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-i nducible, apoptosis-protective protein and subsequently found to be the sec ond 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 int o quiescent NIH/3T3 cells induced S-phase entry as determined by [H-3]-thym idine incorporation. Likewise, overexpression of SAG by either adenovirus i nfection of immortalized human epidermal keratinocytes (Rhek-1) or DNA tran sfection of SY5Y human neuroblastoma cells induced cell proliferation under serum starvation. Because cyclin-dependent kinase inhibitors (CKIs), inclu ding p21, p27, and p57, are degraded through the ubiquitin pathway, we test ed whether SAG-induced cell growth is associated with CKI degradation. Alth ough there was no significant difference in the levels of p21 and p57 betwe en the Vector controls and SAG-overexpressing cells, serum starvation induc ed 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-overexpressi ng SY5Y cells. Significantly, SAG-associated inhibition of p27 accumulation was largely abolished by the treatment with a proteasome inhibitor. In viv o binding of SAG and Skp2, an F-box protein that promotes p27 ubiquitinatio n, was detected, and the binding was enhanced in SAG-overexpressing cells g rown under serum starvation. Thus, SAG-induced growth with serum withdrawal appears to be associated with SAG-mediated p27 degradation. (C) 2001 Wiley -Liss, Inc.