Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1989 Feb 1;108(2):569–578. doi: 10.1083/jcb.108.2.569

Isolation of a new member of the S100 protein family: amino acid sequence, tissue, and subcellular distribution

PMCID: PMC2115452  PMID: 2521861

Abstract

A low molecular mass protein which we term S100L was isolated from bovine lung. S100L possesses many of the properties of brain S100 such as self association, Ca++-binding (2 sites per subunit) with moderate affinity, and exposure of a hydrophobic site upon Ca++-saturation. Antibodies to brain S100 proteins, however, do not cross react with S100L. Tryptic peptides derived from S100L were sequenced revealing similarity to other members of the S100 family. Oligonucleotide probes based on these sequences were used to screen a cDNA library derived from a bovine kidney cell line (MDBK). A 562-nucleotide cDNA was sequenced and found to contain the complete coding region of S100L. The predicted amino acid sequence displays striking similarity, yet is clearly distinct from other members of the S100 protein family. Polyclonal and monoclonal antibodies were raised against S100L and used to determine the tissue and subcellular distribution of this molecule. The S100L protein is expressed at high levels in bovine kidney and lung tissue, low levels in brain and intestine, with intermediate levels in muscle. The MDBK cell line was found to contain both S100L and the calpactin light chain, another member of this protein family. S100L was not found associated with a higher molecular mass subunit in MDBK cells while the calpactin light chain was tightly bound to the calpactin heavy chain. Double label immunofluorescence microscopy confirmed the observation that the calpactin light chain and S100L have a different distribution in these cells.

Full Text

The Full Text of this article is available as a PDF (2.3 MB).

Selected References

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

  1. Barraclough R., Savin J., Dube S. K., Rudland P. S. Molecular cloning and sequence of the gene for p9Ka. A cultured myoepithelial cell protein with strong homology to S-100, a calcium-binding protein. J Mol Biol. 1987 Nov 5;198(1):13–20. doi: 10.1016/0022-2836(87)90453-0. [DOI] [PubMed] [Google Scholar]
  2. Baudier J., Cole R. D. Interactions between the microtubule-associated tau proteins and S100b regulate tau phosphorylation by the Ca2+/calmodulin-dependent protein kinase II. J Biol Chem. 1988 Apr 25;263(12):5876–5883. [PubMed] [Google Scholar]
  3. Baudier J., Holtzscherer C., Gerard D. Zinc-dependent affinity chromatography of the S100b protein on phenyl-Sepharose. A rapid purification method. FEBS Lett. 1982 Nov 8;148(2):231–234. doi: 10.1016/0014-5793(82)80813-2. [DOI] [PubMed] [Google Scholar]
  4. Calabretta B., Battini R., Kaczmarek L., de Riel J. K., Baserga R. Molecular cloning of the cDNA for a growth factor-inducible gene with strong homology to S-100, a calcium-binding protein. J Biol Chem. 1986 Sep 25;261(27):12628–12632. [PubMed] [Google Scholar]
  5. Donato R., Michetti F., Miani N. Soluble and membrane-bound S-100 protein in cerebral cortex synaptosomes. Properties of the S-100 receptor. Brain Res. 1975 Nov 21;98(3):561–573. doi: 10.1016/0006-8993(75)90373-x. [DOI] [PubMed] [Google Scholar]
  6. Dorin J. R., Novak M., Hill R. E., Brock D. J., Secher D. S., van Heyningen V. A clue to the basic defect in cystic fibrosis from cloning the CF antigen gene. Nature. 1987 Apr 9;326(6113):614–617. doi: 10.1038/326614a0. [DOI] [PubMed] [Google Scholar]
  7. Erikson E., Erikson R. L. Identification of a cellular protein substrate phosphorylated by the avian sarcoma virus-transforming gene product. Cell. 1980 Oct;21(3):829–836. doi: 10.1016/0092-8674(80)90446-8. [DOI] [PubMed] [Google Scholar]
  8. Erikson E., Tomasiewicz H. G., Erikson R. L. Biochemical characterization of a 34-kilodalton normal cellular substrate of pp60v-src and an associated 6-kilodalton protein. Mol Cell Biol. 1984 Jan;4(1):77–85. doi: 10.1128/mcb.4.1.77. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ferrari S., Calabretta B., deRiel J. K., Battini R., Ghezzo F., Lauret E., Griffin C., Emanuel B. S., Gurrieri F., Baserga R. Structural and functional analysis of a growth-regulated gene, the human calcyclin. J Biol Chem. 1987 Jun 15;262(17):8325–8332. [PubMed] [Google Scholar]
  10. Geisow M. J., Fritsche U., Hexham J. M., Dash B., Johnson T. A consensus amino-acid sequence repeat in Torpedo and mammalian Ca2+-dependent membrane-binding proteins. Nature. 1986 Apr 17;320(6063):636–638. doi: 10.1038/320636a0. [DOI] [PubMed] [Google Scholar]
  11. Gerke V., Weber K. Calcium-dependent conformational changes in the 36-kDa subunit of intestinal protein I related to the cellular 36-kDa target of Rous sarcoma virus tyrosine kinase. J Biol Chem. 1985 Feb 10;260(3):1688–1695. [PubMed] [Google Scholar]
  12. Gerke V., Weber K. Identity of p36K phosphorylated upon Rous sarcoma virus transformation with a protein purified from brush borders; calcium-dependent binding to non-erythroid spectrin and F-actin. EMBO J. 1984 Jan;3(1):227–233. doi: 10.1002/j.1460-2075.1984.tb01789.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gerke V., Weber K. The regulatory chain in the p36-kd substrate complex of viral tyrosine-specific protein kinases is related in sequence to the S-100 protein of glial cells. EMBO J. 1985 Nov;4(11):2917–2920. doi: 10.1002/j.1460-2075.1985.tb04023.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Glenney J. R., Jr, Boudreau M., Galyean R., Hunter T., Tack B. Association of the S-100-related calpactin I light chain with the NH2-terminal tail of the 36-kDa heavy chain. J Biol Chem. 1986 Aug 15;261(23):10485–10488. [PubMed] [Google Scholar]
  15. Glenney J. R., Jr Phosphorylation of p36 in vitro with pp60src. Regulation by Ca2+ and phospholipid. FEBS Lett. 1985 Nov 11;192(1):79–82. doi: 10.1016/0014-5793(85)80047-8. [DOI] [PubMed] [Google Scholar]
  16. Glenney J. R., Jr, Tack B. F. Amino-terminal sequence of p36 and associated p10: identification of the site of tyrosine phosphorylation and homology with S-100. Proc Natl Acad Sci U S A. 1985 Dec;82(23):7884–7888. doi: 10.1073/pnas.82.23.7884. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Glenney J. R., Jr, Zokas L., Kamps M. P. Monoclonal antibodies to phosphotyrosine. J Immunol Methods. 1988 May 9;109(2):277–285. doi: 10.1016/0022-1759(88)90253-0. [DOI] [PubMed] [Google Scholar]
  18. Glenney J. Antibody probing of western blots which have been stained with india ink. Anal Biochem. 1986 Aug 1;156(2):315–319. doi: 10.1016/0003-2697(86)90259-9. [DOI] [PubMed] [Google Scholar]
  19. Glenney J. Phospholipid-dependent Ca2+ binding by the 36-kDa tyrosine kinase substrate (calpactin) and its 33-kDa core. J Biol Chem. 1986 Jun 5;261(16):7247–7252. [PubMed] [Google Scholar]
  20. Glenney J. Two related but distinct forms of the Mr 36,000 tyrosine kinase substrate (calpactin) that interact with phospholipid and actin in a Ca2+-dependent manner. Proc Natl Acad Sci U S A. 1986 Jun;83(12):4258–4262. doi: 10.1073/pnas.83.12.4258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Goto K., Endo H., Fujiyoshi T. Cloning of the sequences expressed abundantly in established cell lines: identification of a cDNA clone highly homologous to S-100, a calcium binding protein. J Biochem. 1988 Jan;103(1):48–53. doi: 10.1093/oxfordjournals.jbchem.a122237. [DOI] [PubMed] [Google Scholar]
  22. Hagiwara M., Ochiai M., Owada K., Tanaka T., Hidaka H. Modulation of tyrosine phosphorylation of p36 and other substrates by the S-100 protein. J Biol Chem. 1988 May 5;263(13):6438–6441. [PubMed] [Google Scholar]
  23. Hexham J. M., Totty N. F., Waterfield M. D., Crumpton M. J. Homology between the subunits of S100 and a 10kDa polypeptide associated with p36 of pig lymphocytes. Biochem Biophys Res Commun. 1986 Jan 14;134(1):248–254. doi: 10.1016/0006-291x(86)90554-1. [DOI] [PubMed] [Google Scholar]
  24. Hidaka H., Endo T., Kawamoto S., Yamada E., Umekawa H., Tanabe K., Hara K. Purification and characterization of adipose tissue S-100b protein. J Biol Chem. 1983 Feb 25;258(4):2705–2709. [PubMed] [Google Scholar]
  25. Isobe T., Ishioka N., Okuyama T. Structural relation of two S-100 proteins in bovine brain; subunit composition of S-100a protein. Eur J Biochem. 1981 Apr;115(3):469–474. doi: 10.1111/j.1432-1033.1981.tb06225.x. [DOI] [PubMed] [Google Scholar]
  26. Isobe T., Nakajima T., Okuyama T. Reinvestigation of extremely acidic proteins in bovine brain. Biochim Biophys Acta. 1977 Sep 27;494(1):222–232. doi: 10.1016/0005-2795(77)90150-7. [DOI] [PubMed] [Google Scholar]
  27. Isobe T., Okuyama T. The amino-acid sequence of the alpha subunit in bovine brain S-100a protein. Eur J Biochem. 1981 May;116(1):79–86. doi: 10.1111/j.1432-1033.1981.tb05303.x. [DOI] [PubMed] [Google Scholar]
  28. Jackson-Grusby L. L., Swiergiel J., Linzer D. I. A growth-related mRNA in cultured mouse cells encodes a placental calcium binding protein. Nucleic Acids Res. 1987 Aug 25;15(16):6677–6690. doi: 10.1093/nar/15.16.6677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Jensen R., Marshak D. R., Anderson C., Lukas T. J., Watterson D. M. Characterization of human brain S100 protein fraction: amino acid sequence of S100 beta. J Neurochem. 1985 Sep;45(3):700–705. doi: 10.1111/j.1471-4159.1985.tb04048.x. [DOI] [PubMed] [Google Scholar]
  30. Johnsson N., Marriott G., Weber K. p36, the major cytoplasmic substrate of src tyrosine protein kinase, binds to its p11 regulatory subunit via a short amino-terminal amphiphatic helix. EMBO J. 1988 Aug;7(8):2435–2442. doi: 10.1002/j.1460-2075.1988.tb03089.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Johnsson N., Vandekerckhove J., Van Damme J., Weber K. Binding sites for calcium, lipid and p11 on p36, the substrate of retroviral tyrosine-specific protein kinases. FEBS Lett. 1986 Mar 31;198(2):361–364. doi: 10.1016/0014-5793(86)80437-9. [DOI] [PubMed] [Google Scholar]
  32. Kato K., Kimura S. S100ao (alpha alpha) protein is mainly located in the heart and striated muscles. Biochim Biophys Acta. 1985 Oct 17;842(2-3):146–150. doi: 10.1016/0304-4165(85)90196-5. [DOI] [PubMed] [Google Scholar]
  33. Kligman D., Marshak D. R. Purification and characterization of a neurite extension factor from bovine brain. Proc Natl Acad Sci U S A. 1985 Oct;82(20):7136–7139. doi: 10.1073/pnas.82.20.7136. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Kristensen T., Saris C. J., Hunter T., Hicks L. J., Noonan D. J., Glenney J. R., Jr, Tack B. F. Primary structure of bovine calpactin I heavy chain (p36), a major cellular substrate for retroviral protein-tyrosine kinases: homology with the human phospholipase A2 inhibitor lipocortin. Biochemistry. 1986 Aug 12;25(16):4497–4503. doi: 10.1021/bi00364a007. [DOI] [PubMed] [Google Scholar]
  35. Leung I. K., Mani R. S., Kay C. M. Isolation, characterization and metal-ion-binding properties of the alpha-subunit from S-100a protein. Biochem J. 1986 Aug 1;237(3):757–764. doi: 10.1042/bj2370757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Mani R. S., Kay C. M. Isolation and spectral studies on the calcium binding properties of bovine brain S-100a protein. Biochemistry. 1983 Aug 2;22(16):3902–3907. doi: 10.1021/bi00285a027. [DOI] [PubMed] [Google Scholar]
  37. Masiakowski P., Shooter E. M. Nerve growth factor induces the genes for two proteins related to a family of calcium-binding proteins in PC12 cells. Proc Natl Acad Sci U S A. 1988 Feb;85(4):1277–1281. doi: 10.1073/pnas.85.4.1277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Matsudaira P. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem. 1987 Jul 25;262(21):10035–10038. [PubMed] [Google Scholar]
  39. Matus A., Mughal S. Immunohistochemical localisation of S-100 protein in brain. Nature. 1975 Dec 25;258(5537):746–748. doi: 10.1038/258746a0. [DOI] [PubMed] [Google Scholar]
  40. Molin S. O., Rosengren L., Baudier J., Hamberger A., Haglid K. S-100 alpha-like immunoreactivity in tubules of rat kidney. A clue to the function of a "brain-specific" protein. J Histochem Cytochem. 1985 Apr;33(4):367–374. doi: 10.1177/33.4.3884707. [DOI] [PubMed] [Google Scholar]
  41. Molin S. O., Rosengren L., Haglid K., Baudier J., Hamberger A. Differential localization of "brain-specific" S-100 and its subunits in rat salivary glands. J Histochem Cytochem. 1984 Aug;32(8):805–814. doi: 10.1177/32.8.6747272. [DOI] [PubMed] [Google Scholar]
  42. Moore B. W. A soluble protein characteristic of the nervous system. Biochem Biophys Res Commun. 1965 Jun 9;19(6):739–744. doi: 10.1016/0006-291x(65)90320-7. [DOI] [PubMed] [Google Scholar]
  43. Murphy L. C., Murphy L. J., Tsuyuki D., Duckworth M. L., Shiu R. P. Cloning and characterization of a cDNA encoding a highly conserved, putative calcium binding protein, identified by an anti-prolactin receptor antiserum. J Biol Chem. 1988 Feb 15;263(5):2397–2401. [PubMed] [Google Scholar]
  44. Odink K., Cerletti N., Brüggen J., Clerc R. G., Tarcsay L., Zwadlo G., Gerhards G., Schlegel R., Sorg C. Two calcium-binding proteins in infiltrate macrophages of rheumatoid arthritis. Nature. 1987 Nov 5;330(6143):80–82. doi: 10.1038/330080a0. [DOI] [PubMed] [Google Scholar]
  45. Osborn M., Johnsson N., Wehland J., Weber K. The submembranous location of p11 and its interaction with the p36 substrate of pp60 src kinase in situ. Exp Cell Res. 1988 Mar;175(1):81–96. doi: 10.1016/0014-4827(88)90257-1. [DOI] [PubMed] [Google Scholar]
  46. Radke K., Martin G. S. Transformation by Rous sarcoma virus: effects of src gene expression on the synthesis and phosphorylation of cellular polypeptides. Proc Natl Acad Sci U S A. 1979 Oct;76(10):5212–5216. doi: 10.1073/pnas.76.10.5212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Saris C. J., Kristensen T., D'Eustachio P., Hicks L. J., Noonan D. J., Hunter T., Tack B. F. cDNA sequence and tissue distribution of the mRNA for bovine and murine p11, the S100-related light chain of the protein-tyrosine kinase substrate p36 (calpactin I). J Biol Chem. 1987 Aug 5;262(22):10663–10671. [PubMed] [Google Scholar]
  48. Shadle P. J., Weber K. Calcium binding protein from porcine intestine binds to phosphatidylserine vesicles in the presence of calcium. Biochim Biophys Acta. 1987 Mar 12;897(3):502–506. doi: 10.1016/0005-2736(87)90448-2. [DOI] [PubMed] [Google Scholar]
  49. Tanaka T., Umekawa H., Ohmura T., Hidaka H. Calcium-dependent hydrophobic chromatography of calmodulin, S-100 protein and troponin-C. Biochim Biophys Acta. 1984 Jun 14;787(2):158–164. doi: 10.1016/0167-4838(84)90075-x. [DOI] [PubMed] [Google Scholar]
  50. Zimmer D. B., Van Eldik L. J. Analysis of the calcium-modulated proteins, S100 and calmodulin, and their target proteins during C6 glioma cell differentiation. J Cell Biol. 1989 Jan;108(1):141–151. doi: 10.1083/jcb.108.1.141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Zimmer D. B., Van Eldik L. J. Identification of a molecular target for the calcium-modulated protein S100. Fructose-1,6-bisphosphate aldolase. J Biol Chem. 1986 Aug 25;261(24):11424–11428. [PubMed] [Google Scholar]
  52. Zimmer D. B., Van Eldik L. J. Tissue distribution of rat S100 alpha and S100 beta and S100-binding proteins. Am J Physiol. 1987 Mar;252(3 Pt 1):C285–C289. doi: 10.1152/ajpcell.1987.252.3.C285. [DOI] [PubMed] [Google Scholar]
  53. Zokas L., Glenney J. R., Jr The calpactin light chain is tightly linked to the cytoskeletal form of calpactin I: studies using monoclonal antibodies to calpactin subunits. J Cell Biol. 1987 Nov;105(5):2111–2121. doi: 10.1083/jcb.105.5.2111. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES