OCCURRENCE AND ACTIVATION OF CA2+ CALMODULIN-DEPENDENT PROTEIN-KINASE-II AND ITS ENDOGENOUS SUBSTRATES IN BOVINE ADRENAL-MEDULLARY CELLS/

We investigated the presence of and the endogenous substrates for Ca2/calmodulin-dependent protein kinase II (CaM kinase II) in cultured bo vine adrenal medullary cells. By a series of chromatographic steps usi ng DEAE-cellulose, calmodulin affinity, and Sephacryl S-300 columns, w e partially purified two CaM kinases (peaks I and III) and one calmodu lin-binding protein (peak II). Both of the kinases (peaks I and III) s howed broad substrate specificities. Peak I, but not peak III, was imm unoprecipitated with an antibody against rat brain CaM kinase II, sugg esting that peak I is CaM kinase II or a closely associated CaM kinase . Although the anticaldesmon antibody recognized a 77-kDa protein (low molecular mass caldesmon) in crude preparations from the cells, the p rotein in peak II was not immunoblotted with the antibody. The peak II protein was phosphorylated by the CaM kinase in peak I but not by the CaM kinase in peak III. Peak I kinase also phosphorylated purified ty rosine hydroxylase and several proteins from chromaffin granule membra nes. Stimulation of cultured bovine adrenal medullary cells with 56 mM K+ evoked rapid increases in Ca-45(2+) influx and autonomous CaM kina se II activity, both of which were attenuated by the addition of 20 mM MgSO4, an inhibitor of voltage-dependent Ca2+ channels. These results suggest that an isozyme of CaM kinase II exists in adrenal medullary cells and is activated by cell depolarization. Furthermore, the peak I I protein is apparently a novel endogenous substrate for CaM kinase II .