Intracellular localization of the catalytic subunit from cyclic AMP-dependent protein kinase in mutant Chinese hamster ovary cells deficient in this enzyme.

Abstract

A direct cytochemical procedure that specifically locates free catalytic subunits (C) from cAMP-dependent protein kinase has been used to follow the kinetics of kinase dissociation in parental Chinese hamster ovary cells (CHO 10001) and 4 mutant CHO cell lines variously deficient in this enzyme (CHO 10215, 10248, 10260, 10265) (1-5). When cultures of wild-type cells were stimulated with 8BrcAMP a time- and dose-dependent dissociation of kinase was observed. The catalytic unit appeared first in the cytoplasm and nucleolus and with time in the nucleoplasm as well. At peak protein kinase activation (30 min) more than 80% of the cells possessed abundant C in all of these subcellular compartments. The data indicate that both the type I and type II isozymes of the cAMP-dependent protein kinase are localized in similar areas of the cell. Stimulation of the mutant cell lines with 8-BrcAMP revealed that they each contained activatable kinase, determined cytochemically, that paralleled in amount the total assayable protein kinase determined biochemically (1-6). There were differences in the basal (unstimulated) levels of free C in the mutants relative to each other and to wild-type cells. For example, wild-type and mutants 10248, and 10260 had barely detectable cytoplasmic catalytic-subunit in unstimulated cultures whereas mutant 10215 possessed a significant amount of free C. Upon stimulation with 8-BrcAMP, the subcellular distribution of C was in all cases similar; although there were significant quantitative disparities between the wild type and various mutant cell lines. All cell lines examined had roughly equivalent amounts of nucleolar C but differed predominantly in the quantity of cytoplasmic kinase. Specifically, two of the mutant lines (10260 and 10248) contained barely detectable amounts of nucleoplasmic C whereas the other mutants had moderate (10265) to near normal (10215) amounts of nucleoplasmic enzyme. Mutants having only type I isozyme (10248) and those with only type II isozyme (10265) gave similar patterns of free C localization after 8BrcAMP stimulation.