Resolution and Properties of the Catalytic Component and Phosphate Acceptor Proteins of a New Protein Kinase-Substrate Complex from Morris Hepatoma 3924A

Document Type

Article

Publication Date

1-1-1978

Abstract

A self-phosphorylating, polylysine-stimulated protein kinase, which was partially purified from the cytosol of fast-growing Morris hepatoma 3924A, was resolved into a catalytic component and several phosphate acceptor proteins when subjected to a phosphocellulose column. Other chromatographic and electrophoretic procedures were unable to dissociate them. When once dissociated, the catalytic component phosphorylated preferentially casein and nuclear phosphoproteins but not histone and protamine; the component appeared to be one of casein kinases of classical type. With the dissociated phosphate acceptor proteins as substrates, the phosphorylation reaction was stimulated greatly (2-to 10-fold, sometimes more) by the addition of polylysine or H1 histone. These basic polypeptides themselves were not phosphorylated in this reaction. Other histone fractions were less stimulatory. The apparent K a, for polylysine was about 8 μg/ml. When casein was used as phosphate acceptor, less than 2-fold stimulation was observed by the basic polypeptides. The molecular weight of the catalytic component was about 2.1 x 10 5 as estimated by gel filtration. Km for adenosine triphosphate was 4 x 10-6 m. The pH optimum was 7.5 to 8.5. When the dissociated phosphate acceptor proteins were used, in the absence of polylysine the catalytic component showed a biphasic concentration curve for Mg 2+ with optimum concentrations of 50 to 100 mM, whereas in the presence of polylysine the curve was normalized and the reaction proceeded well at lower Mg 2+ concentrations. In contrast, when casein was used as phosphate acceptor, the catalytic component was active at 10 to 75 mM Mg 2+ in both the presence and the absence of polylysine. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the phosphate acceptor proteins showed a major and several minor protein bands; the major protein had an approximate molecular weight of 7 x 10 4. The results indicate that the catalytic component is associated with its own phosphate acceptor proteins and that the phosphorylation of such phosphate acceptor proteins apparently requires another polycationic factor such as polylysine or histone. The naturally occurring polycationic factor in the cytoplasm has not yet been clarified. © 1978, American Association for Cancer Research. All rights reserved.

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