Interaction between simian virus 40 large T antigen and insulin receptor substrate 1 is disrupted by the K1 mutation, resulting in the loss of large T antigen-mediated phosphorylation of Akt.
The cellular kinase Akt is a key controller of cellular metabolism, growth, and proliferation. Many viruses activate Akt due to its beneficial effects on viral replication. We previously showed that wild-type (WT) simian virus 40 (SV40) large T antigen (TAg) inhibits apoptosis via the activation of PI3K/Akt signaling. Here we show that WT TAg expressed from recombinant adenoviruses in U2OS cells induced the phosphorylation of Akt at both T308 and S473. In contrast, Akt phosphorylation was eliminated by the K1 mutation (E107K) within the retinoblastoma protein (Rb) binding motif of TAg. This suggested that Akt phosphorylation may depend on TAg binding to Rb or one of its family members. However, in Rb-negative SAOS2 cells depleted of p107 and p130 by using small hairpin RNAs (shRNAs), WT TAg still mediated Akt phosphorylation. These results suggested that the K1 mutation affects another TAg function. WT-TAg-mediated phosphorylation of Akt was inhibited by a PI3K inhibitor, suggesting that the effects of TAg originated upstream of PI3K; thus, we examined the requirement for insulin receptor substrate 1 (IRS1), which binds and activates PI3K. Depletion of IRS1 by shRNAs abolished the WT-TAg-mediated phosphorylation of Akt. Immunoprecipitation studies showed that the known interaction between TAg and IRS1 is significantly weakened by the K1 mutation. These data indicate that the K1 mutation disrupts not only Rb binding but also IRS1 binding, contributing to the loss of activation of PI3K/Akt signaling.
Yu
Yongjun
Y
Department of Cancer Biology, 314 Biomedical Research Building, 421 Curie Blvd., School of Medicine, University of Pennsylvania, Philadelphia, PA 19104-6142, USA.
Alwine
James C
JC
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000957:Antigens, Viral, Tumor / Q000378:metabolism*; D006801:Humans; D055504:Insulin Receptor Substrate Proteins; D020125:Mutation, Missense; D010766:Phosphorylation; D011485:Protein Binding / Q000235:genetics; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D016160:Retinoblastoma Protein / Q000378:metabolism; D013539:Simian virus 40 / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D000957:Antigens, Viral, Tumor; C526292:IRS1 protein, human; D055504:Insulin Receptor Substrate Proteins; D016160:Retinoblastoma Protein; D051057:Proto-Oncogene Proteins c-akt