Characterization of two cyclic AMP-independent protein kinases and their substrates in AH-66 hepatoma cells

Two cyclic AMP-independent protein kinases(CK 1 and CK 2) in the cytosol fraction of AH-66 hepatoma cells have been purified to homogeneity. CK 1 and CK 2 had molecular weights of 36,000 and 340,000, respectively, as determined by gel filtration. CK 1 consists of a single polypeptide and;CK 2 is composed of three subunits of 43,000, 42,000, and 26,000 molecular weights. CK 1 and the 26,000 molecular weight subunit of CK 2 were autophosphorylated. CK 1 preferentially used ATP as phosphate donor, whereas CK 2 utilized both ATP and GTP. Both enzymes markedly catalyzed the phosphorylation of AH-66 cytosolic proteins with molecular weights of 125,000, 95,000, and 40,000. In order to examine whether these phosphoproteins are specifically present in AH-66 cytosol, CK 1 enzymes purified from AH-66 and liver cytosol were added back to AH-66 and liver cytosol. The CK 1 enzymes from both AH-66 cytosol and liver cytosol markedly catalyzed the phosphorylation of these phosphoproteins in AH-66 cytosol. These phosphoproteins in liver cytosol were less intensely phosphorylated by the addition of the CK 1 enzymes from both sources. From these results we conclude that these phosphoproteins are present in both AH-66 cytosol and liver cytosol,but are highly concentrated in AH-66 cytosol.