Docking groove of SRPK2 governs the binding and phosphorylation of Cp.

(A) Crystal structure of SRPK2ΔNS (PDB ID: 5MYV). A SRPK-specific docking groove is located at the large lobe of the kinase. Active site of the kinase is indicated. Four amino acid residues that are critical for substrate binding and phosphorylation are indicated (right). (B) In vitro GST pull-down assay of GST-tagged CpY132A with SRPK2WT or SRPK2DM. Free GST was used as a control. Samples were resolved by SDS-PAGE and stained by Coomassie Blue. Mutations at the docking groove of SRPK2 abolished the binding of Cp. (C) In vitro radioactive kinase assays with SRPK2WT or SRPK2DM and CpY132A were performed for the indicated time points in the presence of [32P]ATP. Samples were resolved by SDS-PAGE and visualized by autoradiography (upper panels). Radiolabeled protein bands corresponding to phosphorylated CpY132A were excised and quantified by scintillation counting. Data are expressed as mean ± S.D. for three independent experiments (lower panel). (D) In vitro radioactive kinase assays with SRPK2WT or SRPK2DM and CpWT capsid for extended periods of time. The reactions were analyzed by SDS-PAGE and visualized by autoradiography. SRPK2DM failed to phosphorylate CpWT capsid to the same extent as SRPK2WT.