Fig. 4.

Determination of CaMKIIα-binding site in syntaxin. A, Binding visualized using anti-CaMKII antibody. Each recombinant fragment derived from syntaxin 1A as a GST-fusion protein (4 nmol) was immobilized to glutathione Sepharose, incubated with autophosphorylated purified CaMKIIα (5 μg), and then cleaved by PreScission protease like the brain S₂ fraction (see legend to Fig. 1). Eluted fractions were resolved by 10% SDS-PAGE and immunoblotted against anti-CaMKIIα mAb. B, R151G is critical to binding of CaMKII to syntaxin, but mutation in this amino acid did not affect interactions between syntaxin and other syntaxin-binding proteins. Each GST-fusion protein was immobilized as well as those described in A and incubated with each recombinant protein (1 nmol) or with CaMKIIα (5 μg). After PreScission protease digestion, eluted proteins were resolved by 15% SDS-PAGE and immunoblotted. C, Dissociation of CaMKII from syntaxin by CaMKII-binding fragments derived from syntaxin 1A ([145–184]; squares) and glutamate receptors of NR2A ([1349–1461]; circles) and NR2B ([1290–1309];triangles). GST–syntaxin 1A [1–262] (4 nmol) was immobilized with glutathione Sepharose and then incubated with purified autophosphorylated CaMKII (5 μg). After rinsing with PBS, immobilized syntaxin–CaMKII complex was incubated with syntaxin [145–184] for 1 hr at 4°C and centrifuged. In some experiments, various concentrations of NR2A or NR2B fragments were incubated with immobilized syntaxin–CaMKII complex as described above. The supernatant containing released CaMKII was resolved by 10% SDS-PAGE and immunoblotted against anti-CaMKIIα mAb. The amount of the bound CaMKII before incubation with syntaxin fragments [145–184] or derived from glutamate receptors is designated as 100%.