Phosphorylation of brain cytosol proteins. Effects of phospholipids and calmodulin

Calmodulin was removed from brain cytosol by DEAE-52 chromatography or by affinity chromatography employing fluphenazine-Sepharose. The substrates phosphorylated by endogenous protein kinase after chromatography differed depending on the method used, and both chromatographic methods altered the phosphorylation pattern as compared to untreated cytosol. Cytosol, chromatographed on fluphenazine-Sepharose, retained most of the characteristics of untreated cytosol. Both calmodulin and phospholipids increased the phosphorylation of specific but separate brain cytosol proteins in a Ca2+-dependent manner. The effects of phospholipids could be mimicked by the detergent, sodium dodecyl sulfate, and the hydrophobic probe, 8-anilino-1-naphthalenesulfonate. Furthermore, the calmodulin-induced increase in phosphorylation, but not that produced by phospholipids, was blocked by 8-anilino-1-naphthalenesulfonate. These results suggest that the effects of phospholipids may not be due to the presence of a specific phospholipid-sensitive protein kinase in cytosol, but rather to a general interaction of hydrophobic probes with either specific substrate proteins or with the Ca2+-calmodulin-dependent protein kinase itself.