Promotion of S-phase entry and cell growth under serum starvation by SAG/ROC2/Rbx2/Hrt2, an E3 ubiquitin ligase component: association with inhibition of p27 accumulation.
The sensitive-to-apoptosis gene (SAG) was initially identified as a redox-inducible, apoptosis-protective protein and subsequently found to be the second family member of regulator of cullins (ROC)/RING box protein (Rbx)/Hrt, which acts as a component of E3 ubiquitin ligase. We report here that SAG promoted cell growth under serum starvation. Microinjection of SAG mRNA into quiescent NIH/3T3 cells induced S-phase entry as determined by [(3)H]-thymidine incorporation. Likewise, overexpression of SAG by either adenovirus infection of immortalized human epidermal keratinocytes (Rhek-1) or DNA transfection of SY5Y human neuroblastoma cells induced cell proliferation under serum starvation. Because cyclin-dependent kinase inhibitors (CKIs), including p21, p27, and p57, are degraded through the ubiquitin pathway, we tested whether SAG-induced cell growth is associated with CKI degradation. Although there was no significant difference in the levels of p21 and p57 between the vector controls and SAG-overexpressing cells, serum starvation induced 10- to 18-fold accumulation of p27 in control Rhek-1 cells. Accumulation of p27 was remarkably inhibited (only 2 to 5-fold) in SAG-infected cells. Inhibition of p27 accumulation was also observed in stably SAG-overexpressing SY5Y cells. Significantly, SAG-associated inhibition of p27 accumulation was largely abolished by the treatment with a proteasome inhibitor. In vivo binding of SAG and Skp2, an F-box protein that promotes p27 ubiquitination, was detected, and the binding was enhanced in SAG-overexpressing cells grown under serum starvation. Thus, SAG-induced growth with serum withdrawal appears to be associated with SAG-mediated p27 degradation. Mol. Carcinog. 30:37-46, 2001.
Department of Molecular Biology, Pfizer Global Research and Development, Ann Arbor Laboratories, Ann Arbor, Michigan 48105, USA.
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Duan
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Tsvetkov
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Liu
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Song
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Swaroop
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Wen
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Kung
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Zhang
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Sun
D000818:Animals; D018797:Cell Cycle Proteins; D002455:Cell Division / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D016895:Culture Media, Serum-Free; D050760:Cyclin-Dependent Kinase Inhibitor p27; D003546:Cysteine Endopeptidases; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D006801:Humans; D008025:Ligases / Q000378:metabolism*; D051379:Mice; D008845:Microinjections; D008869:Microtubule-Associated Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors; D046988:Proteasome Endopeptidase Complex; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D016196:S Phase; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins; D044767:Ubiquitin-Protein Ligases
C493961:Cdkn1b protein, mouse; D018797:Cell Cycle Proteins; D016895:Culture Media, Serum-Free; D015853:Cysteine Proteinase Inhibitors; D008869:Microtubule-Associated Proteins; D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D044767:Ubiquitin-Protein Ligases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D008025:Ligases