HIV-1 Tat is phosphorylated on S16 and S46 residues in vivo. A, HeLa cells were infected with recombinant adenovirus expressing Flag-tagged Tat as described in Methods. At 48 hours post infection cells were labeled with (32P)-orthophosphate for 2 hours with 1 μM okadaic acid (OA). Lane1, Flag-Tat was immunoprecipitated from whole cell extracts with anti-Flag antibodies and resolved by 15% Tris-Tricine SDS-PAGE. Lane 2, control mock-transfected cells. The picture is an autoradiogram. B, Tat peptides were eluted from the gel shown in panel A by overnight incubation with trypsin and subjected to acid hydrolysis as described in Materials and Methods. The hydrolyzed material was spotted on nitrocellulose plate and examined by two-dimensional thin layer electrophoresis and autoradiography. The indicated positions of non-radioactive phospho-amino acid standards were visualized by staining with 0.5% ninhydrin in ethanol. C, Mutations of S16 and S46 reduce Tat phosphorylation in vivo. 293T cells were transfected with vectors expressing Flag-tagged WT Tat (lane 2), Tat S16A (lane 3), Tat S46A (lane 4) or Tat S16,46A (lane 5). Lane 1, mock transfection. At 48 hours post-transfection the cells were labeled with (32P)-orthophosphate for 2 hours. Whole cell extract was subjected for immunoprecipitation with anti-Flag antibodies, resolved by 15% Tris-Tricine SDS-PAGE, and analyzed by Western blotting with polyclonal anti-Tat antibodies and on Phosphor Imager. Quantification is shown in the lower panel. Position of Tat is indicated by arrow.