IDENTIFICATION AND DIFFERENTIAL EXPRESSION OF 2 FORMS OF REGULATORY SUBUNIT (RII) OF CAMP-DEPENDENT PROTEIN KINASE-II IN FRIEND-ERYTHROLEUKEMIC CELLS - DIFFERENTIATION AND 8-BROMO-CAMP ELICIT A LARGE AND SELECTIVE INCREASE IN THE RATE OF BIOSYNTHESIS OF ONLY ONE TYPE OF RII

Abstract

The concentration of regulatory subunits (R) of type II cAMP-dependent protein kinase increased 4- to 5-fold when [mouse] Friend erythroleukemic cells were either grown in medium containing 0.5 mM 8-bromo-cAMP and 0.2 mM methylisobutylxanthine or stimulated to differentiate. Two species of RII with apparent MW values of 54,000 (RII-54) and 52,000 (RII-52) are expressed in Friend cells. Both forms of RII were covalently labeled with 8-N3-[32P]cAMP, phosphorylated by the catalytic subunit of protein kinase II, and complexed by polyclonal anti-RII IgG. RII-52 and RII-54 were not interconverted by phosphorylation or dephosphorylation. A monoclonal antibody that recognizes an internal site in RII resolved the 2 cAMP-binding proteins by preferentially binding RII-54. The structural diversity suggested by the monoclonal antibody experiment was further examined by comparing 2-dimensional maps of tryptic peptides obtained from metabolically labeled ([35S]met) RII-52 and RII-54. Groups of 35S-labeled peptides that were either uniquely derived from RII-54 or obtained only from RII-52 were readily distinguished, thereby demonstrating that Friend cells produce 2 separate and distinct forms of type II cAMP-binding subunits. The relative rate of synthesis of RII-52 increased 12- to 14-fold during erythroid differentiation and treatment with 8-bromo-cAMP, while the rate of RII-54 synthesis either declined slowly or was unchanged. Thus, 2 homologous forms of RII are subject to different modes of physiological (differentiation) and pharmacological (chronic 8-Br-cAMP) regulation, and the accumulation of total RII results from a selective increase in the rate of biosynthesis of RII-52.