Figure 4.
Effect of CaM-KIIN on GluR1 phosphorylation by CaM-KII and PKC. (A) Kinase reactions, performed at 30°C for 20 min with the indicated mixtures of 0.2 mM [γ 32P]-ATP, 20 nM PKC catalytic fragment (PKM, Calbiochem) or 20 nM CaM-KII, 4 μM GST or 4 μM GST/GluR1816–889 with or without 1 μM CaM-KIINtide, were separated by SDS/PAGE and visualized by autoradiography. GST/GluR1816–889 migrated as a doublet. (B) HEK 293 cells, transfected with GluR1 without or with cotransfected CaM-KII (H282R) were 32P-labeled. Some cells without CaM-KII transfection were stimulated with phorbol 12-tetradecanoate 13-acetate to activate PKC for 20 min. Cells were harvested, and GluR1 was immunoprecipitated, analyzed by SDS/PAGE/autoradiography (Upper), and subjected to GluR1 Western blot (Lower).