Phosphatidylglycerol-modulated protein kinase activity from human spleen. I. Enzyme purification and properties.
Protein kinase P (PK-P) is a phospholipid-modulated protein kinase activity previously described in human and murine cells. This paper details the 3300-fold, high yield purification to electrophoretic homogeneity of protein kinase P from human spleen by a three-step chromatographic process. Physical characterization disclosed a protein of Mr 27,000 (by electrophoresis) or 31,700 (by gel filtration and sedimentation) and pI 5.09. Protein kinase P activity was stimulated by phosphatidylglycerol or phosphatidylinositol, with maximal stimulation observed between 200 and 400 micrograms/ml phospholipid. No stimulation was noted using phosphatidic acid or phosphatidylserine. Histone H2B was the best substrate for demonstrating the protein kinase P phospholipid stimulation. Histone H1 was phosphorylated in a phospholipid independent manner. Vinculin and actin were not substrates. Optimum enzyme activity was observed at approximately 35 degrees C and pH 6.95. PK-P was relatively insensitive to the calmodulin and protein kinase C inhibitors W7 and H7, and to the cAMP-dependent protein kinase inhibitor. Kinetic analysis disclosed complex patterns including optimal rather than Michaelis-Menton kinetics for histone and phospholipid concentration, and a steep activation threshold with respect to histone concentration in the presence of phospholipid. Biphasic kinetics for Mg2+-ATP were observed, with the major stimulatory effect of phospholipid being on Vmax rather than Km. These data suggest a model for the mechanism of activation of protein kinase P by phospholipid entailing a direct three-way interaction between substrate, enzyme, and phospholipid micelles rather than allosteric activation by phospholipid.
Department of Medicine, University of New Mexico Cancer Center, Albuquerque 87131.
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D000255:Adenosine Triphosphate / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D006657:Histones / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D010715:Phosphatidylglycerols / Q000494:pharmacology*; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000302:isolation & purification*; D017346:Protein Serine-Threonine Kinases; D013154:Spleen / Q000187:drug effects / Q000201:enzymology*
D006657:Histones; D010715:Phosphatidylglycerols; D047428:Protein Kinase Inhibitors; D000255:Adenosine Triphosphate; D011494:Protein Kinases; C054566:protein kinase P; D017346:Protein Serine-Threonine Kinases