Uev1A, a ubiquitin conjugating enzyme variant, inhibits stress-induced apoptosis through NF-kappaB activation.

We have previously shown that UEV1 is up-regulated in all tumor cell lines examined and when SV40-transformed human embryonic kidney cells undergo immortalization; however, it is unclear whether and how UEV1 plays a critical role in this process. UEV1A encodes a ubiquitin conjugating enzyme variant, which is required for Ubc13 (ubiquitin conjugating enzyme) catalyzed poly-ubiquitination of target proteins through Lys63-linked chains. One of the target proteins is NEMO/IKKgamma (nuclear factor-kappaB essential modulator/inhibitor of kappaB protein kinase), a regulatory subunit of IkappaB kinase in the NF-kappaB signaling pathway. In this report, we show that constitutive high-level expression of UEV1A alone in cultured human cells was sufficient to cause a significant increase in NF-kappaB activity as well as the expression of its target anti-apoptotic protein, Bcl-2 (B-cell leukemia/lymphoma 2). Overexpression of UEV1A also conferred prolonged cell survival under serum-deprived conditions, and protected cells against apoptosis induced by diverse stressing agents. All of the effects of Uev1A were reversible upon suppression of UEV1 expression by RNA interference. Our observations presented in this report provide evidence that Uev1A is a critical regulatory component in the NF-kappaB signaling pathway in response to environmental stresses and identify UEV1A as a potential proto-oncogene.

Effect of overexpression and nuclear translocation of constitutively active PKB-alpha on cellular survival and proliferation in HepG2 cells.

Protein kinase B (Akt/PKB) is a key component in the PI 3-kinase mediated cell survival pathway and has oncogenic transformation potential. Although the over-expression of PKB-alpha can prevent cell death following growth factor withdrawal, the long-term effects of stable over-expression of PKB-alpha on cell survival in the absence of growth factors remain to be resolved. In the present study, we generated HepG2 cells with stable expression of active PKB-alpha and compared its characteristics with HepG2 cells. Basal as well as insulin-stimulated levels of Ser(473) and Thr(308) phosphorylation in PKB-alpha transfected HepG2 cells were much higher than HepG2 cells. Constitutive expression of active PKB-alpha enabled HepG2 cells to survive up to 96 h without serum in growth media while HepG2 cells fail to survive after 48 h of serum withdrawal. A strong positive correlation (R(2) = 0.71) between cell proliferation and phosphorylated form of PKB-alpha at Thr(308) was observed along with higher levels of phosphorylated 3'-phosphoinositide-dependent kinase-1 (PDK-1). HepG2 cells with constitutive expression of active PKB-alpha also showed higher levels of phosphorylated p65 subunit of nuclear factor-kappaB (NFkappaB) in comparison with HepG2 cells. Predominant nuclear localization of phosphorylated PKB-alpha was observed in stably transfected HepG2 cells. These results indicate that constitutive expression of active PKB-alpha renders HepG2 cells independent of serum based growth factors for survival and proliferation.