Plasmids encoding PKI(1-31), a specific inhibitor of cAMP-stimulated gene expression, inhibit the basal transcriptional activity of some but not all cAMP-regulated DNA response elements in JEG-3 cells.
Plasmids that encode a bioactive amino-terminal fragment of the heat-stable inhibitor of the cAMP-dependent protein kinase, PKI(1-31), were employed to characterize the role of this protein kinase in the control of transcriptional activity mediated by three DNA regulatory elements in the JEG-3 human placental cell line. The 5'-flanking sequence of the human collagenase gene contains the heptameric sequence, 5'-TGAGTCA-3', previously identified as a "phorbol ester" response element. Reporter genes containing either the intact 1.2-kilobase 5'-flanking sequence from the human collagenase gene or just the 7-base pair (bp) response element, when coupled to an enhancerless promoter, each exhibit both cAMP and phorbol ester-stimulated expression in JEG-3 cells. Cotransfection of either construct with plasmids encoding PKI(1-31) inhibits cAMP-stimulated but not basal- or phorbol ester-stimulated expression. Pretreatment of cells with phorbol ester for 1 or 2 days abrogates completely the response to rechallenge with phorbol ester but does not alter the basal expression of either construct; cAMP-stimulated expression, while modestly inhibited, remains vigorous. The 5'-flanking sequence of the human chorionic gonadotropin-alpha subunit (HCG alpha) gene has two copies of the sequence, 5'-TGACGTCA-3', contained in directly adjacent identical 18-bp segments, previously identified as a cAMP-response element. Reporter genes containing either the intact 1.5 kilobase of 5'-flanking sequence from the HCG alpha gene, or just the 36-bp tandem repeat cAMP response element, when coupled to an enhancerless promoter, both exhibit a vigorous cAMP stimulation of expression but no response to phorbol ester in JEG-3 cells. Cotransfection with plasmids encoding PKI(1-31) inhibits both basal and cAMP-stimulated expression in a parallel fashion. The 5'-flanking sequence of the human enkephalin gene mediates cAMP-stimulated expression of reporter genes in both JEG-3 and CV-1 cells. Plasmids encoding PKI(1-31) inhibit the expression that is stimulated by the addition of cAMP analogs in both cell lines; basal expression, however, is inhibited by PKI(1-31) only in the JEG-3 cell line and not in the CV-1 cells. These observations indicate that, in JEG-3 cells, PKI(1-31) is a specific inhibitor of kinase A-mediated gene transcription, but it does not modify kinase C-directed transcription.(ABSTRACT TRUNCATED AT 400 WORDS)
Grove
J R
JR
Howard Hughes Medical Institute Laboratory, Harvard Medical School, Boston, Massachusetts.
Deutsch
P J
PJ
Price
D J
DJ
Habener
J F
JF
Avruch
J
J
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology*; D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology*; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D000242:Cyclic AMP / Q000502:physiology*; D004791:Enzyme Inhibitors; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D010446:Peptide Fragments / Q000235:genetics*; D010957:Plasmids; D011494:Protein Kinases / Q000235:genetics; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013806:Theophylline / Q000031:analogs & derivatives*; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection
D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D010446:Peptide Fragments; C015482:protein kinase modulator; C061814:protein kinase inhibitor peptide (1-31); D015124:8-Bromo Cyclic Adenosine Monophosphate; D013806:Theophylline; D000242:Cyclic AMP; D011494:Protein Kinases; D013755:Tetradecanoylphorbol Acetate; D015056:1-Methyl-3-isobutylxanthine