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. 1985 Jul 1;101(1):207–216. doi: 10.1083/jcb.101.1.207

Calcium-dependent association of a protein complex with the lymphocyte plasma membrane: probable identity with calmodulin-calcineurin

PMCID: PMC2113640  PMID: 2989299

Abstract

A protein complex is shown to participate in a calcium-dependent association with plasma membranes purified either from pig mesenteric lymph node lymphocytes or from human lymphoblastoid cell lines. Plasma membranes prepared in the presence of calcium possess this complex; those prepared in the absence of calcium (5 mM EGTA) do not. The complex associates itself with the inner cytoplasmic surface of the plasma membrane. This complex is referred to as the "acidic protein band" because of its location during migration upon alkaline-urea gel electrophoresis. The complex dissociates from the plasma membrane during electrophoresis on 8-M urea gels, irrespective of calcium levels during electrophoresis; at intermediate urea concentrations (4-6 M), the complex is not dissociated in the presence of calcium. Upon purification of the acidic protein band, SDS acrylamide gel electrophoresis, immunoblotting, and radioimmunoassay techniques suggest that the acidic protein band is composed of at least four peptides (designated 68K, 59K, 20K, 20K): two of these (68K, 20K) are immunopositive for calcineurin and one (20K) is immunopositive for calmodulin. Immunoblots of urea gels also indicate that the calcineurin heavy chain (68K) can also appear at three different locations on the urea gel. Patches and caps induced in human peripheral blood lymphocytes by fluorescein-conjugated goat anti-human IgG are not coincident with the location of calcineurin, which remains distributed throughout the cell.

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Selected References

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  1. Braun J., Fujiwara K., Pollard T. D., Unanue E. R. Two distinct mechanisms for redistribution of lymphocyte surface macromolecules. I. Relationship to cytoplasmic myosin. J Cell Biol. 1978 Nov;79(2 Pt 1):409–418. doi: 10.1083/jcb.79.2.409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Braun J., Sha'afi R. I., Unanue E. R. Crosslinking by ligands to surface immunoglobulin triggers mobilization of intracellular 45Ca2+ in B lymphocytes. J Cell Biol. 1979 Sep;82(3):755–766. doi: 10.1083/jcb.82.3.755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Casnellie J. E., Harrison M. L., Hellstrom K. E., Krebs E. G. A lymphoma protein with an in vitro site of tyrosine phosphorylation homologous to that in pp60src. J Biol Chem. 1982 Dec 10;257(23):13877–13879. [PubMed] [Google Scholar]
  4. Chantler P. D., Szent-Györgyi A. G. Regulatory light-chains and scallop myosin. Full dissociation, reversibility and co-operative effects. J Mol Biol. 1980 Apr 15;138(3):473–492. doi: 10.1016/s0022-2836(80)80013-1. [DOI] [PubMed] [Google Scholar]
  5. Chaplin D. D., Wedner H. J., Parker C. W. Protein phosphorlyation in human peripheral blood lymphocytes. Phosphorylation of endogenous plasma membrane and cytoplasmic proteins. Biochem J. 1979 Aug 15;182(2):537–546. doi: 10.1042/bj1820537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chaplin D. D., Wedner H. J., Parker C. W. Protein phosphorylation in human peripheral blood lymphocytes. Subcellular distribution and partial characterization of adenosine 3':5'-cyclic monophosphate-dependent protein kinase. Biochem J. 1979 Aug 15;182(2):525–536. doi: 10.1042/bj1820525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chavin S. I., Holliman A. Reconstitution of pig lymphocyte plasma membranes from solubilized components, with particular reference to membrane-associated immunoglobulins. Biochem J. 1975 Nov;152(2):267–270. doi: 10.1042/bj1520267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dellagi K., Brouet J. C. Redistribution of intermediate filaments during capping of lymphocyte surface molecules. Nature. 1982 Jul 15;298(5871):284–286. doi: 10.1038/298284a0. [DOI] [PubMed] [Google Scholar]
  9. Earp H. S., Austin K. S., Buessow S. C., Dy R., Gillespie G. Y. Membranes from T and B lymphocytes have different patterns of tyrosine phosphorylation. Proc Natl Acad Sci U S A. 1984 Apr;81(8):2347–2351. doi: 10.1073/pnas.81.8.2347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Flanagan J., Koch G. L. Cross-linked surface Ig attaches to actin. Nature. 1978 May 25;273(5660):278–281. doi: 10.1038/273278a0. [DOI] [PubMed] [Google Scholar]
  11. Gabbiani G., Chaponnier C., Zumbe A., Vassalli P. Actin and tubulin co-cap with surface immunoglobulins in mouse B lymphocytes. Nature. 1977 Oct 20;269(5630):697–698. doi: 10.1038/269697a0. [DOI] [PubMed] [Google Scholar]
  12. Geiger B., Singer S. J. The participation of alpha-actinin in the capping of cell membrane components. Cell. 1979 Jan;16(1):213–222. doi: 10.1016/0092-8674(79)90202-2. [DOI] [PubMed] [Google Scholar]
  13. Hawkes R., Niday E., Gordon J. A dot-immunobinding assay for monoclonal and other antibodies. Anal Biochem. 1982 Jan 1;119(1):142–147. doi: 10.1016/0003-2697(82)90677-7. [DOI] [PubMed] [Google Scholar]
  14. Head J. F., Perry S. V. The interaction of the calcium-binding protein (troponin C) with bivalent cations and the inhibitory protein (troponin I). Biochem J. 1974 Feb;137(2):145–154. doi: 10.1042/bj1370145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hoessli D., Rungger-Brändle E., Jockusch B. M., Gabbiani G. Lymphocyte alpha-actinin. Relationship to cell membrane and co-capping with surface receptors. J Cell Biol. 1980 Feb;84(2):305–314. doi: 10.1083/jcb.84.2.305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Johnson G. D., Nogueira Araujo G. M. A simple method of reducing the fading of immunofluorescence during microscopy. J Immunol Methods. 1981;43(3):349–350. doi: 10.1016/0022-1759(81)90183-6. [DOI] [PubMed] [Google Scholar]
  17. Johnstone A. P., DuBois J. H., Crumpton M. J. Phosphorylated lymphocyte plasma-membrane proteins. Biochem J. 1981 Jan 15;194(1):309–318. doi: 10.1042/bj1940309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Johnstone A. P. Rejoining of DNA strand breaks is an early nuclear event during the stimulation of quiescent lymphocytes. Eur J Biochem. 1984 Apr 16;140(2):401–406. doi: 10.1111/j.1432-1033.1984.tb08116.x. [DOI] [PubMed] [Google Scholar]
  19. Klee C. B., Crouch T. H., Krinks M. H. Calcineurin: a calcium- and calmodulin-binding protein of the nervous system. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6270–6273. doi: 10.1073/pnas.76.12.6270. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  21. Levine J., Willard M. Redistribution of fodrin (a component of the cortical cytoplasm) accompanying capping of cell surface molecules. Proc Natl Acad Sci U S A. 1983 Jan;80(1):191–195. doi: 10.1073/pnas.80.1.191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lichtman A. H., Segel G. B., Lichtman M. A. Total and exchangeable calcium in lymphocytes: effects of PHA and A23187. J Supramol Struct. 1980;14(1):65–75. doi: 10.1002/jss.400140107. [DOI] [PubMed] [Google Scholar]
  23. Maino V. C., Green N. M., Crumpton M. J. The role of calcium ions in initiating transformation of lymphocytes. Nature. 1974 Sep 27;251(5473):324–327. doi: 10.1038/251324b0. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Monneron A., d'Alayer J. Isolation of plasma and nuclear membranes of thymocytes. I. Enzymatic composition and ultrastructure. J Cell Biol. 1978 Apr;77(1):211–231. doi: 10.1083/jcb.77.1.211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Monneron A., d'Alayer J. Isolation of plasma and nuclear membranes of thymocytes. II. Biochemical composition. J Cell Biol. 1978 Apr;77(1):232–245. doi: 10.1083/jcb.77.1.232. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Morrissey J. H. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem. 1981 Nov 1;117(2):307–310. doi: 10.1016/0003-2697(81)90783-1. [DOI] [PubMed] [Google Scholar]
  28. Nelson G. A., Andrews M. L., Karnovsky M. J. Participation of calmodulin in immunoglobulin capping. J Cell Biol. 1982 Dec;95(3):771–780. doi: 10.1083/jcb.95.3.771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Pallen C. J., Wang J. H. Calmodulin-stimulated dephosphorylation of p-nitrophenyl phosphate and free phosphotyrosine by calcineurin. J Biol Chem. 1983 Jul 25;258(14):8550–8553. [PubMed] [Google Scholar]
  30. Pallen C. J., Wang J. H. Regulation of calcineurin by metal ions. Mechanism of activation by Ni2+ and an enhanced response to Ca2+/calmodulin. J Biol Chem. 1984 May 25;259(10):6134–6141. [PubMed] [Google Scholar]
  31. Pober J. S., Guild B. C., Strominger J. L. Phosphorylation in vivo and in vitro of human histocompatibility antigens (HLA-A and HLA-B) in the carboxy-terminal intracellular domain. Proc Natl Acad Sci U S A. 1978 Dec;75(12):6002–6006. doi: 10.1073/pnas.75.12.6002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Pozzan T., Arslan P., Tsien R. Y., Rink T. J. Anti-immunoglobulin, cytoplasmic free calcium, and capping in B lymphocytes. J Cell Biol. 1982 Aug;94(2):335–340. doi: 10.1083/jcb.94.2.335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Stewart A. A., Ingebritsen T. S., Cohen P. The protein phosphatases involved in cellular regulation. 5. Purification and properties of a Ca2+/calmodulin-dependent protein phosphatase (2B) from rabbit skeletal muscle. Eur J Biochem. 1983 May 2;132(2):289–295. doi: 10.1111/j.1432-1033.1983.tb07361.x. [DOI] [PubMed] [Google Scholar]
  34. Stewart A. A., Ingebritsen T. S., Manalan A., Klee C. B., Cohen P. Discovery of a Ca2+- and calmodulin-dependent protein phosphatase: probable identity with calcineurin (CaM-BP80). FEBS Lett. 1982 Jan 11;137(1):80–84. doi: 10.1016/0014-5793(82)80319-0. [DOI] [PubMed] [Google Scholar]
  35. Swarup G., Dasgupta J. D., Garbers D. L. Tyrosine protein kinase activity of rat spleen and other tissues. J Biol Chem. 1983 Sep 10;258(17):10341–10347. [PubMed] [Google Scholar]
  36. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Wallace R. W., Lynch T. J., Tallant E. A., Cheung W. Y. Purification and characterization of an inhibitor protein of brain adenylate cyclase and cyclic nucleotide phosphodiesterase. J Biol Chem. 1979 Jan 25;254(2):377–382. [PubMed] [Google Scholar]
  38. Walsh F. S., Crumpton M. J. Orientation of cell-surface antigens in the lipid bilayer of lymphocyte plasma membrane. Nature. 1977 Sep 22;269(5626):307–311. doi: 10.1038/269307a0. [DOI] [PubMed] [Google Scholar]
  39. Winkler M. A., Merat D. L., Tallant E. A., Hawkins S., Cheung W. Y. Catalytic site of calmodulin-dependent protein phosphatase from bovine brain resides in subunit A. Proc Natl Acad Sci U S A. 1984 May;81(10):3054–3058. doi: 10.1073/pnas.81.10.3054. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Yakara I., Kakimoto-Sameshima F. Microtubule organization of lymphocytes and its modulation by patch and cap formation. Cell. 1978 Sep;15(1):251–259. doi: 10.1016/0092-8674(78)90100-9. [DOI] [PubMed] [Google Scholar]
  41. Yang S. D., Tallant E. A., Cheung W. Y. Calcineurin is a calmodulin-dependent protein phosphatase. Biochem Biophys Res Commun. 1982 Jun 30;106(4):1419–1425. doi: 10.1016/0006-291x(82)91272-4. [DOI] [PubMed] [Google Scholar]

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