Chk1 kinase coordinates cell cycle progression and preserves genome integrity. Here, we show that chemical or genetic ablation of human Chk1 triggered supraphysiological accumulation of the S phase-promoting Cdc25A phosphatase, prevented ionizing radiation (IR)-induced degradation of Cdc25A, and caused radioresistant DNA synthesis (RDS). The basal turnover of Cdc25A operating in unperturbed S phase required Chk1-dependent phosphorylation of serines 123, 178, 278, and 292. IR-induced acceleration of Cdc25A proteolysis correlated with increased phosphate incorporation into these residues generated by a combined action of Chk1 and Chk2 kinases. Finally, phosphorylation of Chk1 by ATM was required to fully accelerate the IR-induced degradation of Cdc25A. Our results provide evidence that the mammalian S phase checkpoint functions via amplification of physiologically operating, Chk1-dependent mechanisms.
- MeSH
- aktivace enzymů MeSH
- ATM protein MeSH
- biologické modely MeSH
- buněčný cyklus * fyziologie účinky záření MeSH
- checkpoint kinasa 2 MeSH
- DNA vazebné proteiny MeSH
- fosfatasy cdc25 * fyziologie účinky záření MeSH
- fosforylace MeSH
- HeLa buňky MeSH
- ionizující záření MeSH
- kinetika MeSH
- lidé MeSH
- nádorové buňky kultivované MeSH
- nádorové supresorové proteiny MeSH
- protein-serin-threoninkinasy fyziologie MeSH
- proteinkinasy * metabolismus MeSH
- proteiny buněčného cyklu MeSH
- replikace DNA účinky záření MeSH
- S fáze účinky záření MeSH
- serin metabolismus MeSH
- signální transdukce MeSH
- Check Tag
- lidé MeSH
The retinoblastoma (pRb)-related p130 pocket protein is a regulator of cell growth and differentiation, and a candidate tumour suppressor. Both pRb and p130 operate through interactions with cellular proteins, including the E2F transcription factors. While such interactions are controlled by phosphorylation of multiple sites of pRb, regulation of p130 remains poorly understood. We now identify 22 in vivo phosphorylation sites of p130, targeted by diverse kinases, and present evidence for three cyclin-dependent kinase 4(6) [Cdk4(6)] specific phosphorylations, which appear critical for controlling the growth-restraining activity of p130. When expressed in U2OS cells, the phosphorylation-deficient mutant p130(Delta)(CDK4), in which the Cdk4 specific sites were mutated to alanine residues, imposed a more sustained G1 arrest than a constitutively active pRb(Delta)(CDK), known to repress all cellular E2F activity. Experiments using p130(Delta)(Cdk4) and another phosphorylation-deficient mutant, p130(PM19A), with 19 phosphorylation sites mutated, revealed that the p130-imposed G1 block reflects cooperative growth-suppressive effects of phosphorylation-regulated E2F binding and phosphorylation-independent sequestration of cyclin E(A)-Cdk2 through the N-terminal cyclin binding motif of p130.
- MeSH
- buněčné linie MeSH
- cyklin D MeSH
- cyklin E metabolismus MeSH
- cyklin-dependentní kinasy metabolismus MeSH
- cykliny metabolismus MeSH
- DNA vazebné proteiny * MeSH
- fosfoproteiny * genetika chemie metabolismus MeSH
- fosforylace MeSH
- G1 fáze * fyziologie MeSH
- lidé MeSH
- mutageneze cílená MeSH
- peptidové mapování MeSH
- protein p130 podobný retinoblastomu MeSH
- proteiny buněčného cyklu * MeSH
- proteiny * MeSH
- retinoblastomový protein * genetika chemie metabolismus MeSH
- transkripční faktor DP1 MeSH
- transkripční faktory E2F MeSH
- transkripční faktory metabolismus MeSH
- transportní proteiny * MeSH
- vazebná místa genetika MeSH
- vazebný protein 1 retinoblastomu MeSH
- Check Tag
- lidé MeSH
p16(INK4a), p15(INK4b), p18(INK4c) and p19(INK4d) comprise a family of cyclin-dependent kinase inhibitors and tumor suppressors. We report that the INK4 proteins share the ability to arrest cells in G1, and interact with CDK4 or CDK6 with similar avidity. In contrast, only p18 and particularly p19 are phosphorylated in vivo, and each of the human INK4 proteins shows unique expression patterns dependent on cell and tissue type, and differentiation stage. Thus, the INK4 proteins harbor redundant as well as non-overlapping properties, suggesting distinct regulatory modes, and diverse roles for the individual INK4 family members in cell cycle control, cellular differentiation, and multistep oncogenesis.
- MeSH
- buněčná diferenciace účinky léků MeSH
- cyklin-dependentní kinasa 4 MeSH
- cyklin-dependentní kinasa 6 MeSH
- cyklin-dependentní kinasy * antagonisté a inhibitory metabolismus MeSH
- fosforylace MeSH
- G1 fáze MeSH
- geny p16 fyziologie MeSH
- inhibitor p15 cyklin-dependentní kinasy MeSH
- inhibitor p16 cyklin-dependentní kinasy MeSH
- inhibitor p18 cyklin-dependentní kinasy MeSH
- inhibitor p19 cyklin-dependentní kinasy MeSH
- inhibitory enzymů * MeSH
- kompetitivní vazba MeSH
- lidé MeSH
- multigenová rodina * genetika MeSH
- nádorové buňky kultivované MeSH
- nádorové supresorové proteiny * MeSH
- orgánová specificita MeSH
- protein-serin-threoninkinasy antagonisté a inhibitory metabolismus MeSH
- proteiny buněčného cyklu * MeSH
- protoonkogenní proteiny * MeSH
- regulace genové exprese u nádorů účinky léků MeSH
- rekombinantní fúzní proteiny genetika MeSH
- stanovení celkové genové exprese MeSH
- teratokarcinom metabolismus patologie MeSH
- transportní proteiny genetika metabolismus MeSH
- tretinoin farmakologie MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
