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. 1995 Jul;15(7):3857–3863. doi: 10.1128/mcb.15.7.3857

Characterization of a mutant calcineurin A alpha gene expressed by EL4 lymphoma cells.

D A Fruman 1, S Y Pai 1, S J Burakoff 1, B E Bierer 1
PMCID: PMC230625  PMID: 7791792

Abstract

The calmodulin-stimulated phosphatase calcineurin plays a critical role in calcium-dependent T-lymphocyte activation pathways. Here, we report the identification of a missense mutation in the calcineurin A alpha gene expressed by EL4 T-lymphoma cells. This mutation changes an evolutionarily conserved aspartic acid to asparagine within the autoinhibitory domain of the calcineurin A alpha protein. A comparison of wild-type and mutant autoinhibitory peptides indicates that this amino acid substitution greatly reduces inhibition of calcineurin phosphatase activity. Additional peptide inhibition studies support a pseudosubstrate model of autoinhibitory function, in which the conserved aspartic acid residue may serve as a molecular mimic of either phosphoserine or phosphothreonine. Expression of the mutant calcineurin appears to affect cellular signal transduction pathways, as EL4 cells can be activated by suboptimal concentrations of calcium ionophore in the presence of phorbol esters. Moreover, this phenotype can be transferred to Jurkat T cells by transfection of the mutated calcineurin gene. These findings implicate a conserved aspartic acid in the mechanism of calcineurin autoinhibition and suggest that mutation of this residue is associated with aberrant calcium-dependent signaling in vivo.

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Selected References

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

  1. Blumenthal D. K., Takio K., Edelman A. M., Charbonneau H., Titani K., Walsh K. A., Krebs E. G. Identification of the calmodulin-binding domain of skeletal muscle myosin light chain kinase. Proc Natl Acad Sci U S A. 1985 May;82(10):3187–3191. doi: 10.1073/pnas.82.10.3187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chused T. M., Wilson H. A., Greenblatt D., Ishida Y., Edison L. J., Tsien R. Y., Finkelman F. D. Flow cytometric analysis of murine splenic B lymphocyte cytosolic free calcium response to anti-IgM and anti-IgD. Cytometry. 1987 Jul;8(4):396–404. doi: 10.1002/cyto.990080409. [DOI] [PubMed] [Google Scholar]
  3. Clipstone N. A., Crabtree G. R. Identification of calcineurin as a key signalling enzyme in T-lymphocyte activation. Nature. 1992 Jun 25;357(6380):695–697. doi: 10.1038/357695a0. [DOI] [PubMed] [Google Scholar]
  4. Cyert M. S., Kunisawa R., Kaim D., Thorner J. Yeast has homologs (CNA1 and CNA2 gene products) of mammalian calcineurin, a calmodulin-regulated phosphoprotein phosphatase. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7376–7380. doi: 10.1073/pnas.88.16.7376. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Donella-Deana A., Krinks M. H., Ruzzene M., Klee C., Pinna L. A. Dephosphorylation of phosphopeptides by calcineurin (protein phosphatase 2B). Eur J Biochem. 1994 Jan 15;219(1-2):109–117. doi: 10.1111/j.1432-1033.1994.tb19920.x. [DOI] [PubMed] [Google Scholar]
  6. Farrar J. J., Fuller-Farrar J., Simon P. L., Hilfiker M. L., Stadler B. M., Farrar W. L. Thymoma production of T cell growth factor (Interleukin 2). J Immunol. 1980 Dec;125(6):2555–2558. [PubMed] [Google Scholar]
  7. Fruman D. A., Klee C. B., Bierer B. E., Burakoff S. J. Calcineurin phosphatase activity in T lymphocytes is inhibited by FK 506 and cyclosporin A. Proc Natl Acad Sci U S A. 1992 May 1;89(9):3686–3690. doi: 10.1073/pnas.89.9.3686. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fruman D. A., Mather P. E., Burakoff S. J., Bierer B. E. Correlation of calcineurin phosphatase activity and programmed cell death in murine T cell hybridomas. Eur J Immunol. 1992 Oct;22(10):2513–2517. doi: 10.1002/eji.1830221008. [DOI] [PubMed] [Google Scholar]
  9. GORER P. A. Studies in antibody response of mice to tumour inoculation. Br J Cancer. 1950 Dec;4(4):372–379. doi: 10.1038/bjc.1950.36. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gillis S., Ferm M. M., Ou W., Smith K. A. T cell growth factor: parameters of production and a quantitative microassay for activity. J Immunol. 1978 Jun;120(6):2027–2032. [PubMed] [Google Scholar]
  11. Guerini D., Klee C. B. Cloning of human calcineurin A: evidence for two isozymes and identification of a polyproline structural domain. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9183–9187. doi: 10.1073/pnas.86.23.9183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hahn W. C., Menzin E., Saito T., Germain R. N., Bierer B. E. The complete sequences of plasmids pFNeo and pMH-Neo: convenient expression vectors for high-level expression of eukaryotic genes in hematopoietic cell lines. Gene. 1993 May 30;127(2):267–268. doi: 10.1016/0378-1119(93)90731-h. [DOI] [PubMed] [Google Scholar]
  13. Hardie G. Pseudosubstrates turn off protein kinases. Nature. 1988 Oct 13;335(6191):592–593. doi: 10.1038/335592a0. [DOI] [PubMed] [Google Scholar]
  14. Hashimoto Y., Perrino B. A., Soderling T. R. Identification of an autoinhibitory domain in calcineurin. J Biol Chem. 1990 Feb 5;265(4):1924–1927. [PubMed] [Google Scholar]
  15. Higuchi S., Tamura J., Giri P. R., Polli J. W., Kincaid R. L. Calmodulin-dependent protein phosphatase from Neurospora crassa. Molecular cloning and expression of recombinant catalytic subunit. J Biol Chem. 1991 Sep 25;266(27):18104–18112. [PubMed] [Google Scholar]
  16. Ikura M., Clore G. M., Gronenborn A. M., Zhu G., Klee C. B., Bax A. Solution structure of a calmodulin-target peptide complex by multidimensional NMR. Science. 1992 May 1;256(5057):632–638. doi: 10.1126/science.1585175. [DOI] [PubMed] [Google Scholar]
  17. Kincaid R. L., Giri P. R., Higuchi S., Tamura J., Dixon S. C., Marietta C. A., Amorese D. A., Martin B. M. Cloning and characterization of molecular isoforms of the catalytic subunit of calcineurin using nonisotopic methods. J Biol Chem. 1990 Jul 5;265(19):11312–11319. [PubMed] [Google Scholar]
  18. Liu J., Albers M. W., Wandless T. J., Luan S., Alberg D. G., Belshaw P. J., Cohen P., MacKintosh C., Klee C. B., Schreiber S. L. Inhibition of T cell signaling by immunophilin-ligand complexes correlates with loss of calcineurin phosphatase activity. Biochemistry. 1992 Apr 28;31(16):3896–3901. doi: 10.1021/bi00131a002. [DOI] [PubMed] [Google Scholar]
  19. Liu J., Farmer J. D., Jr, Lane W. S., Friedman J., Weissman I., Schreiber S. L. Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes. Cell. 1991 Aug 23;66(4):807–815. doi: 10.1016/0092-8674(91)90124-h. [DOI] [PubMed] [Google Scholar]
  20. Manalan A. S., Klee C. B. Activation of calcineurin by limited proteolysis. Proc Natl Acad Sci U S A. 1983 Jul;80(14):4291–4295. doi: 10.1073/pnas.80.14.4291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Milan D., Griffith J., Su M., Price E. R., McKeon F. The latch region of calcineurin B is involved in both immunosuppressant-immunophilin complex docking and phosphatase activation. Cell. 1994 Nov 4;79(3):437–447. doi: 10.1016/0092-8674(94)90253-4. [DOI] [PubMed] [Google Scholar]
  22. Muramatsu T., Kincaid R. L. Molecular cloning and chromosomal mapping of the human gene for the testis-specific catalytic subunit of calmodulin-dependent protein phosphatase (calcineurin A). Biochem Biophys Res Commun. 1992 Oct 15;188(1):265–271. doi: 10.1016/0006-291x(92)92379-c. [DOI] [PubMed] [Google Scholar]
  23. Nolan G. P., Fiering S., Nicolas J. F., Herzenberg L. A. Fluorescence-activated cell analysis and sorting of viable mammalian cells based on beta-D-galactosidase activity after transduction of Escherichia coli lacZ. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2603–2607. doi: 10.1073/pnas.85.8.2603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. O'Keefe S. J., Tamura J., Kincaid R. L., Tocci M. J., O'Neill E. A. FK-506- and CsA-sensitive activation of the interleukin-2 promoter by calcineurin. Nature. 1992 Jun 25;357(6380):692–694. doi: 10.1038/357692a0. [DOI] [PubMed] [Google Scholar]
  25. Paliogianni F., Kincaid R. L., Boumpas D. T. Prostaglandin E2 and other cyclic AMP elevating agents inhibit interleukin 2 gene transcription by counteracting calcineurin-dependent pathways. J Exp Med. 1993 Nov 1;178(5):1813–1817. doi: 10.1084/jem.178.5.1813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Parsons J. N., Wiederrecht G. J., Salowe S., Burbaum J. J., Rokosz L. L., Kincaid R. L., O'Keefe S. J. Regulation of calcineurin phosphatase activity and interaction with the FK-506.FK-506 binding protein complex. J Biol Chem. 1994 Jul 29;269(30):19610–19616. [PubMed] [Google Scholar]
  27. Perrino B. A., Ng L. Y., Soderling T. R. Calcium regulation of calcineurin phosphatase activity by its B subunit and calmodulin. Role of the autoinhibitory domain. J Biol Chem. 1995 Jan 6;270(1):340–346. doi: 10.1074/jbc.270.1.340. [DOI] [PubMed] [Google Scholar]
  28. Sleckman B. P., Peterson A., Jones W. K., Foran J. A., Greenstein J. L., Seed B., Burakoff S. J. Expression and function of CD4 in a murine T-cell hybridoma. Nature. 1987 Jul 23;328(6128):351–353. doi: 10.1038/328351a0. [DOI] [PubMed] [Google Scholar]
  29. Stemmer P. M., Klee C. B. Dual calcium ion regulation of calcineurin by calmodulin and calcineurin B. Biochemistry. 1994 Jun 7;33(22):6859–6866. doi: 10.1021/bi00188a015. [DOI] [PubMed] [Google Scholar]
  30. Stemmer P., Klee C. B. Serine/threonine phosphatases in the nervous system. Curr Opin Neurobiol. 1991 Jun;1(1):53–64. doi: 10.1016/0959-4388(91)90010-5. [DOI] [PubMed] [Google Scholar]
  31. Truneh A., Albert F., Golstein P., Schmitt-Verhulst A. M. Early steps of lymphocyte activation bypassed by synergy between calcium ionophores and phorbol ester. Nature. 1985 Jan 24;313(6000):318–320. doi: 10.1038/313318a0. [DOI] [PubMed] [Google Scholar]
  32. Weiss A., Wiskocil R. L., Stobo J. D. The role of T3 surface molecules in the activation of human T cells: a two-stimulus requirement for IL 2 production reflects events occurring at a pre-translational level. J Immunol. 1984 Jul;133(1):123–128. [PubMed] [Google Scholar]
  33. Woodrow M., Clipstone N. A., Cantrell D. p21ras and calcineurin synergize to regulate the nuclear factor of activated T cells. J Exp Med. 1993 Nov 1;178(5):1517–1522. doi: 10.1084/jem.178.5.1517. [DOI] [PMC free article] [PubMed] [Google Scholar]

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