Cyclic AMP-dependent phosphorylation in intact cells and in cell-free extracts from Chinese hamster ovary cells. Studies with wild type and cyclic AMP-resistant mutants.

Abstract

Cyclic AMP-dependent phosphorylation of endogen o u s proteins was studied in wild type and CAMP-resistant mutants from Chinese hamster ovary cells, both in cell-free extracts and in intact cells.Based on t h e ability of crude extracts to phosphorylate histone, most of the m u t a n t s had altered CAMP-dependent protein kinase, but a f e w appeared to have normal levels of enzyme activity.Experiments carried out with cell-free extracts indicated that, in wild type cells, cAMP strongly enhanced the phosphorylation of f o u r bands of M, = 300,000, 34,000, 26,000, and 17,000; in the three m u t a n t s tested, a similar phosphorylation pattern was obtained but the extent of cAMP effect was only 20 to 35% of wild type.Maximal phosphorylation of the four bands was restored in mutant cell extracts upon addition of exogenous catalytic subunit of CAMP-dependent protein kinase from beef heart, indicating that the defect in CAMP-dependent phosphorylation can be accounted for b y an alteration in CAMP-dependent protein kinase activity in m u t a n t strains.In intact wild type Chinese hamster ovary cells, activation of CAMP-dependent protein kinase by analogs of cAMP (8 bromo cyclic adenosine 3':5', monophosphate), cholera toxin, or methylisobutylxanthine rapidly induced the specific phosphorylation of a 52,000dalton cytosolic protein which when analyzed by twodimensional gel electrophoresis resolved into an array of three to four spots with different charges.These spots did not co-migrate with either of the regulatory subunits of CAMP-dependent protein kinase or with intermediate filament protein.Among the CAMP-resistant mutants tested, three which have reduced amounts of CAMP-dependent protein kinase (both type I a n d type 11) showed CAMP-dependent phosphorylation of the 52,000-dalton protein.Two other strains that are missing type I1 CAMP-dependent protein kinase, but