FIGURE 2.

PKA phosphorylates NS5A on Thr-2332. A, kinetics of NS5A phosphorylation by PKA in vitro were monitored by using gel electrophoresis (top) to identify phosphorylated NS5A (NS5A-P). Care was taken to retain labeled ATP on gel, thus permitting quantitation (bottom) of phosphorylated NS5A, indicating that 1 eq of phosphate was incorporated/molecule of NS5A. B, spectrum of the shown peptide obtained by tandem mass spectrometry indicating phosphorylation on Thr-2332. C, NS5A derivatives having Thr-2332 changed to alanine (T2332A) or glutamic acid (T2232E) were treated with labeled ATP and PKA for 60 min and analyzed as in A. Loss of Thr-2332 prevented phosphorylation, consistent with this residue being the site of phosphorylation. D, an antibody was produced that specifically recognizes Thr(P)-2332-NS5A. Experimental validation of the specificity is shown, using NS5A wild type and the indicated derivatives. Phosphorylation was performed in vitro with PKA or CK2, resolved by gel electrophoresis, and then processed for Western blotting. Top, anti-Thr(P)-2332-NS5A was used; bottom, anti-NS5A was used.