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. 1993 Aug;13(8):4760–4769. doi: 10.1128/mcb.13.8.4760

Identification of the immunophilins capable of mediating inhibition of signal transduction by cyclosporin A and FK506: roles of calcineurin binding and cellular location.

R J Bram 1, D T Hung 1, P K Martin 1, S L Schreiber 1, G R Crabtree 1
PMCID: PMC360102  PMID: 7687744

Abstract

The immunosuppressants cyclosporin A (CsA) and FK506 appear to block T-cell function by inhibiting the calcium-regulated phosphatase calcineurin. While multiple distinct intracellular receptors for these drugs (cyclophilins and FKBPs, collectively immunophilins) have been characterized, the functionally active ones have not been discerned. We found that overexpression of cyclophilin A or B or FKBP12 increased T-cell sensitivity to CsA or FK506, respectively, demonstrating that they are able to mediate the inhibitory effects of their respective immunosuppressants in vivo. In contrast, cyclophilin C, FKBP13, and FKBP25 had no effect. Direct comparison of the Ki of each drug-immunophilin complex for calcineurin in vitro revealed that although calcineurin binding was clearly necessary, it was not sufficient to explain the in vivo activity of the immunophilin. Subcellular localization was shown also to play a role, since gene deletions of cyclophilins B and C which changed their intracellular locations altered their activities significantly. Cyclophilin B has been shown previously to be located within calcium-containing intracellular vesicles; its ability to mediate CsA inhibition implies that certain components of the signal transduction machinery are also spatially restricted within the cell.

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Selected References

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  1. Aitken A., Cohen P., Santikarn S., Williams D. H., Calder A. G., Smith A., Klee C. B. Identification of the NH2-terminal blocking group of calcineurin B as myristic acid. FEBS Lett. 1982 Dec 27;150(2):314–318. doi: 10.1016/0014-5793(82)80759-x. [DOI] [PubMed] [Google Scholar]
  2. Arber S., Krause K. H., Caroni P. s-cyclophilin is retained intracellularly via a unique COOH-terminal sequence and colocalizes with the calcium storage protein calreticulin. J Cell Biol. 1992 Jan;116(1):113–125. doi: 10.1083/jcb.116.1.113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Berger J., Hauber J., Hauber R., Geiger R., Cullen B. R. Secreted placental alkaline phosphatase: a powerful new quantitative indicator of gene expression in eukaryotic cells. Gene. 1988 Jun 15;66(1):1–10. doi: 10.1016/0378-1119(88)90219-3. [DOI] [PubMed] [Google Scholar]
  4. Bierer B. E., Mattila P. S., Standaert R. F., Herzenberg L. A., Burakoff S. J., Crabtree G., Schreiber S. L. Two distinct signal transmission pathways in T lymphocytes are inhibited by complexes formed between an immunophilin and either FK506 or rapamycin. Proc Natl Acad Sci U S A. 1990 Dec;87(23):9231–9235. doi: 10.1073/pnas.87.23.9231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Borel J. F., Feurer C., Gubler H. U., Stähelin H. Biological effects of cyclosporin A: a new antilymphocytic agent. Agents Actions. 1976 Jul;6(4):468–475. doi: 10.1007/BF01973261. [DOI] [PubMed] [Google Scholar]
  6. Chantler P. D. Calcium-dependent association of a protein complex with the lymphocyte plasma membrane: probable identity with calmodulin-calcineurin. J Cell Biol. 1985 Jul;101(1):207–216. doi: 10.1083/jcb.101.1.207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Emmel E. A., Verweij C. L., Durand D. B., Higgins K. M., Lacy E., Crabtree G. R. Cyclosporin A specifically inhibits function of nuclear proteins involved in T cell activation. Science. 1989 Dec 22;246(4937):1617–1620. doi: 10.1126/science.2595372. [DOI] [PubMed] [Google Scholar]
  9. Erlanger B. F. Do we know the site of action of cyclosporin? Immunol Today. 1992 Dec;13(12):487–490. doi: 10.1016/0167-5699(92)90023-Z. [DOI] [PubMed] [Google Scholar]
  10. Fiering S., Northrop J. P., Nolan G. P., Mattila P. S., Crabtree G. R., Herzenberg L. A. Single cell assay of a transcription factor reveals a threshold in transcription activated by signals emanating from the T-cell antigen receptor. Genes Dev. 1990 Oct;4(10):1823–1834. doi: 10.1101/gad.4.10.1823. [DOI] [PubMed] [Google Scholar]
  11. Friedman J., Weissman I. Two cytoplasmic candidates for immunophilin action are revealed by affinity for a new cyclophilin: one in the presence and one in the absence of CsA. Cell. 1991 Aug 23;66(4):799–806. doi: 10.1016/0092-8674(91)90123-g. [DOI] [PubMed] [Google Scholar]
  12. Galat A., Lane W. S., Standaert R. F., Schreiber S. L. A rapamycin-selective 25-kDa immunophilin. Biochemistry. 1992 Mar 3;31(8):2427–2434. doi: 10.1021/bi00123a031. [DOI] [PubMed] [Google Scholar]
  13. Harding M. W., Handschumacher R. E. Cyclophilin, a primary molecular target for cyclosporine. Structural and functional implications. Transplantation. 1988 Aug;46(2 Suppl):29S–35S. doi: 10.1097/00007890-198808001-00006. [DOI] [PubMed] [Google Scholar]
  14. Hasel K. W., Glass J. R., Godbout M., Sutcliffe J. G. An endoplasmic reticulum-specific cyclophilin. Mol Cell Biol. 1991 Jul;11(7):3484–3491. doi: 10.1128/mcb.11.7.3484. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ho S. N., Hunt H. D., Horton R. M., Pullen J. K., Pease L. R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 1989 Apr 15;77(1):51–59. doi: 10.1016/0378-1119(89)90358-2. [DOI] [PubMed] [Google Scholar]
  16. Horton R. M., Hunt H. D., Ho S. N., Pullen J. K., Pease L. R. Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene. 1989 Apr 15;77(1):61–68. doi: 10.1016/0378-1119(89)90359-4. [DOI] [PubMed] [Google Scholar]
  17. Hubbard M. J., Klee C. B. Functional domain structure of calcineurin A: mapping by limited proteolysis. Biochemistry. 1989 Feb 21;28(4):1868–1874. doi: 10.1021/bi00430a066. [DOI] [PubMed] [Google Scholar]
  18. Hung D. T., Schreiber S. L. cDNA cloning of a human 25 kDa FK506 and rapamycin binding protein. Biochem Biophys Res Commun. 1992 Apr 30;184(2):733–738. doi: 10.1016/0006-291x(92)90651-z. [DOI] [PubMed] [Google Scholar]
  19. Jin Y. J., Albers M. W., Lane W. S., Bierer B. E., Schreiber S. L., Burakoff S. J. Molecular cloning of a membrane-associated human FK506- and rapamycin-binding protein, FKBP-13. Proc Natl Acad Sci U S A. 1991 Aug 1;88(15):6677–6681. doi: 10.1073/pnas.88.15.6677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Karttunen J., Shastri N. Measurement of ligand-induced activation in single viable T cells using the lacZ reporter gene. Proc Natl Acad Sci U S A. 1991 May 1;88(9):3972–3976. doi: 10.1073/pnas.88.9.3972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kay J. E., Benzie C. R., Borghetti A. F. Effect of cyclosporin A on lymphocyte activation by the calcium ionophore A23187. Immunology. 1983 Nov;50(3):441–446. [PMC free article] [PubMed] [Google Scholar]
  22. Kolodziej P. A., Young R. A. Epitope tagging and protein surveillance. Methods Enzymol. 1991;194:508–519. doi: 10.1016/0076-6879(91)94038-e. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. 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]
  26. Mattila P. S., Ullman K. S., Fiering S., Emmel E. A., McCutcheon M., Crabtree G. R., Herzenberg L. A. The actions of cyclosporin A and FK506 suggest a novel step in the activation of T lymphocytes. EMBO J. 1990 Dec;9(13):4425–4433. doi: 10.1002/j.1460-2075.1990.tb07893.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Moss S. E., Crompton M. R., Crumpton M. J. Molecular cloning of murine p68, a Ca2+-binding protein of the lipocortin family. Eur J Biochem. 1988 Oct 15;177(1):21–27. doi: 10.1111/j.1432-1033.1988.tb14340.x. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Price E. R., Zydowsky L. D., Jin M. J., Baker C. H., McKeon F. D., Walsh C. T. Human cyclophilin B: a second cyclophilin gene encodes a peptidyl-prolyl isomerase with a signal sequence. Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1903–1907. doi: 10.1073/pnas.88.5.1903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Randak C., Brabletz T., Hergenröther M., Sobotta I., Serfling E. Cyclosporin A suppresses the expression of the interleukin 2 gene by inhibiting the binding of lymphocyte-specific factors to the IL-2 enhancer. EMBO J. 1990 Aug;9(8):2529–2536. doi: 10.1002/j.1460-2075.1990.tb07433.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Schreiber S. L., Crabtree G. R. The mechanism of action of cyclosporin A and FK506. Immunol Today. 1992 Apr;13(4):136–142. doi: 10.1016/0167-5699(92)90111-J. [DOI] [PubMed] [Google Scholar]
  32. Smith D. B., Johnson K. S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. doi: 10.1016/0378-1119(88)90005-4. [DOI] [PubMed] [Google Scholar]
  33. Spitzfaden C., Weber H. P., Braun W., Kallen J., Wider G., Widmer H., Walkinshaw M. D., Wüthrich K. Cyclosporin A-cyclophilin complex formation. A model based on X-ray and NMR data. FEBS Lett. 1992 Apr 6;300(3):291–300. doi: 10.1016/0014-5793(92)80866-f. [DOI] [PubMed] [Google Scholar]
  34. Standaert R. F., Galat A., Verdine G. L., Schreiber S. L. Molecular cloning and overexpression of the human FK506-binding protein FKBP. Nature. 1990 Aug 16;346(6285):671–674. doi: 10.1038/346671a0. [DOI] [PubMed] [Google Scholar]
  35. Takebe Y., Seiki M., Fujisawa J., Hoy P., Yokota K., Arai K., Yoshida M., Arai N. SR alpha promoter: an efficient and versatile mammalian cDNA expression system composed of the simian virus 40 early promoter and the R-U5 segment of human T-cell leukemia virus type 1 long terminal repeat. Mol Cell Biol. 1988 Jan;8(1):466–472. doi: 10.1128/mcb.8.1.466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Ullman K. S., Flanagan W. M., Edwards C. A., Crabtree G. R. Activation of early gene expression in T lymphocytes by Oct-1 and an inducible protein, OAP40. Science. 1991 Oct 25;254(5031):558–562. doi: 10.1126/science.1683003. [DOI] [PubMed] [Google Scholar]
  37. Ullman K. S., Northrop J. P., Admon A., Crabtree G. R. Jun family members are controlled by a calcium-regulated, cyclosporin A-sensitive signaling pathway in activated T lymphocytes. Genes Dev. 1993 Feb;7(2):188–196. doi: 10.1101/gad.7.2.188. [DOI] [PubMed] [Google Scholar]
  38. Verweij C. L., Guidos C., Crabtree G. R. Cell type specificity and activation requirements for NFAT-1 (nuclear factor of activated T-cells) transcriptional activity determined by a new method using transgenic mice to assay transcriptional activity of an individual nuclear factor. J Biol Chem. 1990 Sep 15;265(26):15788–15795. [PubMed] [Google Scholar]

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