REGULATION OF CYCLIC-AMP - DEPENDENT PROTEIN KINASE SUBUNIT LEVELS IN FRIEND ERYTHROLEUKEMIC CELLS (DIFFERENTIATION, PHOSPHORYLATION)

Abstract

Cytosolic extracts of Friend erythroleukemic cells contain both type I and type II cAMP-dependent protein kinases. In control cells the regulatory subunits of the type I and type II protein kinases (R I and R II) were present in approximately equal amounts. After the induction of erythroid differentiation by growth in 2% dimethyl sulfoxide, the total cAMP-binding capacity doubled. The concentration of R II tripled (to 16.6 pmol/mg) while R I content (1.5 pmol/mg) declined to one-third of the control cell level. Treatment of Friend cells with 0.5 mM 8-Br-cAMP and 0.2 mM methylisobutyl xanthine for 2 days did not stimulate cell differentiation. However, the cells accumulated a high level of R II while R I declined, resulting in a distribution of R subunits closely resembling that of differentiated cells.;Cyclic AMP-stimulated histone kinase activity increased in proportion to cAMP-binding activity during differentiation; no such increase was seen following 8-Br-cAMP-treatment. Both R I and R II were fully associated with the catalytic subunit (C) of protein kinase before and after differentiation. In 8-Br-cAMP-treated cells all of R I and 67% of R II were not associated with C. Thus, chronic exposure to 8-Br-cAMP disrupts the coordinate regulation of R and C content.;Two species of R II with apparent Mrs of 54,000 (R II-54) and 52,000 (R II-52) were expressed in Friend cells. R II-52 and R II-54 were not interconverted by phosphorylation or dephosphorylation. A monoclonal antibody resolved the two cAMP-binding proteins by preferentially binding R II-54. Two-dimensional maps of tryptic peptides obtained from R II-52 and R II-54 revealed extensive structural differences. The relative rate of synthesis of R II-52 increased 12 to 14-fold during erythroid differentiation or treatment with 8-Br-cAMP, while the rate of R II-54 synthesis either declined or was unchanged. Thus, the two structurally and functionally homologous forms of R II are subject to markedly different modes of regulation. Net accumulation of total R II results from a large and selective increase in the rate of biosynthesis of R II-52.