A Plasmocytoma Ribosome‐Associated Protein Kinase which Phosphorylates a Specific Protein of the Ribosomal KCl Wash

Abstract

One ribosomal protein kinase activity and 3 soluble protein kinase activities were indentified in [mouse] plasma cell tumors by DEAE-cellulose chromatography. Phosphorylation was shown in vivo and in vitro of a protein fraction from the ribosomal KCl wash which was termed PPx fraction. Phosphorylation of this protein fraction was obtained in vitro with the ribosome-associated protein kinase. For the ribosomal protein kinase, the following characteristics were determined. It is an Mg2+ -dependent enzyme that transfers the .gamma.-phosphate from ATP into phosphoseryl and phosphothreonyl residues of the substrate. It has a wide substrate specificity. Like the soluble protein kinases it catalyzes the phosphorylation of several proteins like histone, phosvitin, casein and ribosomal proteins, but it differs from the main soluble kinases (I, II) by the fact that it catalyzes specifically the phosphorylation at least of 1 of the ribosomal KCl wash proteins. On dodecylsulfate-polyacrylamide gels this protein has a molecular weight of .apprx. 90,000 and it is released from ribosomes under conditions commonly employed for extraction of initiation factors. The ribosome-associated protein kinase is not stimulated by the addition of cyclic AMP. KCl has no effect, NaCl has a weak effect on phosphorylation and Mn2+ and Ca2+ are inhibitors. ADP was a competitive inhibitor. The maximum velocity of the ribosomal protein kinase-catalyzed reaction is 0.65 nmol of 32P incorporated in the KCl wash protein per min and per mg protein. The apparent Km for the ribosomal KCl wash protein as substrate is 0.71 mg/ml and the Km for ATP is 94 .mu.M. The molecular weight of the ribosomal protein kinase, estimated by electrophoresis in polyacrylamide-dodecylsulfate gels, is 60,000 and probably corresponds to a catalytic subunit.