Fig. 2.

CDK2 phosphorylates Tat Ser-16 and PKR phosphorylates Tat Ser-46 in vitro. a Sequence of Flag-tagged HIV-1 Tat indicating peptides that were used for the analysis of Tat phosphorylation in vitro. Potential phosphorylation sites are underscored. Tat Ser-16 and Ser-46 residues are further indicated with superscript numbering. Ser-16 (peptide 12–29); Thr-39 and Thr-40 (peptide 29–45); Ser-46 (peptide 41–57); and Ser-62, Thr-64, Ser-68 and Ser-70 (peptide 57–71). b Phosphorylation of Tat peptides in vitro. Upper panels, Tat peptides (4 µg) were phosphorylated in vitro with recombinant enzymes CDK2/cyclin E (lanes 1–4), CDK9/cyclin T1 (lanes 5–8), and PKR (lanes 9–12) with γ(³²P)ATP as described in Methods. Phosphorylated peptides were resolved on 12% SDS Tris-Tricine gel containing 6 M urea, stained with SimpleBlue SafeStain (Coomassie), dried and analyzed by Phospho Imager. Phosphorylated Tat peptides position indicated with arrows on the right. Lower panel, Coomassie stained gel of Tat peptides showing 20 μg of Tat peptides 12–29, 29–45 and 57–71 and 2 μg of Tat peptide 41–57 resolved on 12% SDS Tris-Tricine gel with urea and stained with SimpleBlue SafeStain (Coomassie). c Relative intensities of the peptides phosphorylated by CDK2/cyclin E, CDK9/cyclin T1 or PKR were quantified with OptiQuant software (Packard)