Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1990 Jul;10(7):3357–3364. doi: 10.1128/mcb.10.7.3357

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

P G Quinn 1, D K Granner 1
PMCID: PMC360759  PMID: 2141384

Abstract

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.

Full text

PDF
3357

Images in this article

3358Image
on p.3358
3360Image
on p.3360
3360Image
on p.3360
3361Image
on p.3361

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abel T., Maniatis T. Gene regulation. Action of leucine zippers. Nature. 1989 Sep 7;341(6237):24–25. doi: 10.1038/341024a0. [DOI] [PubMed] [Google Scholar]
  2. Andrisani O. M., Pot D. A., Zhu Z., Dixon J. E. Three sequence-specific DNA-protein complexes are formed with the same promoter element essential for expression of the rat somatostatin gene. Mol Cell Biol. 1988 May;8(5):1947–1956. doi: 10.1128/mcb.8.5.1947. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Andrisani O. M., Zhu Z. N., Pot D. A., Dixon J. E. In vitro transcription directed from the somatostatin promoter is dependent upon a purified 43-kDa DNA-binding protein. Proc Natl Acad Sci U S A. 1989 Apr;86(7):2181–2185. doi: 10.1073/pnas.86.7.2181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Beale E. G., Chrapkiewicz N. B., Scoble H. A., Metz R. J., Quick D. P., Noble R. L., Donelson J. E., Biemann K., Granner D. K. Rat hepatic cytosolic phosphoenolpyruvate carboxykinase (GTP). Structures of the protein, messenger RNA, and gene. J Biol Chem. 1985 Sep 5;260(19):10748–10760. [PubMed] [Google Scholar]
  5. Becker P. B., Gloss B., Schmid W., Strähle U., Schütz G. In vivo protein-DNA interactions in a glucocorticoid response element require the presence of the hormone. Nature. 1986 Dec 18;324(6098):686–688. doi: 10.1038/324686a0. [DOI] [PubMed] [Google Scholar]
  6. Bokar J. A., Roesler W. J., Vandenbark G. R., Kaetzel D. M., Hanson R. W., Nilson J. H. Characterization of the cAMP responsive elements from the genes for the alpha-subunit of glycoprotein hormones and phosphoenolpyruvate carboxykinase (GTP). Conserved features of nuclear protein binding between tissues and species. J Biol Chem. 1988 Dec 25;263(36):19740–19747. [PubMed] [Google Scholar]
  7. Chu D. T., Granner D. K. The effect of phorbol esters and diacylglycerol on expression of the phosphoenolpyruvate carboxykinase (GTP) gene in rat hepatoma H4IIE cells. J Biol Chem. 1986 Dec 25;261(36):16848–16853. [PubMed] [Google Scholar]
  8. Day R. N., Walder J. A., Maurer R. A. A protein kinase inhibitor gene reduces both basal and multihormone-stimulated prolactin gene transcription. J Biol Chem. 1989 Jan 5;264(1):431–436. [PubMed] [Google Scholar]
  9. Dwarki V. J., Montminy M., Verma I. M. Both the basic region and the 'leucine zipper' domain of the cyclic AMP response element binding (CREB) protein are essential for transcriptional activation. EMBO J. 1990 Jan;9(1):225–232. doi: 10.1002/j.1460-2075.1990.tb08099.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dynan W. S. Modularity in promoters and enhancers. Cell. 1989 Jul 14;58(1):1–4. doi: 10.1016/0092-8674(89)90393-0. [DOI] [PubMed] [Google Scholar]
  11. Fried M., Crothers D. M. Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 1981 Dec 11;9(23):6505–6525. doi: 10.1093/nar/9.23.6505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Frohman M. A., Dush M. K., Martin G. R. Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8998–9002. doi: 10.1073/pnas.85.23.8998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Garner M. M., Revzin A. A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system. Nucleic Acids Res. 1981 Jul 10;9(13):3047–3060. doi: 10.1093/nar/9.13.3047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gonzalez G. A., Montminy M. R. Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at serine 133. Cell. 1989 Nov 17;59(4):675–680. doi: 10.1016/0092-8674(89)90013-5. [DOI] [PubMed] [Google Scholar]
  15. Gorski K., Carneiro M., Schibler U. Tissue-specific in vitro transcription from the mouse albumin promoter. Cell. 1986 Dec 5;47(5):767–776. doi: 10.1016/0092-8674(86)90519-2. [DOI] [PubMed] [Google Scholar]
  16. Hai T. W., Horikoshi M., Roeder R. G., Green M. R. Analysis of the role of the transcription factor ATF in the assembly of a functional preinitiation complex. Cell. 1988 Sep 23;54(7):1043–1051. doi: 10.1016/0092-8674(88)90119-5. [DOI] [PubMed] [Google Scholar]
  17. Hai T. W., Liu F., Allegretto E. A., Karin M., Green M. R. A family of immunologically related transcription factors that includes multiple forms of ATF and AP-1. Genes Dev. 1988 Oct;2(10):1216–1226. doi: 10.1101/gad.2.10.1216. [DOI] [PubMed] [Google Scholar]
  18. Hai T. W., Liu F., Coukos W. J., Green M. R. Transcription factor ATF cDNA clones: an extensive family of leucine zipper proteins able to selectively form DNA-binding heterodimers. Genes Dev. 1989 Dec;3(12B):2083–2090. doi: 10.1101/gad.3.12b.2083. [DOI] [PubMed] [Google Scholar]
  19. Hardy S., Shenk T. Adenoviral control regions activated by E1A and the cAMP response element bind to the same factor. Proc Natl Acad Sci U S A. 1988 Jun;85(12):4171–4175. doi: 10.1073/pnas.85.12.4171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hoeffler J. P., Meyer T. E., Yun Y., Jameson J. L., Habener J. F. Cyclic AMP-responsive DNA-binding protein: structure based on a cloned placental cDNA. Science. 1988 Dec 9;242(4884):1430–1433. doi: 10.1126/science.2974179. [DOI] [PubMed] [Google Scholar]
  21. Horikoshi M., Hai T., Lin Y. S., Green M. R., Roeder R. G. Transcription factor ATF interacts with the TATA factor to facilitate establishment of a preinitiation complex. Cell. 1988 Sep 23;54(7):1033–1042. doi: 10.1016/0092-8674(88)90118-3. [DOI] [PubMed] [Google Scholar]
  22. Hyman S. E., Comb M., Lin Y. S., Pearlberg J., Green M. R., Goodman H. M. A common trans-acting factor is involved in transcriptional regulation of neurotransmitter genes by cyclic AMP. Mol Cell Biol. 1988 Oct;8(10):4225–4233. doi: 10.1128/mcb.8.10.4225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hyman S. E., Comb M., Pearlberg J., Goodman H. M. An AP-2 element acts synergistically with the cyclic AMP- and phorbol ester-inducible enhancer of the human proenkephalin gene. Mol Cell Biol. 1989 Jan;9(1):321–324. doi: 10.1128/mcb.9.1.321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Imagawa M., Chiu R., Karin M. Transcription factor AP-2 mediates induction by two different signal-transduction pathways: protein kinase C and cAMP. Cell. 1987 Oct 23;51(2):251–260. doi: 10.1016/0092-8674(87)90152-8. [DOI] [PubMed] [Google Scholar]
  25. Karnitz L., Poon D., Weil P. A., Chalkley R. Purification and properties of the Rous sarcoma virus internal enhancer binding factor. Mol Cell Biol. 1989 May;9(5):1929–1939. doi: 10.1128/mcb.9.5.1929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kemp B. E., Cheng H. C., Walsh D. A. Peptide inhibitors of cAMP-dependent protein kinase. Methods Enzymol. 1988;159:173–183. doi: 10.1016/0076-6879(88)59018-3. [DOI] [PubMed] [Google Scholar]
  27. Klemm D. J., Roesler W. J., Liu J. S., Park E. A., Hanson R. W. In vitro analysis of promoter elements regulating transcription of the phosphoenolpyruvate carboxykinase (GTP) gene. Mol Cell Biol. 1990 Feb;10(2):480–485. doi: 10.1128/mcb.10.2.480. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lamers W. H., Hanson R. W., Meisner H. M. cAMP stimulates transcription of the gene for cytosolic phosphoenolpyruvate carboxykinase in rat liver nuclei. Proc Natl Acad Sci U S A. 1982 Sep;79(17):5137–5141. doi: 10.1073/pnas.79.17.5137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Landschulz W. H., Johnson P. F., McKnight S. L. The DNA binding domain of the rat liver nuclear protein C/EBP is bipartite. Science. 1989 Mar 31;243(4899):1681–1688. doi: 10.1126/science.2494700. [DOI] [PubMed] [Google Scholar]
  30. Landschulz W. H., Johnson P. F., McKnight S. L. The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. Science. 1988 Jun 24;240(4860):1759–1764. doi: 10.1126/science.3289117. [DOI] [PubMed] [Google Scholar]
  31. Lee C. Q., Miller H. A., Schlichter D., Dong J. N., Wicks W. D. Evidence for a cAMP-dependent nuclear factor capable of interacting with a specific region of a eukaryotic gene. Proc Natl Acad Sci U S A. 1988 Jun;85(12):4223–4227. doi: 10.1073/pnas.85.12.4223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lenardo M. J., Baltimore D. NF-kappa B: a pleiotropic mediator of inducible and tissue-specific gene control. Cell. 1989 Jul 28;58(2):227–229. doi: 10.1016/0092-8674(89)90833-7. [DOI] [PubMed] [Google Scholar]
  33. Maekawa T., Sakura H., Kanei-Ishii C., Sudo T., Yoshimura T., Fujisawa J., Yoshida M., Ishii S. Leucine zipper structure of the protein CRE-BP1 binding to the cyclic AMP response element in brain. EMBO J. 1989 Jul;8(7):2023–2028. doi: 10.1002/j.1460-2075.1989.tb03610.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Maniatis T., Goodbourn S., Fischer J. A. Regulation of inducible and tissue-specific gene expression. Science. 1987 Jun 5;236(4806):1237–1245. doi: 10.1126/science.3296191. [DOI] [PubMed] [Google Scholar]
  35. Mellon P. L., Clegg C. H., Correll L. A., McKnight G. S. Regulation of transcription by cyclic AMP-dependent protein kinase. Proc Natl Acad Sci U S A. 1989 Jul;86(13):4887–4891. doi: 10.1073/pnas.86.13.4887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Mitchell P. J., Tjian R. Transcriptional regulation in mammalian cells by sequence-specific DNA binding proteins. Science. 1989 Jul 28;245(4916):371–378. doi: 10.1126/science.2667136. [DOI] [PubMed] [Google Scholar]
  37. Montminy M. R., Bilezikjian L. M. Binding of a nuclear protein to the cyclic-AMP response element of the somatostatin gene. Nature. 1987 Jul 9;328(6126):175–178. doi: 10.1038/328175a0. [DOI] [PubMed] [Google Scholar]
  38. Montminy M. R., Sevarino K. A., Wagner J. A., Mandel G., Goodman R. H. Identification of a cyclic-AMP-responsive element within the rat somatostatin gene. Proc Natl Acad Sci U S A. 1986 Sep;83(18):6682–6686. doi: 10.1073/pnas.83.18.6682. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Proudfoot N. J., Shander M. H., Manley J. L., Gefter M. L., Maniatis T. Structure and in vitro transcription of human globin genes. Science. 1980 Sep 19;209(4463):1329–1336. doi: 10.1126/science.6158093. [DOI] [PubMed] [Google Scholar]
  40. Quinn P. G., Wong T. W., Magnuson M. A., Shabb J. B., Granner D. K. Identification of basal and cyclic AMP regulatory elements in the promoter of the phosphoenolpyruvate carboxykinase gene. Mol Cell Biol. 1988 Aug;8(8):3467–3475. doi: 10.1128/mcb.8.8.3467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Sasaki K., Cripe T. P., Koch S. R., Andreone T. L., Petersen D. D., Beale E. G., Granner D. K. Multihormonal regulation of phosphoenolpyruvate carboxykinase gene transcription. The dominant role of insulin. J Biol Chem. 1984 Dec 25;259(24):15242–15251. [PubMed] [Google Scholar]
  42. Short J. M., Wynshaw-Boris A., Short H. P., Hanson R. W. Characterization of the phosphoenolpyruvate carboxykinase (GTP) promoter-regulatory region. II. Identification of cAMP and glucocorticoid regulatory domains. J Biol Chem. 1986 Jul 25;261(21):9721–9726. [PubMed] [Google Scholar]
  43. Singh H., LeBowitz J. H., Baldwin A. S., Jr, Sharp P. A. Molecular cloning of an enhancer binding protein: isolation by screening of an expression library with a recognition site DNA. Cell. 1988 Feb 12;52(3):415–423. doi: 10.1016/s0092-8674(88)80034-5. [DOI] [PubMed] [Google Scholar]
  44. Singh H., Sen R., Baltimore D., Sharp P. A. A nuclear factor that binds to a conserved sequence motif in transcriptional control elements of immunoglobulin genes. Nature. 1986 Jan 9;319(6049):154–158. doi: 10.1038/319154a0. [DOI] [PubMed] [Google Scholar]
  45. Yamamoto K. K., Gonzalez G. A., Biggs W. H., 3rd, Montminy M. R. Phosphorylation-induced binding and transcriptional efficacy of nuclear factor CREB. Nature. 1988 Aug 11;334(6182):494–498. doi: 10.1038/334494a0. [DOI] [PubMed] [Google Scholar]
  46. Yamamoto K. K., Gonzalez G. A., Menzel P., Rivier J., Montminy M. R. Characterization of a bipartite activator domain in transcription factor CREB. Cell. 1990 Feb 23;60(4):611–617. doi: 10.1016/0092-8674(90)90664-z. [DOI] [PubMed] [Google Scholar]
  47. Zhu Z., Andrisani O. M., Pot D. A., Dixon J. E. Purification and characterization of a 43-kDa transcription factor required for rat somatostatin gene expression. J Biol Chem. 1989 Apr 15;264(11):6550–6556. [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES