Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1991 Nov 15;280(Pt 1):233–241. doi: 10.1042/bj2800233

The cysteine-rich domain of human proteins, neuronal chimaerin, protein kinase C and diacylglycerol kinase binds zinc. Evidence for the involvement of a zinc-dependent structure in phorbol ester binding.

S Ahmed 1, R Kozma 1, J Lee 1, C Monfries 1, N Harden 1, L Lim 1
PMCID: PMC1130625  PMID: 1660266

Abstract

Diacylglycerol (DG) and its analogue phorbol 12-myristate 13-acetate (PMA) activate the ubiquitous phospholipid/Ca2(+)-dependent protein kinase, protein kinase C (PKC), and cause it to become tightly associated with membranes. DG is produced transiently as it is rapidly metabolized by DG kinase (DGK) to phosphatidic acid. Phorbol esters such as PMA are not metabolized and induced a prolonged membrane association of PKC. Until recently, PKC was the only known phorbol ester receptor. We have shown that a novel brain-specific cDNA, neuronal chimaerin (NC), expressed in Escherichia coli, binds phorbol ester with high affinity, stereospecificity and a phospholipid requirement [Ahmed, Kozma, Monfries, Hall, Lim, Smith & Lim (1990) Biochem. J. 272, 767-773]. The proteins NC, PKC and DGK possess a cysteine-rich domain with the motif HX11/12CX2CXnCX2CX4HX2CX6/7C (where n varies between 12 and 14). The partial motif, CX2CX13CX2C, is present in a number of transcription factors including the steroid hormone receptors and the yeast protein, GAL4, in which zinc plays a structural role of co-ordinating cysteine residues and is essential for DNA binding (protein-nucleic acid interactions). The cysteine-rich domain of NC and PKC is required for phospholipid-dependent phorbol is required for phospholipid-dependent phorbol ester binding, suggesting an involvement of this domain in protein-lipid interactions. We have expressed recombinant NC, PKC and DGK glutathione S-transferase and TrpE fusion proteins in E. coli to investigate the relationship between the cysteine-rich motif, HX11/12CX2CX10-14CX2CX4HX2CX6/7C, zinc and phorbol ester binding. The cysteine-rich domain of NC, PKC and DGK bound 65Zn2+ but only NC and PKC bound [3H]phorbol 12,13-dibutyrate. When NC and PKC were subjected to treatments known to remove metal ions from GAL4 and the human glucocorticoid receptor, phorbol ester binding was inhibited. These data provide evidence for the role of a zinc-dependent structure in phorbol ester binding.

Full text

PDF
233

Images in this article

235Fig. 1.
on p.235
237Fig. 2.
on p.237
238Fig. 3.
on p.238
238Fig. 4.
on p.238

Selected References

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

  1. Ahmed S., Kozma R., Monfries C., Hall C., Lim H. H., Smith P., Lim L. Human brain n-chimaerin cDNA encodes a novel phorbol ester receptor. Biochem J. 1990 Dec 15;272(3):767–773. doi: 10.1042/bj2720767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Akita Y., Ohno S., Konno Y., Yano A., Suzuki K. Expression and properties of two distinct classes of the phorbol ester receptor family, four conventional protein kinase C types, and a novel protein kinase C. J Biol Chem. 1990 Jan 5;265(1):354–362. [PubMed] [Google Scholar]
  3. Bacher N., Zisman Y., Berent E., Livneh E. Isolation and characterization of PKC-L, a new member of the protein kinase C-related gene family specifically expressed in lung, skin, and heart. Mol Cell Biol. 1991 Jan;11(1):126–133. doi: 10.1128/mcb.11.1.126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Barbosa M. S., Lowy D. R., Schiller J. T. Papillomavirus polypeptides E6 and E7 are zinc-binding proteins. J Virol. 1989 Mar;63(3):1404–1407. doi: 10.1128/jvi.63.3.1404-1407.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Beck T. W., Huleihel M., Gunnell M., Bonner T. I., Rapp U. R. The complete coding sequence of the human A-raf-1 oncogene and transforming activity of a human A-raf carrying retrovirus. Nucleic Acids Res. 1987 Jan 26;15(2):595–609. doi: 10.1093/nar/15.2.595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Berg J. M. Potential metal-binding domains in nucleic acid binding proteins. Science. 1986 Apr 25;232(4749):485–487. doi: 10.1126/science.2421409. [DOI] [PubMed] [Google Scholar]
  7. Berg J. M. Zinc fingers and other metal-binding domains. Elements for interactions between macromolecules. J Biol Chem. 1990 Apr 25;265(12):6513–6516. [PubMed] [Google Scholar]
  8. Brown R. S., Sander C., Argos P. The primary structure of transcription factor TFIIIA has 12 consecutive repeats. FEBS Lett. 1985 Jul 8;186(2):271–274. doi: 10.1016/0014-5793(85)80723-7. [DOI] [PubMed] [Google Scholar]
  9. Cazaubon S., Webster C., Camoin L., Strosberg A. D., Parker P. Effector-dependent conformational changes in protein kinase C gamma through epitope mapping with inhibitory monoclonal antibodies. Eur J Biochem. 1990 Dec 27;194(3):799–804. doi: 10.1111/j.1432-1033.1990.tb19472.x. [DOI] [PubMed] [Google Scholar]
  10. Coussens L., Parker P. J., Rhee L., Yang-Feng T. L., Chen E., Waterfield M. D., Francke U., Ullrich A. Multiple, distinct forms of bovine and human protein kinase C suggest diversity in cellular signaling pathways. Science. 1986 Aug 22;233(4766):859–866. doi: 10.1126/science.3755548. [DOI] [PubMed] [Google Scholar]
  11. Csermely P., Szamel M., Resch K., Somogyi J. Zinc can increase the activity of protein kinase C and contributes to its binding to plasma membranes in T lymphocytes. J Biol Chem. 1988 May 15;263(14):6487–6490. [PubMed] [Google Scholar]
  12. Dieckmann C. L., Tzagoloff A. Assembly of the mitochondrial membrane system. CBP6, a yeast nuclear gene necessary for synthesis of cytochrome b. J Biol Chem. 1985 Feb 10;260(3):1513–1520. [PubMed] [Google Scholar]
  13. Diekmann D., Brill S., Garrett M. D., Totty N., Hsuan J., Monfries C., Hall C., Lim L., Hall A. Bcr encodes a GTPase-activating protein for p21rac. Nature. 1991 May 30;351(6325):400–402. doi: 10.1038/351400a0. [DOI] [PubMed] [Google Scholar]
  14. Forbes I. J., Zalewski P. D., Giannakis C., Petkoff H. S., Cowled P. A. Interaction between protein kinase C and regulatory ligand is enhanced by a chelatable pool of cellular zinc. Biochim Biophys Acta. 1990 Jul 12;1053(2-3):113–117. doi: 10.1016/0167-4889(90)90001-t. [DOI] [PubMed] [Google Scholar]
  15. Forbes I. J., Zalewski P. D., Hurst N. P., Giannakis C., Whitehouse M. W. Zinc increases phorbol ester receptors in intact B-cells, neutrophil polymorphs and platelets. FEBS Lett. 1989 Apr 24;247(2):445–447. doi: 10.1016/0014-5793(89)81388-2. [DOI] [PubMed] [Google Scholar]
  16. Freedman L. P., Luisi B. F., Korszun Z. R., Basavappa R., Sigler P. B., Yamamoto K. R. The function and structure of the metal coordination sites within the glucocorticoid receptor DNA binding domain. Nature. 1988 Aug 11;334(6182):543–546. doi: 10.1038/334543a0. [DOI] [PubMed] [Google Scholar]
  17. Gradwohl G., Ménissier de Murcia J. M., Molinete M., Simonin F., Koken M., Hoeijmakers J. H., de Murcia G. The second zinc-finger domain of poly(ADP-ribose) polymerase determines specificity for single-stranded breaks in DNA. Proc Natl Acad Sci U S A. 1990 Apr;87(8):2990–2994. doi: 10.1073/pnas.87.8.2990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Grossman S. R., Laimins L. A. E6 protein of human papillomavirus type 18 binds zinc. Oncogene. 1989 Sep;4(9):1089–1093. [PubMed] [Google Scholar]
  19. Hall C., Monfries C., Smith P., Lim H. H., Kozma R., Ahmed S., Vanniasingham V., Leung T., Lim L. Novel human brain cDNA encoding a 34,000 Mr protein n-chimaerin, related to both the regulatory domain of protein kinase C and BCR, the product of the breakpoint cluster region gene. J Mol Biol. 1990 Jan 5;211(1):11–16. doi: 10.1016/0022-2836(90)90006-8. [DOI] [PubMed] [Google Scholar]
  20. Harada N., Castle B. E., Gorman D. M., Itoh N., Schreurs J., Barrett R. L., Howard M., Miyajima A. Expression cloning of a cDNA encoding the murine interleukin 4 receptor based on ligand binding. Proc Natl Acad Sci U S A. 1990 Feb;87(3):857–861. doi: 10.1073/pnas.87.3.857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Härd T., Kellenbach E., Boelens R., Maler B. A., Dahlman K., Freedman L. P., Carlstedt-Duke J., Yamamoto K. R., Gustafsson J. A., Kaptein R. Solution structure of the glucocorticoid receptor DNA-binding domain. Science. 1990 Jul 13;249(4965):157–160. doi: 10.1126/science.2115209. [DOI] [PubMed] [Google Scholar]
  22. Kadonaga J. T., Carner K. R., Masiarz F. R., Tjian R. Isolation of cDNA encoding transcription factor Sp1 and functional analysis of the DNA binding domain. Cell. 1987 Dec 24;51(6):1079–1090. doi: 10.1016/0092-8674(87)90594-0. [DOI] [PubMed] [Google Scholar]
  23. Kanoh H., Yamada K., Sakane F. Diacylglycerol kinase: a key modulator of signal transduction? Trends Biochem Sci. 1990 Feb;15(2):47–50. doi: 10.1016/0968-0004(90)90172-8. [DOI] [PubMed] [Google Scholar]
  24. Kaplan D. R., Morrison D. K., Wong G., McCormick F., Williams L. T. PDGF beta-receptor stimulates tyrosine phosphorylation of GAP and association of GAP with a signaling complex. Cell. 1990 Apr 6;61(1):125–133. doi: 10.1016/0092-8674(90)90220-9. [DOI] [PubMed] [Google Scholar]
  25. Katzav S., Martin-Zanca D., Barbacid M. vav, a novel human oncogene derived from a locus ubiquitously expressed in hematopoietic cells. EMBO J. 1989 Aug;8(8):2283–2290. doi: 10.1002/j.1460-2075.1989.tb08354.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kikkawa U., Ogita K., Ono Y., Asaoka Y., Shearman M. S., Fujii T., Ase K., Sekiguchi K., Igarashi K., Nishizuka Y. The common structure and activities of four subspecies of rat brain protein kinase C family. FEBS Lett. 1987 Nov 2;223(2):212–216. doi: 10.1016/0014-5793(87)80291-0. [DOI] [PubMed] [Google Scholar]
  27. Knopf J. L., Lee M. H., Sultzman L. A., Kriz R. W., Loomis C. R., Hewick R. M., Bell R. M. Cloning and expression of multiple protein kinase C cDNAs. Cell. 1986 Aug 15;46(4):491–502. doi: 10.1016/0092-8674(86)90874-3. [DOI] [PubMed] [Google Scholar]
  28. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Levin D. E., Fields F. O., Kunisawa R., Bishop J. M., Thorner J. A candidate protein kinase C gene, PKC1, is required for the S. cerevisiae cell cycle. Cell. 1990 Jul 27;62(2):213–224. doi: 10.1016/0092-8674(90)90360-q. [DOI] [PubMed] [Google Scholar]
  31. Maruyama I. N., Brenner S. A phorbol ester/diacylglycerol-binding protein encoded by the unc-13 gene of Caenorhabditis elegans. Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5729–5733. doi: 10.1073/pnas.88.13.5729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Mazen A., Gradwohl G., de Murcia G. Zinc-binding proteins detected by protein blotting. Anal Biochem. 1988 Jul;172(1):39–42. doi: 10.1016/0003-2697(88)90408-3. [DOI] [PubMed] [Google Scholar]
  33. Nagai K., Nakaseko Y., Nasmyth K., Rhodes D. Zinc-finger motifs expressed in E. coli and folded in vitro direct specific binding to DNA. Nature. 1988 Mar 17;332(6161):284–286. doi: 10.1038/332284a0. [DOI] [PubMed] [Google Scholar]
  34. Nagai K., Thøgersen H. C. Synthesis and sequence-specific proteolysis of hybrid proteins produced in Escherichia coli. Methods Enzymol. 1987;153:461–481. doi: 10.1016/0076-6879(87)53072-5. [DOI] [PubMed] [Google Scholar]
  35. Nishizuka Y. Studies and perspectives of protein kinase C. Science. 1986 Jul 18;233(4761):305–312. doi: 10.1126/science.3014651. [DOI] [PubMed] [Google Scholar]
  36. Nishizuka Y. The molecular heterogeneity of protein kinase C and its implications for cellular regulation. Nature. 1988 Aug 25;334(6184):661–665. doi: 10.1038/334661a0. [DOI] [PubMed] [Google Scholar]
  37. Nishizuka Y. The role of protein kinase C in cell surface signal transduction and tumour promotion. Nature. 1984 Apr 19;308(5961):693–698. doi: 10.1038/308693a0. [DOI] [PubMed] [Google Scholar]
  38. Ohno S., Akita Y., Konno Y., Imajoh S., Suzuki K. A novel phorbol ester receptor/protein kinase, nPKC, distantly related to the protein kinase C family. Cell. 1988 Jun 3;53(5):731–741. doi: 10.1016/0092-8674(88)90091-8. [DOI] [PubMed] [Google Scholar]
  39. Ono Y., Fujii T., Igarashi K., Kikkawa U., Ogita K., Nishizuka Y. Nucleotide sequences of cDNAs for alpha and gamma subspecies of rat brain protein kinase C. Nucleic Acids Res. 1988 Jun 10;16(11):5199–5200. doi: 10.1093/nar/16.11.5199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Ono Y., Fujii T., Igarashi K., Kuno T., Tanaka C., Kikkawa U., Nishizuka Y. Phorbol ester binding to protein kinase C requires a cysteine-rich zinc-finger-like sequence. Proc Natl Acad Sci U S A. 1989 Jul;86(13):4868–4871. doi: 10.1073/pnas.86.13.4868. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Ono Y., Fujii T., Ogita K., Kikkawa U., Igarashi K., Nishizuka Y. Protein kinase C zeta subspecies from rat brain: its structure, expression, and properties. Proc Natl Acad Sci U S A. 1989 May;86(9):3099–3103. doi: 10.1073/pnas.86.9.3099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Ono Y., Kikkawa U., Ogita K., Fujii T., Kurokawa T., Asaoka Y., Sekiguchi K., Ase K., Igarashi K., Nishizuka Y. Expression and properties of two types of protein kinase C: alternative splicing from a single gene. Science. 1987 May 29;236(4805):1116–1120. doi: 10.1126/science.3576226. [DOI] [PubMed] [Google Scholar]
  43. Osada S., Mizuno K., Saido T. C., Akita Y., Suzuki K., Kuroki T., Ohno S. A phorbol ester receptor/protein kinase, nPKC eta, a new member of the protein kinase C family predominantly expressed in lung and skin. J Biol Chem. 1990 Dec 25;265(36):22434–22440. [PubMed] [Google Scholar]
  44. Pan T., Coleman J. E. GAL4 transcription factor is not a "zinc finger" but forms a Zn(II)2Cys6 binuclear cluster. Proc Natl Acad Sci U S A. 1990 Mar;87(6):2077–2081. doi: 10.1073/pnas.87.6.2077. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Pan T., Coleman J. E. Structure and function of the Zn(II) binding site within the DNA-binding domain of the GAL4 transcription factor. Proc Natl Acad Sci U S A. 1989 May;86(9):3145–3149. doi: 10.1073/pnas.86.9.3145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Parker P. J., Coussens L., Totty N., Rhee L., Young S., Chen E., Stabel S., Waterfield M. D., Ullrich A. The complete primary structure of protein kinase C--the major phorbol ester receptor. Science. 1986 Aug 22;233(4766):853–859. doi: 10.1126/science.3755547. [DOI] [PubMed] [Google Scholar]
  47. Párraga G., Horvath S., Hood L., Young E. T., Klevit R. E. Spectroscopic studies of wild-type and mutant "zinc finger" peptides: determinants of domain folding and structure. Proc Natl Acad Sci U S A. 1990 Jan;87(1):137–141. doi: 10.1073/pnas.87.1.137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Rapp U. R., Heidecker G., Huleihel M., Cleveland J. L., Choi W. C., Pawson T., Ihle J. N., Anderson W. B. raf family serine/threonine protein kinases in mitogen signal transduction. Cold Spring Harb Symp Quant Biol. 1988;53(Pt 1):173–184. doi: 10.1101/sqb.1988.053.01.023. [DOI] [PubMed] [Google Scholar]
  49. Sakane F., Yamada K., Kanoh H., Yokoyama C., Tanabe T. Porcine diacylglycerol kinase sequence has zinc finger and E-F hand motifs. Nature. 1990 Mar 22;344(6264):345–348. doi: 10.1038/344345a0. [DOI] [PubMed] [Google Scholar]
  50. Schaap D., de Widt J., van der Wal J., Vandekerckhove J., van Damme J., Gussow D., Ploegh H. L., van Blitterswijk W. J., van der Bend R. L. Purification, cDNA-cloning and expression of human diacylglycerol kinase. FEBS Lett. 1990 Nov 26;275(1-2):151–158. doi: 10.1016/0014-5793(90)81461-v. [DOI] [PubMed] [Google Scholar]
  51. Schiff L. A., Nibert M. L., Co M. S., Brown E. G., Fields B. N. Distinct binding sites for zinc and double-stranded RNA in the reovirus outer capsid protein sigma 3. Mol Cell Biol. 1988 Jan;8(1):273–283. doi: 10.1128/mcb.8.1.273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Schiff L. A., Nibert M. L., Fields B. N. Characterization of a zinc blotting technique: evidence that a retroviral gag protein binds zinc. Proc Natl Acad Sci U S A. 1988 Jun;85(12):4195–4199. doi: 10.1073/pnas.85.12.4195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Schwabe J. W., Neuhaus D., Rhodes D. Solution structure of the DNA-binding domain of the oestrogen receptor. Nature. 1990 Nov 29;348(6300):458–461. doi: 10.1038/348458a0. [DOI] [PubMed] [Google Scholar]
  54. 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]
  55. Zahradka P., Ebisuzaki K. Poly(ADP-ribose) polymerase is a zinc metalloenzyme. Eur J Biochem. 1984 Aug 1;142(3):503–509. doi: 10.1111/j.1432-1033.1984.tb08314.x. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES