Skip to main content
NCBI home page
The EMBO Journal logoLink to The EMBO Journal
. 1998 Oct 15;17(20):5913–5922. doi: 10.1093/emboj/17.20.5913

Calcium regulation of Ndr protein kinase mediated by S100 calcium-binding proteins.

T A Millward 1, C W Heizmann 1, B W Schäfer 1, B A Hemmings 1
PMCID: PMC1170919  PMID: 9774336

Abstract

Ndr is a nuclear serine/threonine protein kinase that belongs to a subfamily of kinases identified as being critical for the regulation of cell division and cell morphology. The regulatory mechanisms that control Ndr activity have not been characterized previously. In this paper, we present evidence that Ndr is regulated by EF-hand calcium-binding proteins of the S100 family, in response to changes in the intracellular calcium concentration. In vitro, S100B binds directly to and activates Ndr in a Ca2+-dependent manner. Moreover, Ndr is recovered from cell lysates in anti-S100B immunoprecipitates. The region of Ndr responsible for interaction with Ca2+/S100B is a basic/hydrophobic motif within the N-terminal regulatory domain of Ndr, and activation of Ndr by Ca2+/S100B is inhibited by a synthetic peptide derived from this region. In cultured cells, Ndr is rapidly activated following treatment with Ca2+ ionophore, and this activation is dependent upon the identified Ca2+/S100B-binding domain. Finally, Ndr activity is inhibited by W-7 in melanoma cells overexpressing S100B, but is unaffected by W-7 in melanoma cells that lack S100B. These results suggest that Ndr is regulated at least in part by changes in the intracellular calcium concentration, through binding of S100 proteins to its N-terminal regulatory domain.

Full Text

The Full Text of this article is available as a PDF (368.3 KB).

Selected References

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

  1. Allore R. J., Friend W. C., O'Hanlon D., Neilson K. M., Baumal R., Dunn R. J., Marks A. Cloning and expression of the human S100 beta gene. J Biol Chem. 1990 Sep 15;265(26):15537–15543. [PubMed] [Google Scholar]
  2. Baudier J., Bergeret E., Bertacchi N., Weintraub H., Gagnon J., Garin J. Interactions of myogenic bHLH transcription factors with calcium-binding calmodulin and S100a (alpha alpha) proteins. Biochemistry. 1995 Jun 20;34(24):7834–7846. doi: 10.1021/bi00024a007. [DOI] [PubMed] [Google Scholar]
  3. Baudier J., Briving C., Deinum J., Haglid K., Sörskog L., Wallin M. Effect of S-100 proteins and calmodulin on Ca2+-induced disassembly of brain microtubule proteins in vitro. FEBS Lett. 1982 Oct 18;147(2):165–168. doi: 10.1016/0014-5793(82)81033-8. [DOI] [PubMed] [Google Scholar]
  4. Baudier J., Delphin C., Grunwald D., Khochbin S., Lawrence J. J. Characterization of the tumor suppressor protein p53 as a protein kinase C substrate and a S100b-binding protein. Proc Natl Acad Sci U S A. 1992 Dec 1;89(23):11627–11631. doi: 10.1073/pnas.89.23.11627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Baudier J., Glasser N., Gerard D. Ions binding to S100 proteins. I. Calcium- and zinc-binding properties of bovine brain S100 alpha alpha, S100a (alpha beta), and S100b (beta beta) protein: Zn2+ regulates Ca2+ binding on S100b protein. J Biol Chem. 1986 Jun 25;261(18):8192–8203. [PubMed] [Google Scholar]
  6. Baudier J., Mochly-Rosen D., Newton A., Lee S. H., Koshland D. E., Jr, Cole R. D. Comparison of S100b protein with calmodulin: interactions with melittin and microtubule-associated tau proteins and inhibition of phosphorylation of tau proteins by protein kinase C. Biochemistry. 1987 May 19;26(10):2886–2893. doi: 10.1021/bi00384a033. [DOI] [PubMed] [Google Scholar]
  7. Brook J. D., McCurrach M. E., Harley H. G., Buckler A. J., Church D., Aburatani H., Hunter K., Stanton V. P., Thirion J. P., Hudson T. Molecular basis of myotonic dystrophy: expansion of a trinucleotide (CTG) repeat at the 3' end of a transcript encoding a protein kinase family member. Cell. 1992 Feb 21;68(4):799–808. doi: 10.1016/0092-8674(92)90154-5. [DOI] [PubMed] [Google Scholar]
  8. Brostrom C. O., Brostrom M. A. Calcium-dependent regulation of protein synthesis in intact mammalian cells. Annu Rev Physiol. 1990;52:577–590. doi: 10.1146/annurev.ph.52.030190.003045. [DOI] [PubMed] [Google Scholar]
  9. Böni R., Burg G., Doguoglu A., Ilg E. C., Schäfer B. W., Müller B., Heizmann C. W. Immunohistochemical localization of the Ca2+ binding S100 proteins in normal human skin and melanocytic lesions. Br J Dermatol. 1997 Jul;137(1):39–43. [PubMed] [Google Scholar]
  10. Cocchia D., Michetti F., Donato R. Immunochemical and immuno-cytochemical localization of S-100 antigen in normal human skin. Nature. 1981 Nov 5;294(5836):85–87. doi: 10.1038/294085a0. [DOI] [PubMed] [Google Scholar]
  11. Cochran A. J., Lu H. F., Li P. X., Saxton R., Wen D. R. S-100 protein remains a practical marker for melanocytic and other tumours. Melanoma Res. 1993 Oct;3(5):325–330. doi: 10.1097/00008390-199310000-00004. [DOI] [PubMed] [Google Scholar]
  12. Dignam J. D., Lebovitz R. M., Roeder R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983 Mar 11;11(5):1475–1489. doi: 10.1093/nar/11.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Donato R., Giambanco I., Aisa M. C. Molecular interaction of S-100 proteins with microtubule proteins in vitro. J Neurochem. 1989 Aug;53(2):566–571. doi: 10.1111/j.1471-4159.1989.tb07371.x. [DOI] [PubMed] [Google Scholar]
  14. Fan K. S-100 protein synthesis in cultured glioma cell is G1-phase of cell cycle dependent. Brain Res. 1982 Apr 15;237(2):498–503. doi: 10.1016/0006-8993(82)90462-0. [DOI] [PubMed] [Google Scholar]
  15. Fu Y. H., Friedman D. L., Richards S., Pearlman J. A., Gibbs R. A., Pizzuti A., Ashizawa T., Perryman M. B., Scarlato G., Fenwick R. G., Jr Decreased expression of myotonin-protein kinase messenger RNA and protein in adult form of myotonic dystrophy. Science. 1993 Apr 9;260(5105):235–238. doi: 10.1126/science.8469976. [DOI] [PubMed] [Google Scholar]
  16. Fujii T., Machino K., Andoh H., Satoh T., Kondo Y. Calcium-dependent control of caldesmon-actin interaction by S100 protein. J Biochem. 1990 Jan;107(1):133–137. doi: 10.1093/oxfordjournals.jbchem.a122996. [DOI] [PubMed] [Google Scholar]
  17. Goldberg J., Nairn A. C., Kuriyan J. Structural basis for the autoinhibition of calcium/calmodulin-dependent protein kinase I. Cell. 1996 Mar 22;84(6):875–887. doi: 10.1016/s0092-8674(00)81066-1. [DOI] [PubMed] [Google Scholar]
  18. Grigorian M., Ambartsumian N., Lykkesfeldt A. E., Bastholm L., Elling F., Georgiev G., Lukanidin E. Effect of mts1 (S100A4) expression on the progression of human breast cancer cells. Int J Cancer. 1996 Sep 17;67(6):831–841. doi: 10.1002/(SICI)1097-0215(19960917)67:6<831::AID-IJC13>3.0.CO;2-4. [DOI] [PubMed] [Google Scholar]
  19. Heierhorst J., Mann R. J., Kemp B. E. Interaction of the recombinant S100A1 protein with twitchin kinase, and comparison with other Ca2+-binding proteins. Eur J Biochem. 1997 Oct 1;249(1):127–133. doi: 10.1111/j.1432-1033.1997.00127.x. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Hidaka H., Sasaki Y., Tanaka T., Endo T., Ohno S., Fujii Y., Nagata T. N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, a calmodulin antagonist, inhibits cell proliferation. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4354–4357. doi: 10.1073/pnas.78.7.4354. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hidaka H., Tanaka T. Naphthalenesulfonamides as calmodulin antagonists. Methods Enzymol. 1983;102:185–194. doi: 10.1016/s0076-6879(83)02019-4. [DOI] [PubMed] [Google Scholar]
  23. Ilg E. C., Schäfer B. W., Heizmann C. W. Expression pattern of S100 calcium-binding proteins in human tumors. Int J Cancer. 1996 Nov 4;68(3):325–332. doi: 10.1002/(SICI)1097-0215(19961104)68:3<325::AID-IJC10>3.0.CO;2-7. [DOI] [PubMed] [Google Scholar]
  24. Ivanenkov V. V., Jamieson G. A., Jr, Gruenstein E., Dimlich R. V. Characterization of S-100b binding epitopes. Identification of a novel target, the actin capping protein, CapZ. J Biol Chem. 1995 Jun 16;270(24):14651–14658. doi: 10.1074/jbc.270.24.14651. [DOI] [PubMed] [Google Scholar]
  25. James P., Vorherr T., Carafoli E. Calmodulin-binding domains: just two faced or multi-faceted? Trends Biochem Sci. 1995 Jan;20(1):38–42. doi: 10.1016/s0968-0004(00)88949-5. [DOI] [PubMed] [Google Scholar]
  26. Johnston L. H., Eberly S. L., Chapman J. W., Araki H., Sugino A. The product of the Saccharomyces cerevisiae cell cycle gene DBF2 has homology with protein kinases and is periodically expressed in the cell cycle. Mol Cell Biol. 1990 Apr;10(4):1358–1366. doi: 10.1128/mcb.10.4.1358. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Justice R. W., Zilian O., Woods D. F., Noll M., Bryant P. J. The Drosophila tumor suppressor gene warts encodes a homolog of human myotonic dystrophy kinase and is required for the control of cell shape and proliferation. Genes Dev. 1995 Mar 1;9(5):534–546. doi: 10.1101/gad.9.5.534. [DOI] [PubMed] [Google Scholar]
  28. Kao J. P., Alderton J. M., Tsien R. Y., Steinhardt R. A. Active involvement of Ca2+ in mitotic progression of Swiss 3T3 fibroblasts. J Cell Biol. 1990 Jul;111(1):183–196. doi: 10.1083/jcb.111.1.183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kato K., Kimura S., Haimoto H., Suzuki F. S100a0 (alpha alpha) protein: distribution in muscle tissues of various animals and purification from human pectoral muscle. J Neurochem. 1986 May;46(5):1555–1560. doi: 10.1111/j.1471-4159.1986.tb01776.x. [DOI] [PubMed] [Google Scholar]
  30. Kobe B., Heierhorst J., Feil S. C., Parker M. W., Benian G. M., Weiss K. R., Kemp B. E. Giant protein kinases: domain interactions and structural basis of autoregulation. EMBO J. 1996 Dec 16;15(24):6810–6821. [PMC free article] [PubMed] [Google Scholar]
  31. Koff A., Giordano A., Desai D., Yamashita K., Harper J. W., Elledge S., Nishimoto T., Morgan D. O., Franza B. R., Roberts J. M. Formation and activation of a cyclin E-cdk2 complex during the G1 phase of the human cell cycle. Science. 1992 Sep 18;257(5077):1689–1694. doi: 10.1126/science.1388288. [DOI] [PubMed] [Google Scholar]
  32. Lam M., Dubyak G., Chen L., Nuñez G., Miesfeld R. L., Distelhorst C. W. Evidence that BCL-2 represses apoptosis by regulating endoplasmic reticulum-associated Ca2+ fluxes. Proc Natl Acad Sci U S A. 1994 Jul 5;91(14):6569–6573. doi: 10.1073/pnas.91.14.6569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Leung T., Chen X. Q., Manser E., Lim L. The p160 RhoA-binding kinase ROK alpha is a member of a kinase family and is involved in the reorganization of the cytoskeleton. Mol Cell Biol. 1996 Oct;16(10):5313–5327. doi: 10.1128/mcb.16.10.5313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Lloyd B. H., Platt-Higgins A., Rudland P. S., Barraclough R. Human S100A4 (p9Ka) induces the metastatic phenotype upon benign tumour cells. Oncogene. 1998 Jul 30;17(4):465–473. doi: 10.1038/sj.onc.1201948. [DOI] [PubMed] [Google Scholar]
  35. Marks A., Petsche D., O'Hanlon D., Kwong P. C., Stead R., Dunn R., Baumal R., Liao S. K. S100 protein expression in human melanoma cells: comparison of levels of expression among different cell lines and individual cells in different phases of the cell cycle. Exp Cell Res. 1990 Mar;187(1):59–64. doi: 10.1016/0014-4827(90)90116-r. [DOI] [PubMed] [Google Scholar]
  36. Marshak D. R., Watterson D. M., Van Eldik L. J. Calcium-dependent interaction of S100b, troponin C, and calmodulin with an immobilized phenothiazine. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6793–6797. doi: 10.1073/pnas.78.11.6793. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Millward T., Cron P., Hemmings B. A. Molecular cloning and characterization of a conserved nuclear serine(threonine) protein kinase. Proc Natl Acad Sci U S A. 1995 May 23;92(11):5022–5026. doi: 10.1073/pnas.92.11.5022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. O'Neil K. T., DeGrado W. F. How calmodulin binds its targets: sequence independent recognition of amphiphilic alpha-helices. Trends Biochem Sci. 1990 Feb;15(2):59–64. doi: 10.1016/0968-0004(90)90177-d. [DOI] [PubMed] [Google Scholar]
  39. Okajima T., Tanabe T., Yasuda T. Nonurea sodium dodecyl sulfate-polyacrylamide gel electrophoresis with high-molarity buffers for the separation of proteins and peptides. Anal Biochem. 1993 Jun;211(2):293–300. doi: 10.1006/abio.1993.1272. [DOI] [PubMed] [Google Scholar]
  40. Onions J., Hermann S., Grundström T. Basic helix-loop-helix protein sequences determining differential inhibition by calmodulin and S-100 proteins. J Biol Chem. 1997 Sep 19;272(38):23930–23937. doi: 10.1074/jbc.272.38.23930. [DOI] [PubMed] [Google Scholar]
  41. Pedrocchi M., Schäfer B. W., Durussel I., Cox J. A., Heizmann C. W. Purification and characterization of the recombinant human calcium-binding S100 proteins CAPL and CACY. Biochemistry. 1994 May 31;33(21):6732–6738. doi: 10.1021/bi00187a045. [DOI] [PubMed] [Google Scholar]
  42. Schäfer B. W., Heizmann C. W. The S100 family of EF-hand calcium-binding proteins: functions and pathology. Trends Biochem Sci. 1996 Apr;21(4):134–140. doi: 10.1016/s0968-0004(96)80167-8. [DOI] [PubMed] [Google Scholar]
  43. Schäfer B. W., Wicki R., Engelkamp D., Mattei M. G., Heizmann C. W. Isolation of a YAC clone covering a cluster of nine S100 genes on human chromosome 1q21: rationale for a new nomenclature of the S100 calcium-binding protein family. Genomics. 1995 Feb 10;25(3):638–643. doi: 10.1016/0888-7543(95)80005-7. [DOI] [PubMed] [Google Scholar]
  44. Scotto C., Deloulme J. C., Rousseau D., Chambaz E., Baudier J. Calcium and S100B regulation of p53-dependent cell growth arrest and apoptosis. Mol Cell Biol. 1998 Jul;18(7):4272–4281. doi: 10.1128/mcb.18.7.4272. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Seed B., Aruffo A. Molecular cloning of the CD2 antigen, the T-cell erythrocyte receptor, by a rapid immunoselection procedure. Proc Natl Acad Sci U S A. 1987 May;84(10):3365–3369. doi: 10.1073/pnas.84.10.3365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Selinfreund R. H., Barger S. W., Welsh M. J., Van Eldik L. J. Antisense inhibition of glial S100 beta production results in alterations in cell morphology, cytoskeletal organization, and cell proliferation. J Cell Biol. 1990 Nov;111(5 Pt 1):2021–2028. doi: 10.1083/jcb.111.5.2021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Smith S. P., Barber K. R., Dunn S. D., Shaw G. S. Structural influence of cation binding to recombinant human brain S100b: evidence for calcium-induced exposure of a hydrophobic surface. Biochemistry. 1996 Jul 9;35(27):8805–8814. doi: 10.1021/bi952698c. [DOI] [PubMed] [Google Scholar]
  48. Taylor D. A., Bowman B. F., Stull J. T. Cytoplasmic Ca2+ is a primary determinant for myosin phosphorylation in smooth muscle cells. J Biol Chem. 1989 Apr 15;264(11):6207–6213. [PubMed] [Google Scholar]
  49. Toyn J. H., Johnston L. H. The Dbf2 and Dbf20 protein kinases of budding yeast are activated after the metaphase to anaphase cell cycle transition. EMBO J. 1994 Mar 1;13(5):1103–1113. doi: 10.1002/j.1460-2075.1994.tb06359.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Umekawa H., Endo T., Hidaka H. A rapid separation of bovine brain S-100a and S-100b proteins and related conformation studies. Arch Biochem Biophys. 1983 Nov;227(1):147–153. doi: 10.1016/0003-9861(83)90357-0. [DOI] [PubMed] [Google Scholar]
  51. Verde F., Wiley D. J., Nurse P. Fission yeast orb6, a ser/thr protein kinase related to mammalian rho kinase and myotonic dystrophy kinase, is required for maintenance of cell polarity and coordinates cell morphogenesis with the cell cycle. Proc Natl Acad Sci U S A. 1998 Jun 23;95(13):7526–7531. doi: 10.1073/pnas.95.13.7526. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Werlen G., Belin D., Conne B., Roche E., Lew D. P., Prentki M. Intracellular Ca2+ and the regulation of early response gene expression in HL-60 myeloid leukemia cells. J Biol Chem. 1993 Aug 5;268(22):16596–16601. [PubMed] [Google Scholar]
  53. Wissmann A., Ingles J., McGhee J. D., Mains P. E. Caenorhabditis elegans LET-502 is related to Rho-binding kinases and human myotonic dystrophy kinase and interacts genetically with a homolog of the regulatory subunit of smooth muscle myosin phosphatase to affect cell shape. Genes Dev. 1997 Feb 15;11(4):409–422. doi: 10.1101/gad.11.4.409. [DOI] [PubMed] [Google Scholar]
  54. Xu T., Wang W., Zhang S., Stewart R. A., Yu W. Identifying tumor suppressors in genetic mosaics: the Drosophila lats gene encodes a putative protein kinase. Development. 1995 Apr;121(4):1053–1063. doi: 10.1242/dev.121.4.1053. [DOI] [PubMed] [Google Scholar]
  55. Yarden O., Plamann M., Ebbole D. J., Yanofsky C. cot-1, a gene required for hyphal elongation in Neurospora crassa, encodes a protein kinase. EMBO J. 1992 Jun;11(6):2159–2166. doi: 10.1002/j.1460-2075.1992.tb05275.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Zhang M., Tanaka T., Ikura M. Calcium-induced conformational transition revealed by the solution structure of apo calmodulin. Nat Struct Biol. 1995 Sep;2(9):758–767. doi: 10.1038/nsb0995-758. [DOI] [PubMed] [Google Scholar]
  57. Zimmer D. B., Cornwall E. H., Reynolds P. D., Donald C. M. S100A1 regulates neurite organization, tubulin levels, and proliferation in PC12 cells. J Biol Chem. 1998 Feb 20;273(8):4705–4711. doi: 10.1074/jbc.273.8.4705. [DOI] [PubMed] [Google Scholar]

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

RESOURCES