title: Protein kinase A regulation of cAMP phosphodiesterase expression in rat skeletal myoblasts.
abstract: We have been studying cAMP signaling in L6 myoblasts because of its potential role in regulating the differentiation of these cells into multinucleate myotubes. Previous studies have shown that treatment of L6 myoblasts with cAMP analogs causes an increase in cAMP phosphodiesterase activity. To assess the role of protein kinase A in this cAMP-mediated increase in cAMP phosphodiesterase activity, L6 myoblasts were transfected with a plasmid containing the cDNA for a mutant regulatory subunit of protein kinase A, which functions as a dominant negative inhibitor of this enzyme. The cDNA was under control of the metallothionein promoter in the construct. Induction of the mutant regulatory subunit with Zn2+ decreased cAMP-dependent protein kinase activity by 90%. Zn2+ treatment was also able to completely block the cAMP-mediated increase in phosphodiesterase activity, showing that this effect is mediated by protein kinase A. The activity of the cAMP-induced phosphodiesterase was inhibited by low concentrations of RO 20-1724, showing that it was a member of the type IV low Km cAMP phosphodiesterase family of enzymes. We used the polymerase chain reaction and consensus primers designed to amplify phosphodiesterase sequences to show that L6 myoblasts also contain mRNA for a type IV low Km cAMP phosphodiesterase designated PDE3.1. The levels of this mRNA were increased greatly by treatment with dibutyryl cAMP or forskolin in L6 myoblasts and also in differentiated L6 myotubes. Run-off transcription assays showed that this increase in PDE mRNA was regulated, at least in part, by an increase in the rate of transcription of the PDE3 gene. The induction of PDE3 message by cAMP was blocked when the L6 transfectants were treated with Zn2+ to induce protein kinase A inhibition. Therefore, some of the cAMP-mediated increase in phosphodiesterase activity seen in L6 myoblasts is due to a protein kinase A-mediated increase in PDE3 mRNA. This pathway may serve as a feedback mechanism to modulate the inhibitory effects of cAMP on myogenesis.
authors.affiliation: Department of Biochemistry, University of Western Ontario, London, Canada.
authors.forename: T
authors.initials: T
authors.lastname: Kovala
authors.forename: I A
authors.initials: IA
authors.lastname: Lorimer
authors.forename: A M
authors.initials: AM
authors.lastname: Brickenden
authors.forename: E H
authors.initials: EH
authors.lastname: Ball
authors.forename: B D
authors.initials: BD
authors.lastname: Sanwal
mesh_terms: D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D003994:Bucladesine / Q000494:pharmacology; D002454:Cell Differentiation; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry; D015870:Gene Expression; D008969:Molecular Sequence Data; D009132:Muscles / Q000166:cytology / Q000201:enzymology*; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats
chemical_list: D017931:DNA Primers; D012333:RNA, Messenger; D005576:Colforsin; D003994:Bucladesine; D017868:Cyclic AMP-Dependent Protein Kinases; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D015056:1-Methyl-3-isobutylxanthine