scholarly journals Cyclic AMP-dependent protein kinase regulates transcription of the phosphoenolpyruvate carboxykinase gene but not binding of nuclear factors to the cyclic AMP regulatory element.

1990 ◽  
Vol 10 (7) ◽  
pp. 3357-3364 ◽  
Author(s):  
P G Quinn ◽  
D K Granner
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Rat Liver ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Nuclear Extract ◽  
In Vitro Transcription ◽  
Catalytic Subunit ◽  
Nuclear Factors ◽  
Dependent Protein

We have examined the binding of factors in rat liver nuclear extracts to the phosphoenolpyruvate carboxykinase (PEPCK) gene cyclic AMP (cAMP) response element (CRE) and other CREs and have isolated a rat liver CRE-binding protein (CREBP) cDNA. In addition, we have examined the influence of altering the phosphorylation state of nuclear factors on both CRE binding and in vitro transcription. Specific binding to the PEPCK CRE was measured in a mobility shift assay. CRE sequences of the PEPCK, somatostatin, and glycoprotein hormone alpha subunit genes competed equally for binding of rat liver nuclear factors to the PEPCK CRE, whereas mutant PEPCK CRE sequences did not compete for binding. Oligonucleotides complementary to rat pheochromocytoma CREBP (Gonzalez et al., Nature [London] 337:749-752, 1989) were used to prime rat liver and brain cDNA in the polymerase chain reaction. The predominant CREBP molecule obtained was identical to the rat pheochromocytoma CREBP except for a 14-amino-acid deletion in the N-terminal half that was also present in a human placental cDNA (Hoeffler et al., Science 242:1430-1433, 1988). The regulation of transcription by cAMP was examined by coincubation of rat liver nuclear extract with the purified catalytic subunit of cAMP-dependent protein kinase (protein kinase A). Although binding to the CRE was unaffected, in vitro transcription directed by the PEPCK promoter was stimulated by catalytic subunit, and this effect was blocked by protein kinase inhibitor peptide. In contrast, when nuclear extract was coincubated with phosphatase, there was substantial inhibition of in vitro transcription directed by the PEPCK promoter, but there was no effect on binding to the CRE. The major effects of catalytic subunit were exerted through the CRE, but residual stimulation was evident in promoter fragments containing only the TATA element. These data suggest that factors are bound to the CRE at constitutively high levels and that their capacity for transcriptional activation is regulated by phosphorylation.

Cyclic AMP-dependent protein kinase regulates transcription of the phosphoenolpyruvate carboxykinase gene but not binding of nuclear factors to the cyclic AMP regulatory element

1990 ◽  
Vol 10 (7) ◽  
pp. 3357-3364
Author(s):  
P G Quinn ◽  
D K Granner
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Rat Liver ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Nuclear Extract ◽  
In Vitro Transcription ◽  
Catalytic Subunit ◽  
Nuclear Factors ◽  
Dependent Protein

We have examined the binding of factors in rat liver nuclear extracts to the phosphoenolpyruvate carboxykinase (PEPCK) gene cyclic AMP (cAMP) response element (CRE) and other CREs and have isolated a rat liver CRE-binding protein (CREBP) cDNA. In addition, we have examined the influence of altering the phosphorylation state of nuclear factors on both CRE binding and in vitro transcription. Specific binding to the PEPCK CRE was measured in a mobility shift assay. CRE sequences of the PEPCK, somatostatin, and glycoprotein hormone alpha subunit genes competed equally for binding of rat liver nuclear factors to the PEPCK CRE, whereas mutant PEPCK CRE sequences did not compete for binding. Oligonucleotides complementary to rat pheochromocytoma CREBP (Gonzalez et al., Nature [London] 337:749-752, 1989) were used to prime rat liver and brain cDNA in the polymerase chain reaction. The predominant CREBP molecule obtained was identical to the rat pheochromocytoma CREBP except for a 14-amino-acid deletion in the N-terminal half that was also present in a human placental cDNA (Hoeffler et al., Science 242:1430-1433, 1988). The regulation of transcription by cAMP was examined by coincubation of rat liver nuclear extract with the purified catalytic subunit of cAMP-dependent protein kinase (protein kinase A). Although binding to the CRE was unaffected, in vitro transcription directed by the PEPCK promoter was stimulated by catalytic subunit, and this effect was blocked by protein kinase inhibitor peptide. In contrast, when nuclear extract was coincubated with phosphatase, there was substantial inhibition of in vitro transcription directed by the PEPCK promoter, but there was no effect on binding to the CRE. The major effects of catalytic subunit were exerted through the CRE, but residual stimulation was evident in promoter fragments containing only the TATA element. These data suggest that factors are bound to the CRE at constitutively high levels and that their capacity for transcriptional activation is regulated by phosphorylation.

Multiple hepatic trans-acting factors are required for in vitro transcription of the human alpha-1-antitrypsin gene

1988 ◽  
Vol 8 (10) ◽  
pp. 4362-4369
Author(s):  
Y Li ◽  
R F Shen ◽  
S Y Tsai ◽  
S L Woo
Keyword(s):  
Rat Liver ◽  
Nuclear Extract ◽  
In Vitro Transcription ◽  
Flanking Sequence ◽  
Tissue Specific ◽  
Hepatic Expression ◽  
Nuclear Extracts ◽  
Nuclear Factors ◽  
Alpha 1 Antitrypsin

The human alpha-1-antitrypsin (AAT) gene is expressed in the liver, and its deficiency causes pulmonary emphysema. We have demonstrated that its 5'-flanking region contains cis-acting elements capable of directing proper transcription in the presence of rat liver nuclear extract. The in vitro transcription system is tissue-specific in that the AAT promoter is functional in nuclear extracts prepared from the liver but not from HeLa cells. Experiments in which rat liver and HeLa nuclear extracts were mixed suggested the presence of a specific activator(s) in hepatocytes rather than a repressor(s) in nonproducing cells. Two protected regions were detected in the promoter by DNase I footprinting analysis with rat liver nuclear extracts. Region one spanned -78 to -52 and region two spanned -125 to -100 in the 5'-flanking sequence of the gene. By gel retardation assays with synthetic oligonucleotides, at least two distinct liver nuclear factors were identified, HNF-1 and HNF-2 (hepatocyte nuclear factors), which bound specifically to the first and second region, respectively. We present evidence that HNF-1 and HNF-2 are positively acting, tissue-specific transcription factors that regulate hepatic expression of the human AAT gene.

scholarly journals Antiserum against the catalytic subunit of adenosine 3′:5′-cyclic monophosphate-dependent protein kinase. Reactivity towards various protein kinases

1980 ◽  
Vol 192 (1) ◽  
pp. 223-230 ◽  
Author(s):  
G Schwoch ◽  
A Hamann ◽  
H Hilz
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Protein Kinases ◽  
Rat Liver ◽  
Catalytic Subunit ◽  
Type Ii ◽  
Dependent Protein Kinase ◽  
Subunit C ◽  
Bovine Heart ◽  
Dependent Protein

An antiserum against the catalytic subunit C of cyclic AMP-dependent protein kinase, isolated from bovine heart type II protein kinase, was produced in rabbits. Reaction of the catalytic subunit with antiserum and separation of the immunoglobulin G fraction by Protein A-Sepharose quantitatively removed the enzyme from solutions. Comparative immunotitration of protein kinases showed that the amount of antiserum required to eliminate 50% of the enzymic activity was identical for pure catalytic subunit, and for holoenzymes type I and type II. The reactivity of the holoenzymes with the antiserum was identical in the absence or the presence of dissociating concentrations of cyclic AMP. Most of the holoenzyme (type II) remains intact when bound to the antibodies as shown by quantification of the regulatory subunit in the supernatant of the immunoprecipitate. Titration with the antibodies also revealed the presence of a cyclic AMP-independent histone kinase in bovine heart protein kinase I preparations obtained by DEAE-cellulose chromatography. Cyclic AMP-dependent protein kinase purified from the particulate fraction of bovine heart reacted with the antiserum to the same degree as the soluble enzyme, whereas two cyclic AMP-independent kinases separated from the particle fraction neither reacted with the antiserum nor influenced the reaction of the antibodies with the cyclic AMP-dependent protein kinase. Immunotitration of the protein kinase catalytic subunit C from rat liver revealed that the antibodies had rather similar reactivities towards the rat liver and the bovine heart enzyme. This points to a relatively high degree of homology of the catalytic subunit in mammalian tissues and species. Broad applicability of the antiserum to problems related to cyclic AMP-dependent protein kinases is thus indicated.

scholarly journals Multiple hepatic trans-acting factors are required for in vitro transcription of the human alpha-1-antitrypsin gene.

1988 ◽  
Vol 8 (10) ◽  
pp. 4362-4369 ◽  
Author(s):  
Y Li ◽  
R F Shen ◽  
S Y Tsai ◽  
S L Woo
Keyword(s):  
Rat Liver ◽  
Nuclear Extract ◽  
In Vitro Transcription ◽  
Flanking Sequence ◽  
Tissue Specific ◽  
Hepatic Expression ◽  
Nuclear Extracts ◽  
Nuclear Factors ◽  
Alpha 1 Antitrypsin

The human alpha-1-antitrypsin (AAT) gene is expressed in the liver, and its deficiency causes pulmonary emphysema. We have demonstrated that its 5'-flanking region contains cis-acting elements capable of directing proper transcription in the presence of rat liver nuclear extract. The in vitro transcription system is tissue-specific in that the AAT promoter is functional in nuclear extracts prepared from the liver but not from HeLa cells. Experiments in which rat liver and HeLa nuclear extracts were mixed suggested the presence of a specific activator(s) in hepatocytes rather than a repressor(s) in nonproducing cells. Two protected regions were detected in the promoter by DNase I footprinting analysis with rat liver nuclear extracts. Region one spanned -78 to -52 and region two spanned -125 to -100 in the 5'-flanking sequence of the gene. By gel retardation assays with synthetic oligonucleotides, at least two distinct liver nuclear factors were identified, HNF-1 and HNF-2 (hepatocyte nuclear factors), which bound specifically to the first and second region, respectively. We present evidence that HNF-1 and HNF-2 are positively acting, tissue-specific transcription factors that regulate hepatic expression of the human AAT gene.

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