aPKC phosphorylates HIV-1 Gag. (A) Schematic representation of the AlphaScreen based luminescent system used to screen for human kinases that interact with HIV-1 Gag. Briefly, HIV-1 Gag was incubated with each human protein kinase and protein A-conjugated acceptor beads with anti-FLAG antibody and streptavidin coated donor beads were added and bound to the tagged substrate. Upon laser excitation, the donor beads convert ambient oxygen to singlet oxygen. When a molecular interaction occurs between HIV-1 Gag and a particular kinase, singlet oxygen transfers across to activate the acceptor beads and subsequently emit light at 520–620 nm. (B) HIV-1 Gag interacting kinases identified from a human kinase protein library. (C) Confocal microscopy analysis of V5-aPKC (Alexa 568) and Gag-Flag (Alexa 488) in 293T cells. (D) Immunoprecipitation of Gag with aPKC. 293T cells were transfected with Flag tagged Gag and cell lysates were immunoprecipitated with anti-Flag antibody 48 hours later. Samples were then probed with anti-aPKC and anti-Flag antibodies. Bottom panel shows input for pull-down assay. (E) In vitro phosphorylation of HIV-1 Gag by aPKC. GST, GST tagged aPKC and Gag proteins were expressed and affinity-purified from wheat germ cell-free extract. aPKC-mediated phosphorylation was assessed by incubating recombinant GST or GST-Gag with recombinant active GST-aPKC in the presence of [γ-32P] ATP. The reaction products were analyzed by autoradiography, Coomassie Brilliant Blue (CBB) stain and blotted with GST antibody.