Figure 1. In vitro phosphorylation of lamin A by GST-UL97.
(A) Recombinant His-tagged lamin A was incubated in kinase reaction buffer in the presence of γ-32P-ATP either alone (no kinase), with catalytically deficient GST-UL97 K355Q (K355Q), or with wild-type GST-UL97 (GST97 WT). GST-UL97 K335Q or wild-type GST-UL97 were also incubated in kinase buffer without lamin A. Following termination of kinase reactions, proteins were resolved by SDS-PAGE. Signal from incorporation of 32P was detected by exposure to a phosphorscreen (top panel), and total protein was detected by Coomassie brilliant blue staining (bottom panel). The positions of radiolabeled GST-UL97 (GST97) and lamin A, and Coomassie stained lamin A are indicated. (The amounts of GST-UL97 were too small to see on the stained gel.) (B) UL97 autophosphorylation and labeling of lamin A were quantified following in in vitro kinase reactions in the presence of varying concentrations of maribavir (MBV). Signal detected from 32P incorporation for autophosphorylation of GST-UL97 and phosphorylation of His-tagged lamin A are plotted as a percent of the signal detected in the absence of drug. The results taken together show that UL97 phosphorylates lamin A in vitro.