Characterization of cyclic AMP-resistant Chinese hamster ovary cell mutants lacking type I protein kinase.

Abstract

A group of three mutants of Chinese hamster ovary cells (10260, 10265, and 10223) which are resistant to cyclic AMP (Gottesman, M. M., LeCam, A., Bukowski, M., and Pastan I. (1980) Somatic Cell Genet. 6, 45-61) have been characterized in this work. By genetic analysis, these mutants are all recessive and fall into two complementation groups. Cycl AMP-stimulated protein kinase activity in crude extracts of these mutants using histone as a substrate is decreased to 10 and 7% (complementation group I), and 31% (complementation group II), respectively, of the activity found in wild type extracts. The binding of cyclic [3H]AMP by extracts of all of these mutants is decreased to 30 to 50% of the binding found in wild type extracts. We have used the photoaffinity label 8-azidoadenosine 3':5'-[32P]monophosphate to label the regulatory subunits of type I and type II protein kinase in wild type and mutant extracts analyzed by DEAE-cellulose and Sephadex chromatography. We find that all three mutants lack type I cyclic AMP-dependent protein kinase and have reduced amounts of type II kinase activity. The regulatory subunits of type I and type II kinase are present in both complementation groups. We conclude that type I protein kinase is not needed for normal growth of Chinese hamster ovary cells. The defect in both classes of mutants appears to be in the failure of the catalytic subunit to associate normally with its regulatory subunits.