from budding yeast consists of a single polypeptide (Cak1p), is not a component of TFIIH, and lacks CTD kinase activity. Here we report that Cak1p and p40MO15 have strikingly different substrate specificities. Cak1p preferentially phosphorylated monomeric cdks, whereas p40MO15preferentially phosphorylated cdk/cyclin complexes. Furthermore, p40MO15 only phosphorylated cdk6 bound to cyclin D3, whereas Cak1p recognized monomeric cdk6 and cdk6 bound to cyclin D1, D2, or D3. We also found that cdk inhibitors, including p21CIP1, p27KIP1, p57KIP2, p16INK4a, and p18INK4c, could block phosphorylation by p40MO15 but not phosphorylation by Cak1p. Our results demonstrate that although both Cak1p and p40MO15 activate cdks by phosphorylating the same residue, the structural mechanisms underlying the enzyme-substrate recognition differ greatly. Structural and physiological implications of these findings will be discussed.
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Leisenfelder, Stacey A., and Jennifer F. Moffat. "Varicella-Zoster Virus Infection of Human Foreskin Fibroblast Cells Results in Atypical Cyclin Expression and Cyclin-Dependent Kinase Activity." Journal of Virology 80, no. 11 (June 1, 2006): 5577–87. http://dx.doi.org/10.1128/jvi.00163-06.
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ABSTRACT In its course of human infection, varicella-zoster virus (VZV) infects rarely dividing cells such as dermal fibroblasts, differentiated keratinocytes, mature T cells, and neurons, none of which are actively synthesizing DNA; however, VZV is able to productively infect them and use their machinery to replicate the viral genome. We hypothesized that VZV alters the intracellular environment to favor viral replication by dysregulating cell cycle proteins and kinases. Cyclin-dependent kinases (CDKs) and cyclins displayed a highly unusual profile in VZV-infected confluent fibroblasts: total amounts of CDK1, CDK2, cyclin B1, cyclin D3, and cyclin A protein increased, and kinase activities of CDK2, CDK4, and cyclin B1 were strongly and simultaneously induced. Cyclins B1 and D3 increased as early as 24 h after infection, concurrent with VZV protein synthesis. Confocal microscopy indicated that cyclin D3 overexpression was limited to areas of IE62 production, whereas cyclin B1 expression was irregular across the VZV plaque. Downstream substrates of CDKs, including pRb, p107, and GM130, did not show phosphorylation by immunoblotting, and p21 and p27 protein levels were increased following infection. Finally, although the complement of cyclin expression and high CDK activity indicated a progression through the S and G2 phases of the cell cycle, DNA staining and flow cytometry indicated a possible G1/S blockade in infected cells. These data support earlier studies showing that pharmacological CDK inhibitors can inhibit VZV replication in cultured cells.
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Zhang, H., Y. Xiong, and D. Beach. "Proliferating cell nuclear antigen and p21 are components of multiple cell cycle kinase complexes." Molecular Biology of the Cell 4, no. 9 (September 1993): 897–906. http://dx.doi.org/10.1091/mbc.4.9.897.
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We have recently shown that two proteins, proliferating cell nuclear antigen (PCNA) and p21, are associated with cyclin D. Here we show that PCNA and p21 are common components of a wide variety of cyclin/cyclin-dependent kinase complexes in nontransformed cells. These include kinase complexes containing cyclin A, cyclin B, and cyclin D, associated either with CDC2, CDK2, CDK4, or CDK5. We show that PCNA and p21 form separate quaternary complex with each cyclin/CDK and that these quaternary complexes contain a substantial, if not major, fraction of the cell cycle kinases in asynchronously growing cells. These results suggest that PCNA and p21 may perform a common function for all these kinases.
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Nougarède, Romain, Flavio Della Seta, Patrick Zarzov, and Etienne Schwob. "Hierarchy of S-Phase-Promoting Factors: Yeast Dbf4-Cdc7 Kinase Requires Prior S-Phase Cyclin-Dependent Kinase Activation." Molecular and Cellular Biology 20, no. 11 (June 1, 2000): 3795–806. http://dx.doi.org/10.1128/mcb.20.11.3795-3806.2000.
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ABSTRACT In all eukaryotes, the initiation of DNA synthesis requires the formation of prereplicative complexes (pre-RCs) on replication origins, followed by their activation by two S-T protein kinases, an S-phase cyclin-dependent kinase (S-CDK) and a homologue of yeast Dbf4-Cdc7 kinase (Dbf4p-dependent kinase [DDK]). Here, we show that yeast DDK activity is cell cycle regulated, though less tightly than that of the S-CDK Clb5-Cdk1, and peaks during S phase in correlation with Dbf4p levels. Dbf4p is short-lived throughout the cell cycle, but its instability is accentuated during G1 by the anaphase-promoting complex. Downregulating DDK activity is physiologically important, as joint Cdc7p and Dbf4p overexpression is lethal. Because pre-RC formation is a highly ordered process, we asked whether S-CDK and DDK need also to function in a specific order for the firing of origins. We found that both kinases are activated independently, but we show that DDK can perform its function for DNA replication only after S-CDKs have been activated. Cdc45p, a protein needed for initiation, binds tightly to chromatin only after S-CDK activation (L. Zou and B. Stillman, Science 280:593–596, 1998). We show that Cdc45p is phosphorylated by DDK in vitro, suggesting that it might be one of DDK's critical substrates after S-CDK activation. Linking the origin-bound DDK to the tightly regulated S-CDK in a dependent sequence of events may ensure that DNA replication initiates only at the right time and place.
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CHIARIELLO, Mario, Eliana GOMEZ, and J. Silvio GUTKIND. "Regulation of cyclin-dependent kinase (Cdk) 2 Thr-160 phosphorylation and activity by mitogen-activated protein kinase in late G1 phase." Biochemical Journal 349, no. 3 (July 25, 2000): 869–76. http://dx.doi.org/10.1042/bj3490869.
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Mitogen-activated protein (MAP) kinases, p42MAPK and p44MAPK, are central components of growth-promoting signalling pathways. However, how stimulation of MAP kinases culminates in cell-cycle progression is still poorly understood. Here we show that mitogenic stimulation of NIH 3T3 cells causes a sustained activation of MAP kinases, which lasts until cells begin progressing through the G1/S boundary. Furthermore, we observed that disruption of the MAP-kinase pathway with a selective MEK (MAP kinase/extracellular-signal-regulated protein kinase kinase) inhibitor, PD98059, prevents the activation of cyclin-dependent kinase (Cdk) 2 and DNA synthesis, even when added during late G1 phase, once the known mechanisms by which MAP kinase controls G1 progression, accumulation of G1 cyclins and degradation of Cdk inhibitors have already taken place. Moreover, we provide evidence indicating that MAP kinases control Cdk2 Thr-160 activating phosphorylation and function, possibly by regulating the activity of a Cdk-activating kinase, thus promoting the re-initiation of DNA synthesis. These findings suggest the existence of a novel mechanism whereby signal-transducing pathways converging on MAP kinases can affect the cell-cycle machinery and, ultimately, participate in cell-growth control.
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Swarbrick, Alexander, Christine S. L. Lee, Robert L. Sutherland, and Elizabeth A. Musgrove. "Cooperation of p27Kip1 and p18INK4c in Progestin-Mediated Cell Cycle Arrest in T-47D Breast Cancer Cells." Molecular and Cellular Biology 20, no. 7 (April 1, 2000): 2581–91. http://dx.doi.org/10.1128/mcb.20.7.2581-2591.2000.
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ABSTRACT The steroid hormone progesterone regulates proliferation and differentiation in the mammary gland and uterus by cell cycle phase-specific actions. The long-term effect of progestins on T-47D breast cancer cells is inhibition of cellular proliferation. This is accompanied by decreased G1 cyclin-dependent kinase (CDK) activities, redistribution of the CDK inhibitor p27Kip1among these CDK complexes, and alterations in the elution profile of cyclin E-Cdk2 upon gel filtration chromatography, such that high-molecular-weight complexes predominate. This study aimed to determine the relative contribution of CDK inhibitors to these events. Following progestin treatment, the majority of cyclin E- and D-CDK complexes were bound to p27Kip1 and few were bound to p21Cip1. In vitro, recombinant His6-p27 could quantitatively reproduce the effects on cyclin E-Cdk2 kinase activity and the shift in molecular weight observed following progestin treatment. In contrast, cyclin D-Cdk4 was not inhibited by His6-p27 in vitro or p27Kip1 in vivo. However, an increase in the expression of the Cdk4/6 inhibitor p18INK4c and its extensive association with Cdk4 and Cdk6 were apparent following progestin treatment. Recombinant p18INK4c led to the reassortment of cyclin-CDK-CDK inhibitor complexes in vitro, with consequent decrease in cyclin E-Cdk2 activity. These results suggest a concerted model of progestin action whereby p27Kip1 and p18INK4c cooperate to inhibit cyclin E-Cdk2 and Cdk4. Since similar models have been developed for growth inhibition by transforming growth factor β and during adipogenesis, interaction between the Cip/Kip and INK4 families of inhibitors may be a common theme in physiological growth arrest and differentiation.
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Tian, Jean Q., and Andrea Quaroni. "Involvement of p21(WAF1/Cip1) and p27(Kip1) in intestinal epithelial cell differentiation." American Journal of Physiology-Cell Physiology 276, no. 6 (June 1, 1999): C1245—C1258. http://dx.doi.org/10.1152/ajpcell.1999.276.6.c1245.
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Using the conditionally immortalized human cell line tsFHI, we have investigated the role of cyclin-dependent kinase inhibitors (CKIs) in intestinal epithelial cell differentiation. Expression of cyclins, cyclin-dependent kinases (Cdk), and CKIs was examined under conditions promoting growth, growth arrest, or expression of differentiated traits. Formation of complexes among cell cycle regulatory proteins and their kinase activities were also investigated. The tsFHI cells express three CKIs: p16, p21, and p27. With differentiation, p21 and p27 were strongly induced, but with different kinetics: the p21 increase was rapid but transient and the p27 increase was delayed but sustained. Our results suggest that the function of p16 is primarily to inhibit cyclin D-associated kinases, making tsFHI cells dependent on cyclin E-Cdk2 for pRb phosphorylation and G1/S progression. Furthermore, they indicate that p21 is the main CKI involved in irreversible growth arrest during the early stages of cell differentiation in association with D-type cyclins, cyclin E, and Cdk2, whereas p27 may induce or stabilize expression of differentiated traits acting independently of cyclin-Cdk function.
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Martin, Mathew P., Jane A. Endicott, and Martin E. M. Noble. "Structure-based discovery of cyclin-dependent protein kinase inhibitors." Essays in Biochemistry 61, no. 5 (November 8, 2017): 439–52. http://dx.doi.org/10.1042/ebc20170040.
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The cell fate-determining roles played by members of the cyclin-dependent protein kinase (CDK) family explain why their dysregulation can promote proliferative diseases, and identify them as potential targets for drug discovery in oncology and beyond. After many years of research, the first efficacious CDK inhibitors have now been registered for clinical use in a defined segment of breast cancer. Research is underway to identify inhibitors with appropriate CDK-inhibitory profiles to recapitulate this success in other disease settings. Here, we review the structural data that illustrate the interactions and properties that confer upon inhibitors affinity and/or selectivity toward different CDK family members. We conclude that where CDK inhibitors display selectivity, that selectivity derives from exploiting active site sequence peculiarities and/or from the capacity of the target CDK(s) to access conformations compatible with optimizing inhibitor–target interactions.
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Ross, Karen E., Philipp Kaldis, and Mark J. Solomon. "Activating Phosphorylation of theSaccharomyces cerevisiaeCyclin-dependent Kinase, Cdc28p, Precedes Cyclin Binding." Molecular Biology of the Cell 11, no. 5 (May 2000): 1597–609. http://dx.doi.org/10.1091/mbc.11.5.1597.
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Eukaryotic cell cycle progression is controlled by a family of protein kinases known as cyclin-dependent kinases (Cdks). Two steps are essential for Cdk activation: binding of a cyclin and phosphorylation on a conserved threonine residue by the Cdk-activating kinase (CAK). We have studied the interplay between these regulatory mechanisms during the activation of the major Saccharomyces cerevisiaeCdk, Cdc28p. We found that the majority of Cdc28p was phosphorylated on its activating threonine (Thr-169) throughout the cell cycle. The extent of Thr-169 phosphorylation was similar for monomeric Cdc28p and Cdc28p bound to cyclin. By varying the order of the addition of cyclin and Cak1p, we determined that Cdc28p was activated most efficiently when it was phosphorylated before cyclin binding. Furthermore, we found that a Cdc28pT169Amutant, which cannot be phosphorylated, bound cyclin less well than wild-type Cdc28p in vivo. These results suggest that unphosphorylated Cdc28p may be unable to bind tightly to cyclin. We propose that Cdc28p is normally phosphorylated by Cak1p before it binds cyclin. This activation pathway contrasts with that in higher eukaryotes, in which cyclin binding appears to precede activating phosphorylation.
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Ray, Arpita, Melissa K. James, Stéphane Larochelle, Robert P. Fisher, and Stacy W. Blain. "p27Kip1 Inhibits Cyclin D-Cyclin-Dependent Kinase 4 by Two Independent Modes." Molecular and Cellular Biology 29, no. 4 (December 15, 2008): 986–99. http://dx.doi.org/10.1128/mcb.00898-08.
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ABSTRACT Cell cycle progression is regulated by cyclin-dependent kinases (cdk's), which in turn are regulated by their interactions with stoichiometric inhibitors, such as p27Kip1. Although p27 associates with cyclin D-cyclin-dependent kinase 4 (cdk4) constitutively, whether or not it inhibits this complex is dependent on the absence or presence of a specific tyrosine phosphorylation that converts p27 from a bound inhibitor to a bound noninhibitor under different growth conditions. This phosphorylation occurs within the 3-10 helix of p27 and may dislodge the helix from cdk4's active site to allow ATP binding. Here we show that the interaction of nonphosphorylated p27 with cdk4 also prevents the activating phosphorylation of the T-loop by cyclin H-cdk7, the cdk-activating kinase (CAK). Even though the cyclin H-cdk7 complex is present and active in contact-arrested cells, p27's association with cyclin D-cdk4 prevents T-loop phosphorylation. When p27 is tyrosine phosphorylated in proliferating cells or in vitro with the tyrosine Y kinase Abl, phosphorylation of cdk4 by cyclin H-cdk7 is permitted, even without dissociation of p27. This suggests that upon release from the contact-arrested state, a temporal order for the reactivation of inactive p27-cyclin D-cdk4 complexes must exist: p27 must be Y phosphorylated first, directly permitting cyclin H-cdk7 phosphorylation of residue T172 and the consequent restoration of kinase activity. The non-Y-phosphorylated p27-cyclin D-cdk4 complex could be phosphorylated by purified Csk1, a single-subunit CAK from fission yeast, but was still inactive due to p27's occlusion of the active site. Thus, the two modes by which p27 inhibits cyclin D-cdk4 are independent and may reinforce one another to inhibit kinase activity in contact-arrested cells, while maintaining a reservoir of preformed complex that can be activated rapidly upon cell cycle reentry.
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Saha, P., Q. Eichbaum, E. D. Silberman, B. J. Mayer, and A. Dutta. "p21CIP1 and Cdc25A: competition between an inhibitor and an activator of cyclin-dependent kinases." Molecular and Cellular Biology 17, no. 8 (August 1997): 4338–45. http://dx.doi.org/10.1128/mcb.17.8.4338.
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Cdc25A, a phosphatase essential for G1-S transition, associates with, dephosphorylates, and activates the cell cycle kinase cyclin E-cdk2. p21CIP1 and p27 are cyclin-dependent kinase (cdk) inhibitors induced by growth-suppressive signals such as p53 and transforming growth factor beta (TGF-beta). We have identified a cyclin binding motif near the N terminus of Cdc25A that is similar to the cyclin binding Cy (or RR LFG) motif of the p21CIP1 family of cdk inhibitors and separate from the catalytic domain. Mutations in this motif disrupt the association of Cdc25A with cyclin E- or cyclin A-cdk2 in vitro and in vivo and selectively interfere with the dephosphorylation of cyclin E-cdk2. A peptide based on the Cy motif of p21 competitively disrupts the association of Cdc25A with cyclin-cdks and inhibits the dephosphorylation of the kinase. p21 inhibits Cdc25A-cyclin-cdk2 association and the dephosphorylation of cdk2. Conversely, Cdc25A, which is itself an oncogene up-regulated by the Myc oncogene, associates with cyclin-cdk and protects it from inhibition by p21. Cdc25A also protects DNA replication in Xenopus egg extracts from inhibition by p21. These results describe a mechanism by which the Myc- or Cdc25A-induced oncogenic and p53- or TGF-beta-induced growth-suppressive pathways counterbalance each other by competing for cyclin-cdks.
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Laman, Heike, Dawn Coverley, Torsten Krude, Ronald Laskey, and Nic Jones. "Viral Cyclin–Cyclin-Dependent Kinase 6 Complexes Initiate Nuclear DNA Replication." Molecular and Cellular Biology 21, no. 2 (January 15, 2001): 624–35. http://dx.doi.org/10.1128/mcb.21.2.624-635.2001.
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ABSTRACT The cyclins encoded by Kaposi sarcoma-associated herpesvirus and herpesvirus saimiri are homologs of human D-type cyclins. However, when complexed to cdk6, they have several activities that distinguish them from D-type cyclin-cdk6 complexes, including resistance to cyclin-dependent kinase inhibitors and an enhanced substrate range. We find that viral cyclins interact with and phosphorylate proteins involved in replication initiation. Using mammalian in vitro replication systems, we show that viral cyclin-cdk6 complexes can directly trigger the initiation of DNA synthesis in isolated late-G1-phase nuclei. Viral cyclin-cdk6 complexes share this capacity with cyclin A-cdk2, demonstrating that in addition to functioning as G1-phase cyclin-cdk complexes, they function as S-phase cyclin-cdk complexes.
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NAKAMURA, K., N. YOKOYAMA, and I. IGARASHI. "Cyclin-dependent kinase inhibitors block erythrocyte invasion and intraerythrocytic development of Babesia bovis in vitro." Parasitology 134, no. 10 (July 18, 2007): 1347–53. http://dx.doi.org/10.1017/s0031182007002831.
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SUMMARYCyclin-dependent kinases (CDKs) are essential for the regulation of the eukaryotic cell cycle. A number of chemicals, which selectively inhibit the CDK activities, have been synthesized for the development of anti-cancer drugs. This report describes the inhibitory effect of purine derivatives known to be CDK inhibitors on the asexual growth of Babesia bovis. The 4 compounds, roscovitine, purvalanol A, CGP74514A, and CDK2 Inhibitor II, showed significantly suppressive effects on the in vitro growth of B. bovis. Three (roscovitine, purvalanol A, and CDK2 Inhibitor II) showed an inhibitory effect on the early stages of intraerythrocytic development of B. bovis. CGP74514A (CDK1-specific inhibitor) blocked the erythrocyte invasion by merozoites. Our data suggest the chemotherapeutic potential of the CDK inhibitors for babesiosis, and the target molecules of the compounds would participate in the process of successful erythrocyte invasion or intraerythrocytic development of B. bovis.
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Hirai, H., M. F. Roussel, J. Y. Kato, R. A. Ashmun, and C. J. Sherr. "Novel INK4 proteins, p19 and p18, are specific inhibitors of the cyclin D-dependent kinases CDK4 and CDK6." Molecular and Cellular Biology 15, no. 5 (May 1995): 2672–81. http://dx.doi.org/10.1128/mcb.15.5.2672.
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Cyclin D-dependent kinases act as mitogen-responsive, rate-limiting controllers of G1 phase progression in mammalian cells. Two novel members of the mouse INK4 gene family, p19 and p18, that specifically inhibit the kinase activities of CDK4 and CDK6, but do not affect those of cyclin E-CDK2, cyclin A-CDK2, or cyclin B-CDC2, were isolated. Like the previously described human INK4 polypeptides, p16INK4a/MTS1 and p15INK4b/MTS2, mouse p19 and p18 are primarily composed of tandemly repeated ankyrin motifs, each ca. 32 amino acids in length, p19 and p18 bind directly to CDK4 and CDK6, whether untethered or in complexes with D cyclins, and can inhibit the activity of cyclin D-bound cyclin-dependent kinases (CDKs). Although neither protein interacts with D cyclins or displaces them from preassembled cyclin D-CDK complexes in vitro, both form complexes with CDKs at the expense of cyclins in vivo, suggesting that they may also interfere with cyclin-CDK assembly. In proliferating macrophages, p19 mRNA and protein are periodically expressed with a nadir in G1 phase and maximal synthesis during S phase, consistent with the possibility that INK4 proteins limit the activities of CDKs once cells exit G1 phase. However, introduction of a vector encoding p19 into mouse NIH 3T3 cells leads to constitutive p19 synthesis, inhibits cyclin D1-CDK4 activity in vivo, and induces G1 phase arrest.
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Niiya, Fumihiko, Xiaozhen Xie, Kyung S. Lee, Hiroki Inoue, and Toru Miki. "Inhibition of Cyclin-dependent Kinase 1 Induces Cytokinesis without Chromosome Segregation in an ECT2 and MgcRacGAP-dependent Manner." Journal of Biological Chemistry 280, no. 43 (August 23, 2005): 36502–9. http://dx.doi.org/10.1074/jbc.m508007200.
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Cleavage furrow formation marks the onset of cell division during early anaphase. The small GTPase RhoA and its regulators ECT2 and MgcRacGAP have been implicated in furrow ingression in mammalian cells, but the signaling upstream of these molecules remains unclear. We now show that the inhibition of cyclin-dependent kinase (Cdk)1 is sufficient to initiate cytokinesis. When mitotically synchronized cells were treated with the Cdk-specific inhibitor BMI-1026, the initiation of cytokinesis was induced precociously before chromosomal separation. Cytokinesis was also induced by the Cdk1-specific inhibitor purvalanol A but not by Cdk2/Cdk5- or Cdk4-specific inhibitors. Consistent with initiation of precocious cytokinesis by Cdk1 inhibition, introduction of anti-Cdk1 monoclonal antibody resulted in cells with aberrant nuclei. Depolymerization of mitotic spindles by nocodazole inhibited BMI-1026-induced precocious cytokinesis. However, in the presence of a low concentration of nocodazole, BMI-1026 induced excessive membrane blebbing, which appeared to be caused by formation of ectopic cleavage furrows. Depletion of ECT2 or MgcRacGAP by RNA interference abolished both of the phenotypes (precocious furrowing after nocodazole release and excessive blebbing in the presence of nocodazole). RNA interference of RhoA or expression of dominant-negative RhoA efficiently reduced both phenotypes. RhoA was localized at the cleavage furrow or at the necks of blebs. We propose that Cdk1 inactivation is sufficient to activate a signaling pathway leading to cytokinesis, which emanates from mitotic spindles and is regulated by ECT2, MgcRacGAP, and RhoA. Chemical induction of cytokinesis will be a valuable tool to study the initiation mechanism of cytokinesis.
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Ko, L. J., S. Y. Shieh, X. Chen, L. Jayaraman, K. Tamai, Y. Taya, C. Prives, and Z. Q. Pan. "p53 is phosphorylated by CDK7-cyclin H in a p36MAT1-dependent manner." Molecular and Cellular Biology 17, no. 12 (December 1997): 7220–29. http://dx.doi.org/10.1128/mcb.17.12.7220.
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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.
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Shou, W., and W. G. Dunphy. "Cell cycle control by Xenopus p28Kix1, a developmentally regulated inhibitor of cyclin-dependent kinases." Molecular Biology of the Cell 7, no. 3 (March 1996): 457–69. http://dx.doi.org/10.1091/mbc.7.3.457.
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We have isolated Xenopus p28Kix1, a member of the p21CIP1/p27KIP1/p57KIP2 family of cyclin-dependent kinase (Cdk) inhibitors. Members of this family negatively regulate cell cycle progression in mammalian cells by inhibiting the activities of Cdks. p28 shows significant sequence homology with p21, p27, and p57 in its N-terminal region, where the Cdk inhibition domain is known to reside. In contrast, the C-terminal domain of p28 is distinct from that of p21, p27, and p57. In co-immunoprecipitation experiments, p28 was found to be associated with Cdk2, cyclin E, and cyclin A, but not the Cdc2/cyclin B complex in Xenopus egg extracts. Xenopus p28 associates with the proliferating cell nuclear antigen, but with a substantially lower affinity than human p21. In kinase assays with recombinant Cdks, p28 inhibits pre-activated Cdk2/cyclin E and Cdk2/cyclin A, but not Cdc2/cyclin B. However, at high concentrations, p28 does prevent the activation of Cdc2/cyclin B by the Cdk-activating kinase. Consistent with the role of p28 as a Cdk inhibitor, recombinant p28 elicits an inhibition of both DNA replication and mitosis upon addition to egg extracts, indicating that it can regulate multiple cell cycle transitions. The level of p28 protein shows a dramatic developmental profile: it is low in Xenopus oocytes, eggs, and embryos up to stage 11, but increases approximately 100-fold between stages 12 and 13, and remains high thereafter. The induction of p28 expression temporally coincides with late gastrulation. Thus, although p28 may play only a limited role during the early embryonic cleavages, it may function later in development to establish a somatic type of cell cycle. Taken together, our results indicate that Xenopus p28 is a new member of the p21/p27/p57 class of Cdk inhibitors, and that it may play a role in developmental processes.
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Franklin, David S., Virginia L. Godfrey, Deborah A. O'Brien, Chuxia Deng, and Yue Xiong. "Functional Collaboration between Different Cyclin-Dependent Kinase Inhibitors Suppresses Tumor Growth with Distinct Tissue Specificity." Molecular and Cellular Biology 20, no. 16 (August 15, 2000): 6147–58. http://dx.doi.org/10.1128/mcb.20.16.6147-6158.2000.
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ABSTRACT The presence of two families of seven distinct mammalian cyclin-dependent kinase (CDK) inhibitor genes is thought to mediate the complexity of connecting a variety of cellular processes to the cell cycle control pathway. The distinct pattern of tissue expression of CDK inhibitor genes suggests that they may function as tumor suppressors with different tissue specificities. To test this hypothesis, we have characterized two strains of double mutant mice lacking either p18INK4c and p27KIP1 or p18INK4cand p21CIP1/WAF1. Loss of both p18 and p27 function resulted in the spontaneous development by 3 months of age of at least eight different types of hyperplastic tissues and/or tumors in the pituitary, adrenals, thyroid, parathyroid, testes, pancreas, duodenum, and stomach. Six of these hyperplastic tissues and tumors were in endocrine organs, and several types of tumors routinely developed within the same animal, a phenotype reminiscent of that seen in combined human multiple endocrine neoplasia syndromes. The p18-p21 double null mice, on the other hand, developed pituitary adenomas, multifocal gastric neuroendocrine hyperplasia, and lung bronchioalveolar tumors later in life. G1 CDK2 and CDK4 kinase activities were increased in both normal and neoplastic tissues derived from mice lacking individual CDK inhibitors and were synergistically stimulated by the simultaneous loss of two CDK inhibitors. This indicates that an increase in G1 CDK kinase activity is a critical step during but is not sufficient for tumor growth. Our results suggest that functional collaborations between distinct CDK inhibitor genes are tissue specific and confer yet another level of regulation in cell growth control and tumor suppression.
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Bockstaele, Laurence, Xavier Bisteau, Sabine Paternot, and Pierre P. Roger. "Differential Regulation of Cyclin-Dependent Kinase 4 (CDK4) and CDK6, Evidence that CDK4 Might Not Be Activated by CDK7, and Design of a CDK6 Activating Mutation." Molecular and Cellular Biology 29, no. 15 (June 1, 2009): 4188–200. http://dx.doi.org/10.1128/mcb.01823-08.
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ABSTRACT The homologous cyclin-dependent kinases (CDK) CDK4 and CDK6 integrate mitogenic and oncogenic signaling cascades with the cell cycle. Their activation requires binding to a D-type cyclin and then T-loop phosphorylation at T172 and T177 (respectively) by the only CDK-activating kinase identified in animal cells, cyclin H-CDK7. At odds with the existing data showing the constitutive activity of CDK7, we have recently identified the T172 phosphorylation of cyclin D-bound CDK4 as a crucial cell cycle regulatory target. Here we show that T172 phosphorylation of CDK4 is conditioned by its unique proline 173 residue. In contrast to CDK4, CDK6 does not contain such a proline and, unexpectedly, remained poorly phosphorylated and active in a variety of cells. Mutations of proline 173 did not adversely affect CDK4 activation by CDK7, but in cells they abolished CDK4 T172 phosphorylation and activity. Conversely, substituting a proline for the corresponding residue of CDK6 enforced its complete, apparently cyclin-independent T177 phosphorylation and dramatically increased its activity. These results lead us to propose that CDK4 might not be phosphorylated by CDK7 in intact cells but is more likely phosphorylated by another, presumably proline-directed kinase(s). Moreover, they provide a new model of a potentially oncogenic activating mutation of a CDK.
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Ruas, Margarida, Fiona Gregory, Rebecca Jones, Robert Poolman, Maria Starborg, Janice Rowe, Sharon Brookes, and Gordon Peters. "CDK4 and CDK6 Delay Senescence by Kinase-Dependent and p16INK4a-Independent Mechanisms." Molecular and Cellular Biology 27, no. 12 (April 9, 2007): 4273–82. http://dx.doi.org/10.1128/mcb.02286-06.
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ABSTRACT Replicative senescence of human diploid fibroblasts (HDFs) is largely implemented by the cyclin-dependent kinase (CDK) inhibitors p16INK4a and p21CIP1. Their accumulation results in a loss of CDK2 activity, and cells arrest with the retinoblastoma protein (pRb) in its hypophosphorylated state. It has become standard practice to bypass the effects of p16INK4a by overexpressing CDK4 or a variant form that is unable to bind to INK4 proteins. Although CDK4 and CDK6 and their INK4-insensitive variants can extend the life span of HDFs, they also cause a substantial increase in the levels of endogenous p16INK4a. Here we show that CDK4 and CDK6 can extend the life span of HDFs that have inactivating mutations in both alleles of INK4a or in which INK4a levels are repressed, indicating that overexpression of CDK4/6 is not equivalent to ablation of p16INK4a. However, catalytically inactive versions of these kinases are unable to extend the replicative life span, suggesting that the impact of ectopic CDK4/6 depends on their ability to phosphorylate as yet unidentified substrates rather than to sequester CDK inhibitors. Since p16INK4a deficiency, CDK4 expression, and p53 or p21CIP1 ablation have additive effects on replicative life span, our results underscore the idea that senescence is an integrated response to diverse signals.
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Reynard, Gregory J., William Reynolds, Rati Verma, and Raymond J. Deshaies. "Cks1 Is Required for G1Cyclin–Cyclin-Dependent Kinase Activity in Budding Yeast." Molecular and Cellular Biology 20, no. 16 (August 15, 2000): 5858–64. http://dx.doi.org/10.1128/mcb.20.16.5858-5864.2000.
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ABSTRACT p13suc1 (Cks) proteins have been implicated in the regulation of cyclin-dependent kinase (CDK) activity. However, the mechanism by which Cks influences the function of cyclin-CDK complexes has remained elusive. We show here that Cks1 is required for the protein kinase activity of budding yeast G1 cyclin-CDK complexes. Cln2 and Cdc28 subunits coexpressed in baculovirus-infected insect cells fail to exhibit protein kinase activity towards multiple substrates in the absence of Cks1. Cks1 can both stabilize Cln2-Cdc28 complexes and activate intact complexes in vitro, suggesting that it plays multiple roles in the biogenesis of active G1cyclin-CDK complexes. In contrast, Cdc28 forms stable, active complexes with the B-type cyclins Clb4 and Clb5 regardless of whether Cks1 is present. The levels of Cln2-Cdc28 and Cln3-Cdc28 protein kinase activity are severely reduced in cks1-38 cell extracts. Moreover, phosphorylation of G1 cyclins, which depends on Cdc28 activity, is reduced in cks1-38 cells. The role of Cks1 in promoting G1 cyclin-CDK protein kinase activity both in vitro and in vivo provides a simple molecular rationale for the essential role of CKS1 in progression through G1 phase in budding yeast.
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Aprelikova, Olga, Yue Xiong, and Edison T. Liu. "Both p16 and p21 Families of Cyclin-dependent Kinase (CDK) Inhibitors Block the Phosphorylation of Cyclin-dependent Kinases by the CDK-activating Kinase." Journal of Biological Chemistry 270, no. 31 (August 4, 1995): 18195–97. http://dx.doi.org/10.1074/jbc.270.31.18195.
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Major, Michael L., Rita Lepe, and Robert H. Costa. "Forkhead Box M1B Transcriptional Activity Requires Binding of Cdk-Cyclin Complexes for Phosphorylation-Dependent Recruitment of p300/CBP Coactivators." Molecular and Cellular Biology 24, no. 7 (April 1, 2004): 2649–61. http://dx.doi.org/10.1128/mcb.24.7.2649-2661.2004.
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ABSTRACT Previous liver regeneration studies demonstrated that the mouse forkhead box M1B (FoxM1B) transcription factor regulates hepatocyte proliferation through expression of cell cycle genes that stimulate cyclin-dependent kinase 2 (Cdk2) and Cdk1 activity. In this study, we demonstrated that disruption of the FoxM1B Cdk1/2 phosphorylation site at Thr residue 596 significantly reduced both FoxM1B transcriptional activity and Cdk phosphorylation of the FoxM1B T596A mutant protein in vivo. Retention of this FoxM1B 596 Cdk phosphorylation site was found to be essential for recruiting the histone acetyltransferase CREB binding protein (CBP) to the FoxM1B transcriptional activation domain. Consistent with these findings, dominant negative Cdk1 protein significantly reduced FoxM1B transcriptional activity and inhibited FoxM1B recruitment of the CBP coactivator protein. Likewise, Cdc25B-mediated stimulation of Cdk activity together with elevated levels of the CBP coactivator protein provided a 6.2-fold synergistic increase in FoxM1B transcriptional activity. Furthermore, mutation of the FoxM1B Leu 641 residue within an LXL motif (residues 639 to 641) inhibited recruitment of Cdk-cyclin complexes and caused significant reduction in both FoxM1B transcriptional activity and in vivo Cdk phosphorylation of the FoxM1B Thr 596 residue. We demonstrated that FoxM1B transcriptional activity requires binding of either S-phase or M-phase Cdk-cyclin complexes to mediate efficient Cdk phosphorylation of the FoxM1B Thr 596 residue, which is essential for recruitment of p300/CBP coactivator proteins.
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BARBERIS, Matteo, Luca DE GIOIA, Maria RUZZENE, Stefania SARNO, Paola COCCETTI, Piercarlo FANTUCCI, Marco VANONI, and Lilia ALBERGHINA. "The yeast cyclin-dependent kinase inhibitor Sic1 and mammalian p27Kip1 are functional homologues with a structurally conserved inhibitory domain." Biochemical Journal 387, no. 3 (April 26, 2005): 639–47. http://dx.doi.org/10.1042/bj20041299.
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In Saccharomyces cerevisiae, Sic1, an inhibitor of Cdk (cyclin-dependent kinase), blocks the activity of S-Cdk1 (Cdk1/Clb5,6) kinase that is required for DNA replication. Deletion of Sic1 causes premature DNA replication from fewer origins, extension of the S phase and inefficient separation of sister chromatids during anaphase. Despite the well-documented relevance of Sic1 inhibition of S-Cdk1 for cell cycle control and genome instability, the molecular mechanism by which Sic1 inhibits S-Cdk1 activity remains obscure. In this paper, we show that Sic1 is functionally and structurally related to the mammalian Cki (Cdk inhibitor) p27Kip1 of the Kip/Cip family. A molecular model of the inhibitory domain of Sic1 bound to the Cdk2–cyclin A complex suggested that the yeast inhibitor might productively interface with the mammalian Cdk2–cyclin A complex. Consistent with this, Sic1 is able to bind to, and strongly inhibit the kinase activity of, the Cdk2–cyclin A complex. In addition, comparison of the different inhibitory patterns obtained using histone H1 or GST (glutathione S-transferase)–pRb (retinoblastoma protein) fusion protein as substrate (the latter of which recognizes both the docking site and the catalytic site of Cdk2–cyclin A) offers interesting suggestions for the inhibitory mechanism of Sic1. Finally, overexpression of the KIP1 gene in vivo in Saccharomyces cerevisiae, like overexpression of the related SIC1 gene, rescues the cell cycle-related phenotype of a sic1Δ strain. Taken together, these findings strongly indicate that budding yeast Sic1 and mammalian p27Kip1 are functional homologues with a structurally conserved inhibitory domain.
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Nugiel, David A., Ana-Marie Etzkorn, Anup Vidwans, Pamela A. Benfield, Michael Boisclair, Catherine R. Burton, Sarah Cox, Phillip M. Czerniak, Deborah Doleniak, and Steven P. Seitz. "Indenopyrazoles as Novel Cyclin Dependent Kinase (CDK) Inhibitors." Journal of Medicinal Chemistry 44, no. 9 (April 2001): 1334–36. http://dx.doi.org/10.1021/jm0100032.
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Sánchez-Martínez, Concepción, Lawrence M. Gelbert, María José Lallena, and Alfonso de Dios. "Cyclin dependent kinase (CDK) inhibitors as anticancer drugs." Bioorganic & Medicinal Chemistry Letters 25, no. 17 (September 2015): 3420–35. http://dx.doi.org/10.1016/j.bmcl.2015.05.100.
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