FIGURE 6.
CK2 phosphorylates CAPS in vivo. A, CAPS phosphorylation was inhibited in PC12 cells treated with a specific CK2 inhibitor. PC12 cells were labeled with [32P]orthophosphate for 1 h in the absence or presence of 60 μm tetrabromo-2-benzotriazole (TBB). CAPS immunoprecipitates were analyzed by 7.5% SDS-PAGE and autoradiography or Western blotting with a general CAPS antibody (Ab). Densitometric scanning of autoradiogram and Western blot was used to assess relative phosphorylation. Values are the mean of triplicate determinations with the indicated S.D. Tetrabromo-2-benzotriazole inhibited CAPS phosphorylation by 70%. B, CAPS dephosphorylation was promoted by Ca2+ influx in synaptosomes. Mouse brain synaptosomes were incubated under control conditions (♦) or stimulated with high K+ (▲) for the indicated times. Synaptosomes were hypotonically lysed, and supernatant fractions were analyzed by Western blotting with Ser(P)-5 and -6 phosphospecific antibody or general CAPS antibody. For the graphic representation the signal at the zero time point is set as 100%, and values are the mean of duplicate determination with indicated ranges.