Multiplicity of Adenosine 3′,5′-Monophosphate-dependent Protein Kinases from Rat Liver and Mode of Action of Nucleoside 3′,5′-Monophosphate

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Abstract Two fractions of adenosine 3',5'-monophosphate (cyclic AMP)-dependent protein kinase (protein kinase B1 and B2) and one fraction of cyclic AMP-independent protein kinase (protein kinase B0) are resolved and partially purified from rat liver cytosol treated with diisopropyl fluorophosphate by ammonium sulfate fractionation followed by DEAE-Sephadex and hydroxylapatite column chromatography. Analysis with gel filtration on a Sephadex column reveals that protein kinase B0 shows a single peak with themolecular weight of approximately 3 x 104, whereas protein kinase B1 is a mixture of two, major and minor, species of cyclic AMP-dependent protein kinases with the molecular weights of 1.8 x 105 and 1.2 x 105, respectively. However, the minor species appears to be an artifact due to proteolysis during the isolation procedure. Protein kinase B2 is also a mixture of multiple cyclic AMP-dependent protein kinases with the molecular weights ranging from 1.0 x 105 up to more than 2.0 x 105. This heterogeneity does not seem to represent simply artifact due to proteolysis during the isolation procedure. Each of the cyclic AMP-dependent protein kinases is shown to be an inactive form of protein kinase, the activity of which is inhibited by association of regulatory protein (R-protein). R-protein per se is catalytically inactive. Cyclic AMP activates such inactive form by binding to the R-protein molecule in an allosteric manner, resulting in the release of active protein kinase. All active protein kinases thus released from both protein kinase B1 and B2 are indistinguishable from protein kinase B0, and show the molecular weight of 3 x 104. The active protein kinases show similar kinetic properties and behave in an identical manner upon chromatography on a hydroxylapatite column. These kinases are not stimulated nor inhibited by the cyclic nucleotide, and phosphorylate the same specific seryl and threonyl residues of histone. An available evidence suggests that the multiple cyclic AMP-dependent protein kinases differ from each other in their associated R-proteins, although the exact identity of the active protein kinases may be explored by further investigations. Protein kinase B1 is shown to be reconstituted from protein kinase B0 and each of the R-proteins with the molecular weights of 1.5 x 105 and 9 x 104, although the latter R-protein is probably an artifact due to proteolysis. These R-proteins are partially purified from rat liver cytosol. However, attempts to reconstitute protein kinase B2 have been unsuccessful. Most of the R-protein is unstable and decomposed to a smaller protein with the molecular weight of about 4 x 104. Protein kinase B1 and B2 show closely similar properties, but their affinities for cyclic AMP are slightly different.