Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1988 Jan;7(1):21–27. doi: 10.1002/j.1460-2075.1988.tb02779.x

Primary structure of the human, membrane-associated Ca2+-binding protein p68 a novel member of a protein family.

M R Crompton 1, R J Owens 1, N F Totty 1, S E Moss 1, M D Waterfield 1, M J Crumpton 1
PMCID: PMC454210  PMID: 3258820

Abstract

cDNA clones encoding human 'p68', a membrane-associated Ca2+-binding protein, were isolated from a lambda gt11 expression library of the human T-leukaemia cell line J6, by using a rabbit antiserum against denatured purified lymphocyte p68, and from a liver cDNA library by using 32P-labelled p68 cDNA fragments. The amino acid sequence of p68, deduced from the sequences of overlapping cDNA clones, is described. The results show that p68 is closely related to a family of proteins which includes intracellular substrates of the EGF receptor and pp60src tyrosine kinases. The p68 amino acid sequence is internally repetitive, being constructed from eight repeats of varying lengths, each of which contains a highly conserved sequence. Multiple copies of the latter sequence are also present in the related proteins p36, lipocortin I and protein II. We discuss how the common structural features of these proteins may reflect common functions and, furthermore, how the eight repeat structure of p68 may have evolved. The sequences of independent cDNAs suggest that alternatively-spliced mRNAs could encode different p68 protein species. This suggestion is consistent with the observation that purified p68 migrates as a closely-spaced doublet when analysed by SDS-PAGE.

Full text

PDF
21

Images in this article

22Image
on p.22
24Image
on p.24

Selected References

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

  1. Apgar J. R., Herrmann S. H., Robinson J. M., Mescher M. F. Triton X-100 extraction of P815 tumor cells: evidence for a plasma membrane skeleton structure. J Cell Biol. 1985 May;100(5):1369–1378. doi: 10.1083/jcb.100.5.1369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Apgar J. R., Mescher M. F. Agorins: major structural proteins of the plasma membrane skeleton of P815 tumor cells. J Cell Biol. 1986 Aug;103(2):351–360. doi: 10.1083/jcb.103.2.351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bentley D. R. Primary structure of human complement component C2. Homology to two unrelated protein families. Biochem J. 1986 Oct 15;239(2):339–345. doi: 10.1042/bj2390339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Birnstiel M. L., Busslinger M., Strub K. Transcription termination and 3' processing: the end is in site! Cell. 1985 Jun;41(2):349–359. doi: 10.1016/s0092-8674(85)80007-6. [DOI] [PubMed] [Google Scholar]
  5. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  6. Church G. M., Gilbert W. Genomic sequencing. Proc Natl Acad Sci U S A. 1984 Apr;81(7):1991–1995. doi: 10.1073/pnas.81.7.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cirino G., Flower R. J., Browning J. L., Sinclair L. K., Pepinsky R. B. Recombinant human lipocortin 1 inhibits thromboxane release from guinea-pig isolated perfused lung. Nature. 1987 Jul 16;328(6127):270–272. doi: 10.1038/328270a0. [DOI] [PubMed] [Google Scholar]
  8. Creutz C. E., Dowling L. G., Sando J. J., Villar-Palasi C., Whipple J. H., Zaks W. J. Characterization of the chromobindins. Soluble proteins that bind to the chromaffin granule membrane in the presence of Ca2+. J Biol Chem. 1983 Dec 10;258(23):14664–14674. [PubMed] [Google Scholar]
  9. Creutz C. E., Zaks W. J., Hamman H. C., Crane S., Martin W. H., Gould K. L., Oddie K. M., Parsons S. J. Identification of chromaffin granule-binding proteins. Relationship of the chromobindins to calelectrin, synhibin, and the tyrosine kinase substrates p35 and p36. J Biol Chem. 1987 Feb 5;262(4):1860–1868. [PubMed] [Google Scholar]
  10. Davidson F. F., Dennis E. A., Powell M., Glenney J. R., Jr Inhibition of phospholipase A2 by "lipocortins" and calpactins. An effect of binding to substrate phospholipids. J Biol Chem. 1987 Feb 5;262(4):1698–1705. [PubMed] [Google Scholar]
  11. Davies A. A., Crumpton M. J. Identification of calcium-binding proteins associated with the lymphocyte plasma membrane. Biochem Biophys Res Commun. 1985 Apr 30;128(2):571–577. doi: 10.1016/0006-291x(85)90084-1. [DOI] [PubMed] [Google Scholar]
  12. Davies A. A., Wigglesworth N. M., Allan D., Owens R. J., Crumpton M. J. Nonidet P-40 extraction of lymphocyte plasma membrane. Characterization of the insoluble residue. Biochem J. 1984 Apr 1;219(1):301–308. doi: 10.1042/bj2190301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Geisow M., Childs J., Dash B., Harris A., Panayotou G., Südhof T., Walker J. H. Cellular distribution of three mammalian Ca2+-binding proteins related to Torpedo calelectrin. EMBO J. 1984 Dec 1;3(12):2969–2974. doi: 10.1002/j.1460-2075.1984.tb02242.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. 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]
  20. Glenney J. R., Jr, Tack B., Powell M. A. Calpactins: two distinct Ca++-regulated phospholipid- and actin-binding proteins isolated from lung and placenta. J Cell Biol. 1987 Mar;104(3):503–511. doi: 10.1083/jcb.104.3.503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Gould K. L., Woodgett J. R., Isacke C. M., Hunter T. The protein-tyrosine kinase substrate p36 is also a substrate for protein kinase C in vitro and in vivo. Mol Cell Biol. 1986 Jul;6(7):2738–2744. doi: 10.1128/mcb.6.7.2738. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hewick R. M., Hunkapiller M. W., Hood L. E., Dreyer W. J. A gas-liquid solid phase peptide and protein sequenator. J Biol Chem. 1981 Aug 10;256(15):7990–7997. [PubMed] [Google Scholar]
  24. Higgins R. C., Dahmus M. E. Rapid visualization of protein bands in preparative SDS-polyacrylamide gels. Anal Biochem. 1979 Mar;93(2):257–260. doi: 10.1016/s0003-2697(79)80148-7. [DOI] [PubMed] [Google Scholar]
  25. Hirata F., Matsuda K., Notsu Y., Hattori T., del Carmine R. Phosphorylation at a tyrosine residue of lipomodulin in mitogen-stimulated murine thymocytes. Proc Natl Acad Sci U S A. 1984 Aug;81(15):4717–4721. doi: 10.1073/pnas.81.15.4717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Huang K. S., Wallner B. P., Mattaliano R. J., Tizard R., Burne C., Frey A., Hession C., McGray P., Sinclair L. K., Chow E. P. Two human 35 kd inhibitors of phospholipase A2 are related to substrates of pp60v-src and of the epidermal growth factor receptor/kinase. Cell. 1986 Jul 18;46(2):191–199. doi: 10.1016/0092-8674(86)90736-1. [DOI] [PubMed] [Google Scholar]
  27. Kozak M. Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell. 1986 Jan 31;44(2):283–292. doi: 10.1016/0092-8674(86)90762-2. [DOI] [PubMed] [Google Scholar]
  28. Kretsinger R. H. The informational role of calcium in the cytosol. Adv Cyclic Nucleotide Res. 1979;11:1–26. [PubMed] [Google Scholar]
  29. Krissansen G. W., Owen M. J., Verbi W., Crumpton M. J. Primary structure of the T3 gamma subunit of the T3/T cell antigen receptor complex deduced from cDNA sequences: evolution of the T3 gamma and delta subunits. EMBO J. 1986 Aug;5(8):1799–1808. doi: 10.1002/j.1460-2075.1986.tb04429.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Mescher M. F., Jose M. J., Balk S. P. Actin-containing matrix associated with the plasma membrane of murine tumour and lymphoid cells. Nature. 1981 Jan 15;289(5794):139–144. doi: 10.1038/289139a0. [DOI] [PubMed] [Google Scholar]
  32. Moore P. B., Kraus-Friedmann N., Dedman J. R. Unique calcium-dependent hydrophobic binding proteins: possible independent mediators of intracellular calcium distinct from calmodulin. J Cell Sci. 1984 Dec;72:121–133. doi: 10.1242/jcs.72.1.121. [DOI] [PubMed] [Google Scholar]
  33. Owens R. J., Crumpton M. J. Isolation and characterization of a novel 68,000-Mr Ca2+-binding protein of lymphocyte plasma membrane. Biochem J. 1984 Apr 1;219(1):309–316. doi: 10.1042/bj2190309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Owens R. J., Gallagher C. J., Crumpton M. J. Cellular distribution of p68, a new calcium-binding protein from lymphocytes. EMBO J. 1984 May;3(5):945–952. doi: 10.1002/j.1460-2075.1984.tb01912.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Pepinsky R. B., Sinclair L. K., Browning J. L., Mattaliano R. J., Smart J. E., Chow E. P., Falbel T., Ribolini A., Garwin J. L., Wallner B. P. Purification and partial sequence analysis of a 37-kDa protein that inhibits phospholipase A2 activity from rat peritoneal exudates. J Biol Chem. 1986 Mar 25;261(9):4239–4246. [PubMed] [Google Scholar]
  36. Pepinsky R. B., Sinclair L. K. Epidermal growth factor-dependent phosphorylation of lipocortin. Nature. 1986 May 1;321(6065):81–84. doi: 10.1038/321081a0. [DOI] [PubMed] [Google Scholar]
  37. Radke K., Gilmore T., Martin G. S. Transformation by Rous sarcoma virus: a cellular substrate for transformation-specific protein phosphorylation contains phosphotyrosine. Cell. 1980 Oct;21(3):821–828. doi: 10.1016/0092-8674(80)90445-6. [DOI] [PubMed] [Google Scholar]
  38. Raeymaekers L., Wuytack F., Casteels R. Isolation of calelectrin-like proteins associated with smooth muscle plasma membranes. Biochem Biophys Res Commun. 1985 Oct 30;132(2):526–532. doi: 10.1016/0006-291x(85)91165-9. [DOI] [PubMed] [Google Scholar]
  39. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Saris C. J., Tack B. F., Kristensen T., Glenney J. R., Jr, Hunter T. The cDNA sequence for the protein-tyrosine kinase substrate p36 (calpactin I heavy chain) reveals a multidomain protein with internal repeats. Cell. 1986 Jul 18;46(2):201–212. doi: 10.1016/0092-8674(86)90737-3. [DOI] [PubMed] [Google Scholar]
  41. Schlaepfer D. D., Haigler H. T. Characterization of Ca2+-dependent phospholipid binding and phosphorylation of lipocortin I. J Biol Chem. 1987 May 15;262(14):6931–6937. [PubMed] [Google Scholar]
  42. Shadle P. J., Gerke V., Weber K. Three Ca2+-binding proteins from porcine liver and intestine differ immunologically and physicochemically and are distinct in Ca2+ affinities. J Biol Chem. 1985 Dec 25;260(30):16354–16360. [PubMed] [Google Scholar]
  43. 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]
  44. Smith V. L., Dedman J. R. An immunological comparison of several novel calcium-binding proteins. J Biol Chem. 1986 Dec 5;261(34):15815–15818. [PubMed] [Google Scholar]
  45. Südhof T. C., Ebbecke M., Walker J. H., Fritsche U., Boustead C. Isolation of mammalian calelectrins: a new class of ubiquitous Ca2+-regulated proteins. Biochemistry. 1984 Mar 13;23(6):1103–1109. doi: 10.1021/bi00301a010. [DOI] [PubMed] [Google Scholar]
  46. Tufty R. M., Kretsinger R. H. Troponin and parvalbumin calcium binding regions predicted in myosin light chain and T4 lysozyme. Science. 1975 Jan 17;187(4172):167–169. doi: 10.1126/science.1111094. [DOI] [PubMed] [Google Scholar]
  47. Wallner B. P., Mattaliano R. J., Hession C., Cate R. L., Tizard R., Sinclair L. K., Foeller C., Chow E. P., Browing J. L., Ramachandran K. L. Cloning and expression of human lipocortin, a phospholipase A2 inhibitor with potential anti-inflammatory activity. Nature. 1986 Mar 6;320(6057):77–81. doi: 10.1038/320077a0. [DOI] [PubMed] [Google Scholar]
  48. Weber K., Johnsson N., Plessmann U., Van P. N., Söling H. D., Ampe C., Vandekerckhove J. The amino acid sequence of protein II and its phosphorylation site for protein kinase C; the domain structure Ca2+-modulated lipid binding proteins. EMBO J. 1987 Jun;6(6):1599–1604. doi: 10.1002/j.1460-2075.1987.tb02406.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Weeks A., Briggs L. GLUCOSE AND ALKALIES IN SURGERY. Cal State J Med. 1922 Mar;20(3):100–102. [PMC free article] [PubMed] [Google Scholar]
  50. Wilbur W. J., Lipman D. J. Rapid similarity searches of nucleic acid and protein data banks. Proc Natl Acad Sci U S A. 1983 Feb;80(3):726–730. doi: 10.1073/pnas.80.3.726. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES