In response to DNA damage, a cell can be forced to permanently exit the cell cycle and become senescent. Senescence provides an early barrier against tumor development by preventing proliferation of cells with damaged DNA. By studying single cells, we show that Cdk activity persists after DNA damage until terminal cell cycle exit. This low level of Cdk activity not only allows cell cycle progression, but also promotes cell cycle exit at a decision point in G2 phase. We find that residual Cdk1/2 activity is required for efficient p21 production, allowing for nuclear sequestration of Cyclin B1, subsequent APC/CCdh1 -dependent degradation of mitotic inducers and induction of senescence. We suggest that the same activity that triggers mitosis in an unperturbed cell cycle enforces senescence in the presence of DNA damage, ensuring a robust response when most needed.

• Klíčová slova
• Cdk1, Cdk2, DNA damage response, G2 phase, cell cycle, checkpoint recovery, p21, senescence,
• MeSH
• analýza jednotlivých buněk MeSH
• buněčné linie MeSH
• CD antigeny MeSH
• chinoliny farmakologie   MeSH
• cyklin B1 genetika   metabolismus   MeSH
• cyklin-dependentní kinasa 2 antagonisté a inhibitory   genetika   metabolismus   MeSH
• epitelové buňky cytologie   účinky léků   enzymologie   MeSH
• etoposid farmakologie   MeSH
• inhibitor p21 cyklin-dependentní kinasy genetika   metabolismus   MeSH
• kadheriny genetika   metabolismus   MeSH
• kontrolní body fáze G2 buněčného cyklu účinky léků   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• osteoblasty cytologie   účinky léků   enzymologie   MeSH
• poškození DNA MeSH
• proteinkinasa CDC2 antagonisté a inhibitory   genetika   metabolismus   MeSH
• pteridiny farmakologie   MeSH
• puriny farmakologie   MeSH
• regulace genové exprese MeSH
• retinální pigmentový epitel cytologie   účinky léků   enzymologie   MeSH
• signální transdukce MeSH
• stárnutí buněk účinky léků   MeSH
• thiazoly farmakologie   MeSH
• velikost buňky MeSH
• viabilita buněk účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 4-(6-cyclohexylmethoxy-9H-purin-2-ylamino)-N,N-diethylbenzamide MeSH Prohlížeč
• BI 2536 MeSH Prohlížeč
• CCNB1 protein, human MeSH Prohlížeč
• CD antigeny MeSH
• CDH1 protein, human MeSH Prohlížeč
• CDK1 protein, human MeSH Prohlížeč
• CDK2 protein, human MeSH Prohlížeč
• chinoliny MeSH
• cyklin B1 MeSH
• cyklin-dependentní kinasa 2 MeSH
• etoposid MeSH
• inhibitor p21 cyklin-dependentní kinasy MeSH
• kadheriny MeSH
• proteinkinasa CDC2 MeSH
• pteridiny MeSH
• puriny MeSH
• RO 3306 MeSH Prohlížeč
• thiazoly MeSH

Cell cycle control must be modified at meiosis to allow two divisions to follow a single round of DNA replication, resulting in ploidy reduction. The mechanisms that ensure meiosis termination at the end of the second and not at the end of first division are poorly understood. We show here that Arabidopsis thaliana TDM1, which has been previously shown to be essential for meiotic termination, interacts directly with the Anaphase-Promoting Complex. Further, mutations in TDM1 in a conserved putative Cyclin-Dependant Kinase (CDK) phosphorylation site (T16-P17) dominantly provoked premature meiosis termination after the first division, and the production of diploid spores and gametes. The CDKA;1-CYCA1.2/TAM complex, which is required to prevent premature meiotic exit, phosphorylated TDM1 at T16 in vitro. Finally, while CYCA1;2/TAM was previously shown to be expressed only at meiosis I, TDM1 is present throughout meiosis. These data, together with epistasis analysis, lead us to propose that TDM1 is an APC/C component whose function is to ensure meiosis termination at the end of meiosis II, and whose activity is inhibited at meiosis I by CDKA;1-TAM-mediated phosphorylation to prevent premature meiotic exit. This provides a molecular mechanism for the differential decision of performing an additional round of division, or not, at the end of meiosis I and II, respectively.

• MeSH
• anafázi podporující komplex metabolismus   MeSH
• Arabidopsis cytologie   genetika   MeSH
• biologické modely MeSH
• chromozomy rostlin genetika   MeSH
• cykliny genetika   metabolismus   MeSH
• dominantní geny MeSH
• fosforylace MeSH
• fosfothreonin metabolismus   MeSH
• genetická epistáze MeSH
• genetické testování MeSH
• meióza * MeSH
• mutace genetika   MeSH
• podjednotky proteinů metabolismus   MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• tetraploidie MeSH
• tubulin metabolismus   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• anafázi podporující komplex MeSH
• cykliny MeSH
• fosfothreonin MeSH
• podjednotky proteinů MeSH
• proteiny huseníčku MeSH
• TDM1 protein, Arabidopsis MeSH Prohlížeč
• tubulin MeSH

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.

• MeSH
• cyklin-dependentní kinasy chemie   metabolismus   MeSH
• cykliny chemie   metabolismus   MeSH
• ELISA MeSH
• HeLa buňky MeSH
• hmotnostní spektrometrie MeSH
• imunoprecipitace MeSH
• konformace proteinů MeSH
• krystalizace MeSH
• lidé MeSH
• molekulární modely * MeSH
• multiproteinové komplexy chemie   metabolismus   MeSH
• substrátová specifita MeSH
• western blotting MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• CCNK protein, human MeSH Prohlížeč
• CDK12 protein, human MeSH Prohlížeč
• cyklin-dependentní kinasy MeSH
• cykliny MeSH
• multiproteinové komplexy MeSH

Deregulation of cyclin-dependent kinases (CDKs) has been associated with many cancer types and has evoked an interest in chemical inhibitors with possible therapeutic benefit. While most known inhibitors display broad selectivity towards multiple CDKs, recent work highlights CDK9 as the critical target responsible for the anticancer activity of clinically evaluated drugs. In this review, we discuss recent findings provided by structural biologists that may allow further development of highly specific inhibitors of CDK9 towards applications in cancer therapy. We also highlight the role of CDK9 in inflammatory processes and diseases.

• MeSH
• antitumorózní látky farmakologie   MeSH
• cyklin-dependentní kinasa 9 antagonisté a inhibitory   metabolismus   MeSH
• inhibitory proteinkinas farmakologie   MeSH
• lidé MeSH
• nádory farmakoterapie   enzymologie   MeSH
• racionální návrh léčiv MeSH
• systémy cílené aplikace léků * MeSH
• zánět enzymologie   patologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• antitumorózní látky MeSH
• cyklin-dependentní kinasa 9 MeSH
• inhibitory proteinkinas MeSH

The structures of fully active cyclin-dependent kinase-2 (CDK2) complexed with ATP and peptide substrate, CDK2 after the catalytic reaction, and CDK2 inhibited by phosphorylation at Thr14/Tyr15 were studied using molecular dynamics (MD) simulations. The structural details of the CDK2 catalytic site and CDK2 substrate binding box were described. Comparison of MD simulations of inhibited complexes of CDK2 was used to help understand the role of inhibitory phosphorylation at Thr14/Tyr15. Phosphorylation at Thr14/Tyr15 causes ATP misalignment for the phosphate-group transfer, changes in the Mg(2+) coordination sphere, and changes in the H-bond network formed by CDK2 catalytic residues (Asp127, Lys129, Asn132). The inhibitory phosphorylation causes the G-loop to shift from the ATP binding site, which leads to opening of the CDK2 substrate binding box, thus probably weakening substrate binding. All these effects explain the decrease in kinase activity observed after inhibitory phosphorylation at Thr14/Tyr15 in the G-loop. Interaction of the peptide substrate, and the phosphorylated peptide product, with CDK2 was also studied and compared. These results broaden hypotheses drawn from our previous MD studies as to why a basic residue (Arg/Lys) is preferred at the P(+2) substrate position.

• MeSH
• cyklin-dependentní kinasa 2 antagonisté a inhibitory   chemie   metabolismus   MeSH
• fosforylace MeSH
• katalytická doména MeSH
• lidé MeSH
• sekundární struktura proteinů MeSH
• threonin chemie   metabolismus   MeSH
• tyrosin chemie   metabolismus   MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cyklin-dependentní kinasa 2 MeSH
• threonin MeSH
• tyrosin MeSH

Molecular dynamics (MD) simulations were used to explain structural details of cyclin-dependent kinase-2 (CDK2) inhibition by phosphorylation at T14 and/or Y15 located in the glycine-rich loop (G-loop). Ten-nanosecond-long simulations of fully active CDK2 in a complex with a short peptide (HHASPRK) substrate and of CDK2 inhibited by phosphorylation of T14 and/or Y15 were produced. The inhibitory phosphorylations at T14 and/or Y15 show namely an ATP misalignment and a G-loop shift (~5 A) causing the opening of the substrate binding box. The biological functions of the G-loop and GxGxxG motif evolutionary conservation in protein kinases are discussed. The position of the ATP gamma-phosphate relative to the phosphorylation site (S/T) of the peptide substrate in the active CDK2 is described and compared with inhibited forms of CDK2. The MD results clearly provide an explanation previously not known as to why a basic residue (R/K) is preferred at the P(2) position in phosphorylated S/T peptide substrates.

• MeSH
• adenosintrifosfát chemie   MeSH
• aminokyselinové motivy MeSH
• časové faktory MeSH
• cyklin-dependentní kinasa 2 MeSH
• fosfáty chemie   MeSH
• fosforylace MeSH
• hořčík chemie   MeSH
• inhibitory enzymů chemie   MeSH
• ionty MeSH
• kinasy CDC2-CDC28 antagonisté a inhibitory   chemie   MeSH
• konformace proteinů MeSH
• lidé MeSH
• molekulární modely MeSH
• peptidy chemie   MeSH
• rentgenové záření MeSH
• software MeSH
• stereoizomerie MeSH
• terciární struktura proteinů MeSH
• threonin chemie   MeSH
• tyrosin chemie   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosintrifosfát MeSH
• CDK2 protein, human MeSH Prohlížeč
• cyklin-dependentní kinasa 2 MeSH
• fosfáty MeSH
• hořčík MeSH
• inhibitory enzymů MeSH
• ionty MeSH
• kinasy CDC2-CDC28 MeSH
• peptidy MeSH
• threonin MeSH
• tyrosin MeSH

Nanoseconds long molecular dynamics (MD) trajectories of differently active complexes of human cyclin-dependent kinase 2 (inactive CDK2/ATP, semiactive CDK2/Cyclin A/ATP, fully active pT160-CDK2/Cyclin A/ATP, inhibited pT14-; pY15-; and pT14,pY15,pT160-CDK2/Cyclin A/ATP) were compared. The MD simulations results of CDK2 inhibition by phosphorylation at T14 and/or Y15 sites provide insight into the structural aspects of CDK2 deactivation. The inhibitory sites are localized in the glycine-rich loop (G-loop) positioned opposite the activation T-loop. Phosphorylation of T14 and both inhibitory sites T14 and Y15 together causes ATP misalignment for phosphorylation and G-loop conformational change. This conformational change leads to the opening of the CDK2 substrate binding box. The phosphorylated Y15 residue negatively affects substrate binding or its correct alignment for ATP terminal phospho-group transfer to the CDK2 substrate. The MD simulations of the CDK2 activation process provide results in agreement with previous X-ray data.

• MeSH
• aktivace enzymů MeSH
• cyklin-dependentní kinasa 2 MeSH
• fosforylace MeSH
• fosfotyrosin metabolismus   MeSH
• glycin metabolismus   MeSH
• kinasy CDC2-CDC28 antagonisté a inhibitory   chemie   metabolismus   MeSH
• molekulární modely MeSH
• sekundární struktura proteinů MeSH
• terciární struktura proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cyklin-dependentní kinasa 2 MeSH
• fosfotyrosin MeSH
• glycin MeSH
• kinasy CDC2-CDC28 MeSH