Cyclic nucleotide-independent protein kinases bound to cytoplasmic and nuclear polyribosomes in non-infected and adenovirus-infected HeLa cells

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Abstract
Cyclic nucleotide-independent protein kinase activity bound to cytoplasmic and nuclear polyribosomes from noninfected and adenovirus-infected HeLa [human cervical carcinoma] cells was compared. The enzymes catalyzed the incorporation of phosphate from .gamma.-32P-labeled ATP or GTP into acid-precipitable material in the absence of exogenous substrates. Their activity was not affected by cyclic[c]AMP or cGMP and was not inhibited by a cyclic nucleotide-dependent protein kinase-inhibitor protein. The kinases are tightly bound to polyribosomes from infected and noninfected cells, since treatment with 0.5 M-NaCl did not dissociate the activity. The enzymes and the enzyme-associated endogenous substrates of cytoplasmic polyribosomes are significantly different from those of the nucleus, and adenovirus infection of the cells did not alter the nature of the enzymes or the substrates at 18-20 h after infection. Nuclear kinases catalyzed 3- to 4-fold more phosphate incorporation than did the cytoplasmic kinases. They did not phosphorylate endogenous substrates in the cytoplasmic preparations, and vice versa, which suggests that such substrates for cytoplasmic and nuclear kinases are specific. Polyacrylamide-gel electrophoresis of the phosphorylated proteins revealed the presence of a higher number of endogenous substrates in the nuclear preparation. The nuclear kinases phosphorylated all histones from HeLa cells, but the cytoplasmic ones phosphorylated predominantly the histone of MW 12,000. Bovine heart kinase phosphorylated several low-MW cytoplasmic proteins and no nuclear proteins. With a DEAE-cellulose column either enzyme activity could be resolved into a number of peaks. The substrate specificities of these peaks indicate that there are at least 2 different forms of the enzyme in each preparation of polyribosomes.