Figure 2

WNV MTase domain is phosphorylated at Ser38 by PKG in vitro . (a) WNV MTase and full-length (FL) NS5 were bacterially expressed and purified. MTase (Lane 1 and 2) and full-length NS5 (Lane 3 and 4) were used as substrates in an in vitro kinase assay with PKGIα enzyme and γ-³²P ATP. The upper panel is an autoradiograpHshowing relative levels of ³²P incorporation, indicative of protein phosphorylation. The lower panel is the same SDS-PAGE gel, Coomassie-stained to demonstrate equal protein loading. (b) Bacterially expressed and purified WNV MTase was incubated with bovine lung PKGIα, cGMP, and non-radiolabelled ATP. The sample was separated on 10% SDS-PAGE, and the 33kD band corresponding to the MTase was excised for mass spectrometric analysis. The sample was subjected to an in-gel tryptic digest, and half of the sample was treated with 20 units of phosphatase. The MALDI-TOF spectrum of the sample that was not treated with phosphatase is shown, with the monoisotopic peak of the singly phosphorylated peptide corresponding to aa Ser38-Lys45 with a mass of 948.64 Da indicated. (c) MALDI-TOF spectrum of the sample after treatment with phosphatase. The peak corresponding to the Ser38-Lys45 peptide is shown with an approximately 80 Da decrease in mass, as compared to (b). (d) LC-MS/MS ionizations of the phosphorylated peptide corresponding to aa Glu30-Lys41 showing the specific phosphorylation of the Ser38 residue. The peptide was further purified through a C-18 HPLC column before fragmentation. The mass was calculated for post-translational modifications with an addition of 79.9 for one phosphate in the peptide sequences. Major fragment ions are labeled with their corresponding b (C-terminal) and y (N-terminal) ions and their charge state.