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. 1996 Oct;16(10):5546–5556. doi: 10.1128/mcb.16.10.5546

Induction of bcl-2 expression by phosphorylated CREB proteins during B-cell activation and rescue from apoptosis.

B E Wilson 1, E Mochon 1, L M Boxer 1
PMCID: PMC231554  PMID: 8816467

Abstract

Engagement of surface immunoglobulin on mature B cells leads to rescue from apoptosis and to proliferation. Levels of bcl-2 mRNA and protein increase with cross-linking of surface immunoglobulin. We have located the major positive regulatory region for control of bcl-2 expression in B cells in the 5'-flanking region. The positive region can be divided into an upstream and a downstream regulatory region. The downstream regulatory region contains a cyclic AMP-responsive element (CRE). We show by antibody supershift experiments and UV cross-linking followed by denaturing polyacrylamide gel electrophoresis that both CREB and ATF family members bind to this region in vitro. Mutations of the CRE site that result in loss of CREB binding also lead to loss of functional activity of the bcl-2 promoter in transient-transfection assays. The presence of an active CRE site in the bcl-2 promoter implies that the regulation of bcl-2 expression is linked to a signal transduction pathway in B cells. Treatment of the mature B-cell line BAL-17 with either anti-immunoglobulin M or phorbol 12-myristate 13-acetate leads to an increase in bcl-2 expression that is mediated by the CRE site. Treatment of the more immature B-cell line, Ramos, with phorbol esters rescues the cells from calcium-dependent apoptosis. bcl-2 expression is increased following phorbol ester treatment, and the increased expression is dependent on the CRE site. These stimuli result in phosphorylation of CREB at serine 133. The phosphorylation of CREB that results in activation is mediated by protein kinase C rather than by protein kinase A. Although the CRE site is necessary, optimal induction of bcl-2 expression requires participation of the upstream regulatory element, suggesting that phosphorylation of CREB alters its interaction with the upstream regulatory element. The CRE site in the bcl-2 promoter appears to play a major role in the induction of bcl-2 expression during the activation of mature B cells and during the rescue of immature B cells from apoptosis. It is possible that the CRE site is responsible for induction of bcl-2 expression in other cell types, particularly those in which protein kinase C is involved.

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Selected References

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  1. Barton K., Muthusamy N., Chanyangam M., Fischer C., Clendenin C., Leiden J. M. Defective thymocyte proliferation and IL-2 production in transgenic mice expressing a dominant-negative form of CREB. Nature. 1996 Jan 4;379(6560):81–85. doi: 10.1038/379081a0. [DOI] [PubMed] [Google Scholar]
  2. Bonnefoy-Berard N., Genestier L., Flacher M., Revillard J. P. The phosphoprotein phosphatase calcineurin controls calcium-dependent apoptosis in B cell lines. Eur J Immunol. 1994 Feb;24(2):325–329. doi: 10.1002/eji.1830240208. [DOI] [PubMed] [Google Scholar]
  3. Brunswick M., Samelson L. E., Mond J. J. Surface immunoglobulin crosslinking activates a tyrosine kinase pathway in B cells that is independent of protein kinase C. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1311–1314. doi: 10.1073/pnas.88.4.1311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Burkhardt A. L., Brunswick M., Bolen J. B., Mond J. J. Anti-immunoglobulin stimulation of B lymphocytes activates src-related protein-tyrosine kinases. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7410–7414. doi: 10.1073/pnas.88.16.7410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cambier J. C., Justement L. B., Newell M. K., Chen Z. Z., Harris L. K., Sandoval V. M., Klemsz M. J., Ransom J. T. Transmembrane signals and intracellular "second messengers" in the regulation of quiescent B-lymphocyte activation. Immunol Rev. 1987 Feb;95:37–57. doi: 10.1111/j.1600-065x.1987.tb00499.x. [DOI] [PubMed] [Google Scholar]
  6. Campbell M. A., Sefton B. M. Protein tyrosine phosphorylation is induced in murine B lymphocytes in response to stimulation with anti-immunoglobulin. EMBO J. 1990 Jul;9(7):2125–2131. doi: 10.1002/j.1460-2075.1990.tb07381.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chaouchi N., Vazquez A., Galanaud P., Leprince C. B cell antigen receptor-mediated apoptosis. Importance of accessory molecules CD19 and CD22, and of surface IgM cross-linking. J Immunol. 1995 Apr 1;154(7):3096–3104. [PubMed] [Google Scholar]
  8. Chen H. M., Boxer L. M. Pi 1 binding sites are negative regulators of bcl-2 expression in pre-B cells. Mol Cell Biol. 1995 Jul;15(7):3840–3847. doi: 10.1128/mcb.15.7.3840. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chen Z. Z., Coggeshall K. M., Cambier J. C. Translocation of protein kinase C during membrane immunoglobulin-mediated transmembrane signaling in B lymphocytes. J Immunol. 1986 Mar 15;136(6):2300–2304. [PubMed] [Google Scholar]
  10. Chiles T. C., Liu J. L., Rothstein T. L. Cross-linking of surface Ig receptors on murine B lymphocytes stimulates the expression of nuclear tetradecanoyl phorbol acetate-response element-binding proteins. J Immunol. 1991 Mar 15;146(6):1730–1735. [PubMed] [Google Scholar]
  11. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  12. Cohen J. J. Programmed cell death in the immune system. Adv Immunol. 1991;50:55–85. doi: 10.1016/s0065-2776(08)60822-6. [DOI] [PubMed] [Google Scholar]
  13. Cuende E., Alés-Martínez J. E., Ding L., Gónzalez-García M., Martínez C., Nunez G. Programmed cell death by bcl-2-dependent and independent mechanisms in B lymphoma cells. EMBO J. 1993 Apr;12(4):1555–1560. doi: 10.1002/j.1460-2075.1993.tb05799.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Feuerstein N., Huang D., Hinrichs S. H., Orten D. J., Aiyar N., Prystowsky M. B. Regulation of cAMP-responsive enhancer binding proteins during cell cycle progression in T lymphocytes stimulated by IL-2. J Immunol. 1995 Jan 1;154(1):68–79. [PubMed] [Google Scholar]
  15. Forsgren A., Svedjelund A., Wigzell H. Lymphocyte stimulation by protein A of Staphylococcus aureus. Eur J Immunol. 1976 Mar;6(3):207–213. doi: 10.1002/eji.1830060312. [DOI] [PubMed] [Google Scholar]
  16. Gauss G. H., Lieber M. R. DEAE-dextran enhances electroporation of mammalian cells. Nucleic Acids Res. 1992 Dec 25;20(24):6739–6740. doi: 10.1093/nar/20.24.6739. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Genestier L., Bonnefoy-Berard N., Rouault J. P., Flacher M., Revillard J. P. Tumor necrosis factor-alpha up-regulates Bcl-2 expression and decreases calcium-dependent apoptosis in human B cell lines. Int Immunol. 1995 Apr;7(4):533–540. doi: 10.1093/intimm/7.4.533. [DOI] [PubMed] [Google Scholar]
  18. Gold M. R., Jakway J. P., DeFranco A. L. Involvement of a guanine-nucleotide-binding component in membrane IgM-stimulated phosphoinositide breakdown. J Immunol. 1987 Dec 1;139(11):3604–3613. [PubMed] [Google Scholar]
  19. Gold M. R., Law D. A., DeFranco A. L. Stimulation of protein tyrosine phosphorylation by the B-lymphocyte antigen receptor. Nature. 1990 Jun 28;345(6278):810–813. doi: 10.1038/345810a0. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Graninger W. B., Seto M., Boutain B., Goldman P., Korsmeyer S. J. Expression of Bcl-2 and Bcl-2-Ig fusion transcripts in normal and neoplastic cells. J Clin Invest. 1987 Nov;80(5):1512–1515. doi: 10.1172/JCI113235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Gregory C. D., Dive C., Henderson S., Smith C. A., Williams G. T., Gordon J., Rickinson A. B. Activation of Epstein-Barr virus latent genes protects human B cells from death by apoptosis. Nature. 1991 Feb 14;349(6310):612–614. doi: 10.1038/349612a0. [DOI] [PubMed] [Google Scholar]
  23. Hanson R. D., Grisolano J. L., Ley T. J. Consensus AP-1 and CRE motifs upstream from the human cytotoxic serine protease B (CSP-B/CGL-1) gene synergize to activate transcription. Blood. 1993 Nov 1;82(9):2749–2757. [PubMed] [Google Scholar]
  24. Harnett M. M., Klaus G. G. G protein coupling of antigen receptor-stimulated polyphosphoinositide hydrolysis in B cells. J Immunol. 1988 May 1;140(9):3135–3139. [PubMed] [Google Scholar]
  25. Hartley S. B., Cooke M. P., Fulcher D. A., Harris A. W., Cory S., Basten A., Goodnow C. C. Elimination of self-reactive B lymphocytes proceeds in two stages: arrested development and cell death. Cell. 1993 Feb 12;72(3):325–335. doi: 10.1016/0092-8674(93)90111-3. [DOI] [PubMed] [Google Scholar]
  26. Huang L. M., Jeang K. T. Increased spacing between Sp1 and TATAA renders human immunodeficiency virus type 1 replication defective: implication for Tat function. J Virol. 1993 Dec;67(12):6937–6944. doi: 10.1128/jvi.67.12.6937-6944.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Knox K. A., Finney M., Milner A. E., Gregory C. D., Wakelam M. J., Michell R. H., Gordon J. Second-messenger pathways involved in the regulation of survival in germinal-centre B cells and in Burkitt lymphoma lines. Int J Cancer. 1992 Dec 2;52(6):959–966. doi: 10.1002/ijc.2910520622. [DOI] [PubMed] [Google Scholar]
  28. Krajewski W., Lee K. A. A monomeric derivative of the cellular transcription factor CREB functions as a constitutive activator. Mol Cell Biol. 1994 Nov;14(11):7204–7210. doi: 10.1128/mcb.14.11.7204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lalli E., Sassone-Corsi P. Signal transduction and gene regulation: the nuclear response to cAMP. J Biol Chem. 1994 Jul 1;269(26):17359–17362. [PubMed] [Google Scholar]
  30. Lane P. J., Ledbetter J. A., McConnell F. M., Draves K., Deans J., Schieven G. L., Clark E. A. The role of tyrosine phosphorylation in signal transduction through surface Ig in human B cells. Inhibition of tyrosine phosphorylation prevents intracellular calcium release. J Immunol. 1991 Jan 15;146(2):715–722. [PubMed] [Google Scholar]
  31. Lee K. A., Masson N. Transcriptional regulation by CREB and its relatives. Biochim Biophys Acta. 1993 Sep 23;1174(3):221–233. doi: 10.1016/0167-4781(93)90191-f. [DOI] [PubMed] [Google Scholar]
  32. Levy Y., Brouet J. C. Interleukin-10 prevents spontaneous death of germinal center B cells by induction of the bcl-2 protein. J Clin Invest. 1994 Jan;93(1):424–428. doi: 10.1172/JCI116977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Liu J. L., Chiles T. C., Sen R. J., Rothstein T. L. Inducible nuclear expression of NF-kappa B in primary B cells stimulated through the surface Ig receptor. J Immunol. 1991 Mar 1;146(5):1685–1691. [PubMed] [Google Scholar]
  34. Liu Y. J., Mason D. Y., Johnson G. D., Abbot S., Gregory C. D., Hardie D. L., Gordon J., MacLennan I. C. Germinal center cells express bcl-2 protein after activation by signals which prevent their entry into apoptosis. Eur J Immunol. 1991 Aug;21(8):1905–1910. doi: 10.1002/eji.1830210819. [DOI] [PubMed] [Google Scholar]
  35. Meyer T. E., Habener J. F. Cyclic adenosine 3',5'-monophosphate response element binding protein (CREB) and related transcription-activating deoxyribonucleic acid-binding proteins. Endocr Rev. 1993 Jun;14(3):269–290. doi: 10.1210/edrv-14-3-269. [DOI] [PubMed] [Google Scholar]
  36. Miyashita T., Harigai M., Hanada M., Reed J. C. Identification of a p53-dependent negative response element in the bcl-2 gene. Cancer Res. 1994 Jun 15;54(12):3131–3135. [PubMed] [Google Scholar]
  37. Miyazaki T., Liu Z. J., Kawahara A., Minami Y., Yamada K., Tsujimoto Y., Barsoumian E. L., Permutter R. M., Taniguchi T. Three distinct IL-2 signaling pathways mediated by bcl-2, c-myc, and lck cooperate in hematopoietic cell proliferation. Cell. 1995 Apr 21;81(2):223–231. doi: 10.1016/0092-8674(95)90332-1. [DOI] [PubMed] [Google Scholar]
  38. Monroe J. G., Haldar S. Involvement of a specific guanine nucleotide binding protein in receptor immunoglobulin stimulated inositol phospholipid hydrolysis. Biochim Biophys Acta. 1989 Oct 9;1013(3):273–278. doi: 10.1016/0167-4889(89)90146-8. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. Negrini M., Silini E., Kozak C., Tsujimoto Y., Croce C. M. Molecular analysis of mbcl-2: structure and expression of the murine gene homologous to the human gene involved in follicular lymphoma. Cell. 1987 May 22;49(4):455–463. doi: 10.1016/0092-8674(87)90448-x. [DOI] [PubMed] [Google Scholar]
  41. Ning Z. Q., Norton J. D., Johnson D., Murphy J. J. Early gene signalling-dependent and -independent induction of apoptosis in Ramos human B cells can be inhibited by over-expression of Bcl-2. Biochem Biophys Res Commun. 1995 Oct 4;215(1):23–29. doi: 10.1006/bbrc.1995.2429. [DOI] [PubMed] [Google Scholar]
  42. Parnes J. R., Seidman J. G. Structure of wild-type and mutant mouse beta 2-microglobulin genes. Cell. 1982 Jun;29(2):661–669. doi: 10.1016/0092-8674(82)90182-9. [DOI] [PubMed] [Google Scholar]
  43. Quinn P. G. Distinct activation domains within cAMP response element-binding protein (CREB) mediate basal and cAMP-stimulated transcription. J Biol Chem. 1993 Aug 15;268(23):16999–17009. [PubMed] [Google Scholar]
  44. Reed J. C., Tsujimoto Y., Alpers J. D., Croce C. M., Nowell P. C. Regulation of bcl-2 proto-oncogene expression during normal human lymphocyte proliferation. Science. 1987 Jun 5;236(4806):1295–1299. doi: 10.1126/science.3495884. [DOI] [PubMed] [Google Scholar]
  45. Reith W., Siegrist C. A., Durand B., Barras E., Mach B. Function of major histocompatibility complex class II promoters requires cooperative binding between factors RFX and NF-Y. Proc Natl Acad Sci U S A. 1994 Jan 18;91(2):554–558. doi: 10.1073/pnas.91.2.554. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Rinaudo M. S., Su K., Falk L. A., Halder S., Mufson R. A. Human interleukin-3 receptor modulates bcl-2 mRNA and protein levels through protein kinase C in TF-1 cells. Blood. 1995 Jul 1;86(1):80–88. [PubMed] [Google Scholar]
  47. Romagnani S., Giudizi M. G., Biagiotti R., Almerigogna F., Maggi E., Del Prete G., Ricci M. Surface immunoglobulins are involved in the interaction of protein A with human B cells and in the triggering of B cell proliferation induced by protein A-containing Staphylococcus aureus. J Immunol. 1981 Oct;127(4):1307–1313. [PubMed] [Google Scholar]
  48. Schanke J. T., Van Ness B. G. Organization of the transcription factor binding sites in the kappa Ig intron enhancer. Effects of position, orientation, and spacing. J Immunol. 1994 Nov 15;153(10):4565–4572. [PubMed] [Google Scholar]
  49. Seto M., Jaeger U., Hockett R. D., Graninger W., Bennett S., Goldman P., Korsmeyer S. J. Alternative promoters and exons, somatic mutation and deregulation of the Bcl-2-Ig fusion gene in lymphoma. EMBO J. 1988 Jan;7(1):123–131. doi: 10.1002/j.1460-2075.1988.tb02791.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Struthers R. S., Vale W. W., Arias C., Sawchenko P. E., Montminy M. R. Somatotroph hypoplasia and dwarfism in transgenic mice expressing a non-phosphorylatable CREB mutant. Nature. 1991 Apr 18;350(6319):622–624. doi: 10.1038/350622a0. [DOI] [PubMed] [Google Scholar]
  51. Tsubata T., Wu J., Honjo T. B-cell apoptosis induced by antigen receptor crosslinking is blocked by a T-cell signal through CD40. Nature. 1993 Aug 12;364(6438):645–648. doi: 10.1038/364645a0. [DOI] [PubMed] [Google Scholar]
  52. Valentine M. A., Licciardi K. A. Rescue from anti-IgM-induced programmed cell death by the B cell surface proteins CD20 and CD40. Eur J Immunol. 1992 Dec;22(12):3141–3148. doi: 10.1002/eji.1830221217. [DOI] [PubMed] [Google Scholar]
  53. Venkataraman L., Francis D. A., Wang Z., Liu J., Rothstein T. L., Sen R. Cyclosporin-A sensitive induction of NF-AT in murine B cells. Immunity. 1994 Jun;1(3):189–196. doi: 10.1016/1074-7613(94)90097-3. [DOI] [PubMed] [Google Scholar]
  54. Wang G., Yeh H. I., Lin J. J. Characterization of cis-regulating elements and trans-activating factors of the rat cardiac troponin T gene. J Biol Chem. 1994 Dec 2;269(48):30595–30603. [PubMed] [Google Scholar]
  55. Xie H., Chiles T. C., Rothstein T. L. Induction of CREB activity via the surface Ig receptor of B cells. J Immunol. 1993 Jul 15;151(2):880–889. [PubMed] [Google Scholar]
  56. Xie H., Rothstein T. L. Protein kinase C mediates activation of nuclear cAMP response element-binding protein (CREB) in B lymphocytes stimulated through surface Ig. J Immunol. 1995 Feb 15;154(4):1717–1723. [PubMed] [Google Scholar]
  57. Xing L., Quinn P. G. Three distinct regions within the constitutive activation domain of cAMP regulatory element-binding protein (CREB) are required for transcription activation. J Biol Chem. 1994 Nov 18;269(46):28732–28736. [PubMed] [Google Scholar]
  58. 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]
  59. Yamanashi Y., Fukui Y., Wongsasant B., Kinoshita Y., Ichimori Y., Toyoshima K., Yamamoto T. Activation of Src-like protein-tyrosine kinase Lyn and its association with phosphatidylinositol 3-kinase upon B-cell antigen receptor-mediated signaling. Proc Natl Acad Sci U S A. 1992 Feb 1;89(3):1118–1122. doi: 10.1073/pnas.89.3.1118. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Young R. L., Korsmeyer S. J. A negative regulatory element in the bcl-2 5'-untranslated region inhibits expression from an upstream promoter. Mol Cell Biol. 1993 Jun;13(6):3686–3697. doi: 10.1128/mcb.13.6.3686. [DOI] [PMC free article] [PubMed] [Google Scholar]

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