Effective cell cycle completion requires both Myc and E2F activities. However, whether these two activities interact to regulate cell survival remains to be tested. Here we have analysed survival of inducible c-Myc-overexpressing cell lines derived from U2OS human osteosarcoma cells, which carry wild-type pRb and p53 and are deficient for p16 and ARF expression. Induced U2OS-Myc cells neither underwent apoptosis spontaneously nor upon reconstitution of the ARF-p53 axis and/or serum-starvation. However, they died massively when concomitantly exposed to inhibitors of E2F activity, including a constitutively active pRb (RbDeltacdk) mutant, p16, a stable p27 (p27T187A) mutant, a dominant-negative (dn) CDK2, or dnDP-1. Similar apoptotic effect was observed upon down-modulation of endogenous E2Fs through overexpression of E2F binding site oligonucleotides in U2OS-Myc cells, upon expression of RbDeltacdk or dnDP-1 in the Myc-amplified HL-60 (ARF-; p53-) human leukemia cells, and upon co-transfection of Myc and RbDeltacdk in SAOS-2 (ARF+; p53-) human osteosarcoma cells but not in human primary fibroblasts. Consistent with these results, a dnp53 mutant did not abrogate the Myc-induced apoptotic phenotype, which instead strictly depended on caspase-3-like proteases and on Myc transcriptional activity. Our data indicate that in contrast to normal cells, Myc-overexpressing human cancer cells need E2F activity for their survival, regardless of their ARF and p53 status, a notion that may have important implications for antineoplastic treatment strategies.
- MeSH
- apoptóza * fyziologie MeSH
- buněčné dělení fyziologie MeSH
- buněčný cyklus fyziologie MeSH
- časové faktory MeSH
- DNA vazebné proteiny * MeSH
- dominantní geny fyziologie MeSH
- down regulace MeSH
- genetické vektory genetika MeSH
- imunoblotting MeSH
- inhibitor p27 cyklin-dependentní kinasy MeSH
- inhibitory enzymů farmakologie MeSH
- inhibitory kaspas MeSH
- kaspasa 3 MeSH
- kaspasy metabolismus MeSH
- kultivované buňky MeSH
- lidé MeSH
- nádorové supresorové proteiny metabolismus MeSH
- nádorový supresorový protein p14ARF * metabolismus MeSH
- nádorový supresorový protein p53 metabolismus MeSH
- nádory kostí * metabolismus patologie MeSH
- oligonukleotidy * farmakologie MeSH
- osteosarkom * metabolismus patologie MeSH
- poly(ADP-ribosa)-polymerasy metabolismus MeSH
- proteiny buněčného cyklu metabolismus MeSH
- protoonkogenní proteiny c-myc * metabolismus MeSH
- retinoblastomový protein farmakologie metabolismus MeSH
- transfekce MeSH
- transkripční faktory E2F MeSH
- transkripční faktory genetika metabolismus MeSH
- upregulace MeSH
- western blotting MeSH
- Check Tag
- lidé MeSH
The "pocket proteins" pRb (retinoblastoma tumor suppressor protein), p107, and p130 regulate cell proliferation via phosphorylation-sensitive interactions with E2F transcription factors and other proteins. We previously identified 22 in vivo phosphorylation sites in human p130, including three sites selectively targeted by cyclin D-Cdk4(6) kinases. Here we assessed the effects of alanine substitution at the individual or combined Cdk4(6)-specific sites in p130, compared with homologous sites in p107 (Thr(369)/Ser(650)/Ser(964)). In U-2-OS cells, the triple p107(DeltaCdk4)* mutant strongly inhibited E2F-4 activity and imposed a G(1) arrest resistant to cyclin D1 coexpression. In contrast, the p130(DeltaCdk4) mutant still responded to cyclin D1, suggesting the existence of additional phosphorylation sites critical for E2F-4 regulation. Extensive mutagenesis, sensitive E2F reporter assays, and cell cycle analyses allowed the identification of six such residues (serines 413, 639, 662, 1044, 1080, and 1112) that, in addition to the Cdk4-specific sites, are necessary and sufficient for the regulation of E2F-4 and the cell cycle by p130. Surprisingly, 12 of the in vivo phosphorylation sites seem dispensable for E2F regulation and probably modulate other functions of p130. These results further elucidate the complex regulation of p130 and provide a molecular mechanism to explain the differential control of p107 and p130 by cyclin-dependent kinases.
- MeSH
- biologické modely MeSH
- buněčné dělení MeSH
- buněčné linie MeSH
- buněčný cyklus MeSH
- cyklin D1 metabolismus MeSH
- DNA vazebné proteiny * metabolismus MeSH
- fosfoproteiny * metabolismus MeSH
- fosforylace MeSH
- G1 fáze MeSH
- glutathiontransferasa metabolismus MeSH
- imunohistochemie MeSH
- jaderné proteiny * metabolismus MeSH
- lidé MeSH
- mutace MeSH
- peptidové mapování MeSH
- peptidy chemie MeSH
- plazmidy metabolismus MeSH
- protein p107 podobný retinoblastomu MeSH
- protein p130 podobný retinoblastomu MeSH
- proteiny * MeSH
- průtoková cytometrie MeSH
- rekombinantní fúzní proteiny metabolismus MeSH
- terciární struktura proteinů MeSH
- transkripční faktor E2F4 MeSH
- transkripční faktory * metabolismus MeSH
- vazebná místa 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
- MeSH
- artritida experimentální * MeSH
- chemické techniky analytické * metody využití MeSH
- experimenty na zvířatech MeSH
- glykosaminoglykany diagnostické užití metabolismus MeSH
- histocytochemie * metody využití MeSH
- histologické techniky metody využití MeSH
- kolagen účinky léků MeSH
- králíci MeSH
- modely nemocí na zvířatech MeSH
- osteoartróza MeSH
- papain diagnostické užití metabolismus MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- zvířata MeSH
