aPKC phosphorylates HIV-1 Gag on Ser487. (A) GST-p6, GST-p6S461A or GST-p6S487A proteins were expressed and affinity-purified from wheat germ cell-free extracts. aPKC-mediated phosphorylation was assessed by incubating recombinant GST-p6, GST-p6S461A or GST-p6S487A with recombinant active GST-aPKC in the presence of [γ-32P] ATP for 60 min at 37°C. The reaction products were analyzed by autoradiography and Coomassie Brilliant Blue (CBB) staining. (B) Assessment of phospho-Ser487 antibody specificity and binding efficiency using the AlphaScreen system. Biotin tagged peptides were incubated with phospho-Ser487 specific antibodies for. The graph shows the luminescence signal intensity which represents antibody binding efficiency. Each value is the mean of three independent experiments. (C) 293T cells were transfected with wild type Gag-pol and either V5 tagged wild type aPKC (WT) or kinase-negative aPKC (Kn). Whole-cell extracts were prepared, and equal amounts of proteins for each sample were separated by SDS-PAGE and subjected to western blot analysis using indicated antibodies. (D) Effects of aPKC inhibitor on the phosphorylation of Gag-Ser487. 293T cells were transfected with Gag-pol expression vector and cultured in the presence of the indicated concentrations of aPKC inhibitor. Clarified cell lysates were prepared and equal amounts of proteins were subjected to SDS-PAGE, followed by western blot analysis using indicated antibodies. Densitometry analysis of the p55Gag (pS487)/p55Gag bands in the western blot is shown in the bottom panel. (E) Effects of aPKC inhibitor on viability of 293T cells. 293T cells were treated with aPKC inhibitor (2 or 5 μM) or 1 μM Staurosporine (STS) and analysed for cell viability using trypan blue exclusion at 48 hours. (F) Effects of kinase specific inhibitors on the phosphorylation of Gag-Ser487. 293T cells were transfected with Gag-pol expression vector and treated with inhibitors against cPKC, AKT, CDK and PI3K. Cell lysates were prepared and analyzed as described in (D).