Identification and differential expression of two forms of regulatory subunits (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.
The concentration of regulatory subunits (R) of type II cAMP-dependent protein kinase increased 4- to 5-fold when 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 Mr values of 54,000 (RII-54) and 52,000 (RII-52) are expressed in Friend cells. Both forms of RII were (a) covalently labeled with 8-N3-[32P]cAMP, (b) phosphorylated by the catalytic subunit of protein kinase II, and (c) complexed by polyclonal anti-RII IgGs. RII-52 and RII-54 were not interconverted by phosphorylation or dephosphorylation. A monoclonal antibody that recognizes an internal site in RII resolved the two cAMP-binding proteins by preferentially binding RII-54. The structural diversity suggested by the monoclonal antibody experiment was further examined by comparing two-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 two 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, two 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 observed in the present and previous (Schwartz, D. A., and Rubin, C. S. (1983) J. Biol. Chem. 258, 777-784) studies results from a selective increase in the rate of biosynthesis of RII-52.
Schwartz
D A
DA
Rubin
C S
CS
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D002454:Cell Differentiation; D002999:Clone Cells / Q000378:metabolism; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D005622:Friend murine leukemia virus; D047908:Intracellular Signaling Peptides and Proteins; D004915:Leukemia, Erythroblastic, Acute / Q000378:metabolism*; D051379:Mice; D010766:Phosphorylation
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; C015482:protein kinase modulator; D015124:8-Bromo Cyclic Adenosine Monophosphate; D015056:1-Methyl-3-isobutylxanthine; D004121:Dimethyl Sulfoxide