CK2 phosphorylates the Na_v1.2 ankyrin-binding motif and regulates its interaction with MBD-ank. (A–C) The Na_v1.2 ankyrin-binding motif is phosphorylated by CK2 in vitro. (A) Schematic representation of GST-Na_v1.2 linker II-III constructs. The position of the ankyrin-binding motif is indicated by gray boxes. Numbers refer to the position of corresponding amino acid residue in Na_v1.2. (B) The constructs were subjected to in vitro CK2 phosphorylation followed by SDS-PAGE. Coomassie brilliant blue staining (CBB) and ³²P incorporation revealed by autoradiography are shown. (C) The effect of site-directed serine mutations on CK2 phosphorylation of GST-Na_v1.2 II-III. CBB staining (bottom) and ³²P incorporation revealed by autoradiography (top) are shown. Numbers to the right of the gel blots indicate the molecular mass of standard markers (in kD). (D–F) SPR analysis of the interaction between the Na_v1.2 ankyrin-binding motif and MBD-ank. Typical sensorgrams are illustrated. Increasing concentrations of MBD-ankB ranging from 0.1 to 250 nM were injected over immobilized GST-Na_v1.2 1,080–1,203 (D), GST-Na_v1.2 989–1,133 (E), and GST-Na_v1.2 1,080–1,203 E1111A (F) before (top) and after in situ CK2 phosphorylation (bottom) of the immobilized constructs.