Promotion of S-Phase entry and cell growth under serum starvation by SAC/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 SAC mRNA into quiescent NIH/3T3 cells induced S-phase entry as determined by [H-3]-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. (C) 2001 Wiley-Liss, Inc.

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