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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 [3H]‐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. © 2001 Wiley‐Liss, Inc.