Regulation of the expression of the regulatory subunit of cAMP-dependent protein kinase II beta in Friend erythroleukemic cells. Evidence for posttranscriptional control and a central role for the C subunit.
Friend erythroleukemic cells provide a system for studying the regulation of the expression of regulatory (R) and catalytic (C) subunit isoforms of cAMP-dependent protein kinases. Friend cells contain RI alpha, two RII subunits previously designated RII-52 and RII-54, and C alpha. When the cells are treated with 0.2 mM methylisobutylxanthine (MIX) and either 20 microM forskolin or 0.5 mM 8-Br-cAMP, RI alpha content declines 50-75% because of a large decrease in the t1/2 value for the dissociated RI alpha subunit; RII-54 expression is invariant, but the amount and rate of synthesis of RII-52 increases 10-15-fold (Schwartz, D. A., and Rubin, C. S. (1985) J. Biol. Chem. 260, 6296-6303). We now demonstrate that RII-52 and RII-54 correspond to RII beta and RII alpha, respectively. When cAMP levels are elevated in Friend cells the abundance of the 3.3-kilobase RII beta mRNA increases 25-30-fold in parallel with the rate of RII beta subunit synthesis indicating that pretranslational control is operative. Other R and C mRNAs are not markedly induced. Surprisingly, the rate of transcriptional initiation of the RII beta gene and the stability of RII beta mRNA are not altered during RII beta induction. Rather, the induction of RII beta mRNA is associated with the accumulation of major (3.4 kilobases) and minor (4 kilobases) RII beta pre-mRNAs in the nucleus. It appears that the cAMP signal-transduction system alters a nuclear protein(s) such that either the proportion of RII beta pre-mRNAs that are processed to mature mRNAs and are exported to the cytoplasm is greatly increased or the nuclear precursors are stabilized. Thus, regulation is exerted at a posttranscriptional level. In order to establish directly a causal role for C in RII beta induction and to rule out artifacts introduced by the use of drugs such as forskolin, MIX, and cAMP analogs we stably transfected Friend cells with a vector containing C alpha cDNA under the regulation of the zinc-activated metallothionein I promoter. The addition of 0.15 mM ZnSO4 caused the accumulation of dissociated C subunits and the selective induction of RII beta.
Gross
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Department of Molecular Pharmacology, Atran Laboratories, Albert Einstein College of Medicine, Bronx, New York 10461.
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Rubin
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D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000494:pharmacology*; D004247:DNA / Q000235:genetics; D005622:Friend murine leukemia virus; D005786:Gene Expression Regulation; D006207:Half-Life; D007700:Kinetics; D004915:Leukemia, Erythroblastic, Acute / Q000378:metabolism*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D011494:Protein Kinases / Q000235:genetics*; D012322:RNA Precursors / Q000096:biosynthesis; D012333:RNA, Messenger / Q000096:biosynthesis; D015398:Signal Transduction; D014158:Transcription, Genetic; D014162:Transfection
D046911:Macromolecular Substances; D009842:Oligopeptides; D012322:RNA Precursors; D012333:RNA, Messenger; D005576:Colforsin; D015124:8-Bromo Cyclic Adenosine Monophosphate; C037642:kemptide; D004247:DNA; D000242:Cyclic AMP; D011494:Protein Kinases; D015056:1-Methyl-3-isobutylxanthine