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. 1996 Jan;16(1):37–44. doi: 10.1128/mcb.16.1.37

p95vav associates with the nuclear protein Ku-70.

F Romero 1, C Dargemont 1, F Pozo 1, W H Reeves 1, J Camonis 1, S Gisselbrecht 1, S Fischer 1
PMCID: PMC230976  PMID: 8524317

Abstract

The proto-oncogene vav is expressed solely in hematopoietic cells and plays an important role in cell signaling, although little is known about the proteins involved in these pathways. To gain further information, the Src homology 2 (SH2) and 3 (SH3) domains of Vav were used to screen a lymphoid cell cDNA library by the yeast two-hybrid system. Among the positive clones, we detected a nuclear protein, Ku-70, which is the DNA-binding element of the DNA-dependent protein kinase. In Jurkat and UT7 cells, Vav is partially localized in the nuclei, as judged from immunofluorescence and confocal microscopy studies. By using glutathione S-transferase fusion proteins derived from Ku-70 and coimmunoprecipitation experiments with lysates prepared from human thymocytes and Jurkat and UT7 cells, we show that Vav associates with Ku-70. The interaction of Vav with Ku-70 requires only the 150-residue carboxy-terminal portion of Ku-70, which binds to the 25 carboxy-terminal residues of the carboxy SH3 domain of Vav. A proline-to-leucine mutation in the carboxy SH3 of Vav that blocks interaction with proline-rich sequences does not modify the binding of Ku-70, which lacks this motif. Therefore, the interaction of Vav with Ku-70 may be a novel form of protein-protein interaction. The potential role of Vav/Ku-70 complexes is discussed.

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Selected References

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  1. Alai M., Mui A. L., Cutler R. L., Bustelo X. R., Barbacid M., Krystal G. Steel factor stimulates the tyrosine phosphorylation of the proto-oncogene product, p95vav, in human hemopoietic cells. J Biol Chem. 1992 Sep 5;267(25):18021–18025. [PubMed] [Google Scholar]
  2. Benichou S., Bomsel M., Bodéus M., Durand H., Douté M., Letourneur F., Camonis J., Benarous R. Physical interaction of the HIV-1 Nef protein with beta-COP, a component of non-clathrin-coated vesicles essential for membrane traffic. J Biol Chem. 1994 Dec 2;269(48):30073–30076. [PubMed] [Google Scholar]
  3. Blier P. R., Griffith A. J., Craft J., Hardin J. A. Binding of Ku protein to DNA. Measurement of affinity for ends and demonstration of binding to nicks. J Biol Chem. 1993 Apr 5;268(10):7594–7601. [PubMed] [Google Scholar]
  4. Boguski M. S., McCormick F. Proteins regulating Ras and its relatives. Nature. 1993 Dec 16;366(6456):643–654. doi: 10.1038/366643a0. [DOI] [PubMed] [Google Scholar]
  5. Breeden L., Nasmyth K. Regulation of the yeast HO gene. Cold Spring Harb Symp Quant Biol. 1985;50:643–650. doi: 10.1101/sqb.1985.050.01.078. [DOI] [PubMed] [Google Scholar]
  6. Bustelo X. R., Barbacid M. Tyrosine phosphorylation of the vav proto-oncogene product in activated B cells. Science. 1992 May 22;256(5060):1196–1199. doi: 10.1126/science.256.5060.1196. [DOI] [PubMed] [Google Scholar]
  7. Bustelo X. R., Ledbetter J. A., Barbacid M. Product of vav proto-oncogene defines a new class of tyrosine protein kinase substrates. Nature. 1992 Mar 5;356(6364):68–71. doi: 10.1038/356068a0. [DOI] [PubMed] [Google Scholar]
  8. Bustelo X. R., Suen K. L., Leftheris K., Meyers C. A., Barbacid M. Vav cooperates with Ras to transform rodent fibroblasts but is not a Ras GDP/GTP exchange factor. Oncogene. 1994 Aug;9(8):2405–2413. [PubMed] [Google Scholar]
  9. Bustelo X. R., Suen K. L., Michael W. M., Dreyfuss G., Barbacid M. Association of the vav proto-oncogene product with poly(rC)-specific RNA-binding proteins. Mol Cell Biol. 1995 Mar;15(3):1324–1332. doi: 10.1128/mcb.15.3.1324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Clark S. G., Stern M. J., Horvitz H. R. C. elegans cell-signalling gene sem-5 encodes a protein with SH2 and SH3 domains. Nature. 1992 Mar 26;356(6367):340–344. doi: 10.1038/356340a0. [DOI] [PubMed] [Google Scholar]
  11. Clevenger C. V., Ngo W., Sokol D. L., Luger S. M., Gewirtz A. M. Vav is necessary for prolactin-stimulated proliferation and is translocated into the nucleus of a T-cell line. J Biol Chem. 1995 Jun 2;270(22):13246–13253. doi: 10.1074/jbc.270.22.13246. [DOI] [PubMed] [Google Scholar]
  12. Coppola J., Bryant S., Koda T., Conway D., Barbacid M. Mechanism of activation of the vav protooncogene. Cell Growth Differ. 1991 Feb;2(2):95–105. [PubMed] [Google Scholar]
  13. Dosil M., Wang S., Lemischka I. R. Mitogenic signalling and substrate specificity of the Flk2/Flt3 receptor tyrosine kinase in fibroblasts and interleukin 3-dependent hematopoietic cells. Mol Cell Biol. 1993 Oct;13(10):6572–6585. doi: 10.1128/mcb.13.10.6572. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dusanter-Fourt I., Casadevall N., Lacombe C., Muller O., Billat C., Fischer S., Mayeux P. Erythropoietin induces the tyrosine phosphorylation of its own receptor in human erythropoietin-responsive cells. J Biol Chem. 1992 May 25;267(15):10670–10675. [PubMed] [Google Scholar]
  15. Dvir A., Stein L. Y., Calore B. L., Dynan W. S. Purification and characterization of a template-associated protein kinase that phosphorylates RNA polymerase II. J Biol Chem. 1993 May 15;268(14):10440–10447. [PubMed] [Google Scholar]
  16. Eck M. J., Atwell S. K., Shoelson S. E., Harrison S. C. Structure of the regulatory domains of the Src-family tyrosine kinase Lck. Nature. 1994 Apr 21;368(6473):764–769. doi: 10.1038/368764a0. [DOI] [PubMed] [Google Scholar]
  17. Evans G. A., Howard O. M., Erwin R., Farrar W. L. Interleukin-2 induces tyrosine phosphorylation of the vav proto-oncogene product in human T cells: lack of requirement for the tyrosine kinase lck. Biochem J. 1993 Sep 1;294(Pt 2):339–342. doi: 10.1042/bj2940339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Falzon M., Fewell J. W., Kuff E. L. EBP-80, a transcription factor closely resembling the human autoantigen Ku, recognizes single- to double-strand transitions in DNA. J Biol Chem. 1993 May 15;268(14):10546–10552. [PubMed] [Google Scholar]
  19. Feldmann H., Winnacker E. L. A putative homologue of the human autoantigen Ku from Saccharomyces cerevisiae. J Biol Chem. 1993 Jun 15;268(17):12895–12900. [PubMed] [Google Scholar]
  20. Fields S., Song O. A novel genetic system to detect protein-protein interactions. Nature. 1989 Jul 20;340(6230):245–246. doi: 10.1038/340245a0. [DOI] [PubMed] [Google Scholar]
  21. Fischer K. D., Zmuldzinas A., Gardner S., Barbacid M., Bernstein A., Guidos C. Defective T-cell receptor signalling and positive selection of Vav-deficient CD4+ CD8+ thymocytes. Nature. 1995 Mar 30;374(6521):474–477. doi: 10.1038/374474a0. [DOI] [PubMed] [Google Scholar]
  22. Francoeur A. M., Peebles C. L., Gompper P. T., Tan E. M. Identification of Ki (Ku, p70/p80) autoantigens and analysis of anti-Ki autoantibody reactivity. J Immunol. 1986 Mar 1;136(5):1648–1653. [PubMed] [Google Scholar]
  23. Getts R. C., Stamato T. D. Absence of a Ku-like DNA end binding activity in the xrs double-strand DNA repair-deficient mutant. J Biol Chem. 1994 Jun 10;269(23):15981–15984. [PubMed] [Google Scholar]
  24. Griffith A. J., Blier P. R., Mimori T., Hardin J. A. Ku polypeptides synthesized in vitro assemble into complexes which recognize ends of double-stranded DNA. J Biol Chem. 1992 Jan 5;267(1):331–338. [PubMed] [Google Scholar]
  25. Gulbins E., Coggeshall K. M., Baier G., Katzav S., Burn P., Altman A. Tyrosine kinase-stimulated guanine nucleotide exchange activity of Vav in T cell activation. Science. 1993 May 7;260(5109):822–825. doi: 10.1126/science.8484124. [DOI] [PubMed] [Google Scholar]
  26. Gulbins E., Coggeshall K. M., Baier G., Telford D., Langlet C., Baier-Bitterlich G., Bonnefoy-Berard N., Burn P., Wittinghofer A., Altman A. Direct stimulation of Vav guanine nucleotide exchange activity for Ras by phorbol esters and diglycerides. Mol Cell Biol. 1994 Jul;14(7):4749–4758. doi: 10.1128/mcb.14.7.4749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Gulbins E., Coggeshall K. M., Langlet C., Baier G., Bonnefoy-Berard N., Burn P., Wittinghofer A., Katzav S., Altman A. Activation of Ras in vitro and in intact fibroblasts by the Vav guanine nucleotide exchange protein. Mol Cell Biol. 1994 Feb;14(2):906–913. doi: 10.1128/mcb.14.2.906. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Gulbins E., Langlet C., Baier G., Bonnefoy-Berard N., Herbert E., Altman A., Coggeshall K. M. Tyrosine phosphorylation and activation of Vav GTP/GDP exchange activity in antigen receptor-triggered B cells. J Immunol. 1994 Mar 1;152(5):2123–2129. [PubMed] [Google Scholar]
  29. Hannon G. J., Demetrick D., Beach D. Isolation of the Rb-related p130 through its interaction with CDK2 and cyclins. Genes Dev. 1993 Dec;7(12A):2378–2391. doi: 10.1101/gad.7.12a.2378. [DOI] [PubMed] [Google Scholar]
  30. Higashiura M., Shimizu Y., Tanimoto M., Morita T., Yagura T. Immunolocalization of Ku-proteins (p80/p70): localization of p70 to nucleoli and periphery of both interphase nuclei and metaphase chromosomes. Exp Cell Res. 1992 Aug;201(2):444–451. doi: 10.1016/0014-4827(92)90293-h. [DOI] [PubMed] [Google Scholar]
  31. Hobert O., Jallal B., Schlessinger J., Ullrich A. Novel signaling pathway suggested by SH3 domain-mediated p95vav/heterogeneous ribonucleoprotein K interaction. J Biol Chem. 1994 Aug 12;269(32):20225–20228. [PubMed] [Google Scholar]
  32. Hu P., Margolis B., Schlessinger J. Vav: a potential link between tyrosine kinases and ras-like GTPases in hematopoietic cell signaling. Bioessays. 1993 Mar;15(3):179–183. doi: 10.1002/bies.950150306. [DOI] [PubMed] [Google Scholar]
  33. Katzav S., Cleveland J. L., Heslop H. E., Pulido D. Loss of the amino-terminal helix-loop-helix domain of the vav proto-oncogene activates its transforming potential. Mol Cell Biol. 1991 Apr;11(4):1912–1920. doi: 10.1128/mcb.11.4.1912. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. Katzav S., Sutherland M., Packham G., Yi T., Weiss A. The protein tyrosine kinase ZAP-70 can associate with the SH2 domain of proto-Vav. J Biol Chem. 1994 Dec 23;269(51):32579–32585. [PubMed] [Google Scholar]
  36. Khosravi-Far R., Chrzanowska-Wodnicka M., Solski P. A., Eva A., Burridge K., Der C. J. Dbl and Vav mediate transformation via mitogen-activated protein kinase pathways that are distinct from those activated by oncogenic Ras. Mol Cell Biol. 1994 Oct;14(10):6848–6857. doi: 10.1128/mcb.14.10.6848. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Knuth M. W., Gunderson S. I., Thompson N. E., Strasheim L. A., Burgess R. R. Purification and characterization of proximal sequence element-binding protein 1, a transcription activating protein related to Ku and TREF that binds the proximal sequence element of the human U1 promoter. J Biol Chem. 1990 Oct 15;265(29):17911–17920. [PubMed] [Google Scholar]
  38. Kuhn A., Gottlieb T. M., Jackson S. P., Grummt I. DNA-dependent protein kinase: a potent inhibitor of transcription by RNA polymerase I. Genes Dev. 1995 Jan 15;9(2):193–203. doi: 10.1101/gad.9.2.193. [DOI] [PubMed] [Google Scholar]
  39. Lees-Miller S. P., Chen Y. R., Anderson C. W. Human cells contain a DNA-activated protein kinase that phosphorylates simian virus 40 T antigen, mouse p53, and the human Ku autoantigen. Mol Cell Biol. 1990 Dec;10(12):6472–6481. doi: 10.1128/mcb.10.12.6472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Lowenstein E. J., Daly R. J., Batzer A. G., Li W., Margolis B., Lammers R., Ullrich A., Skolnik E. Y., Bar-Sagi D., Schlessinger J. The SH2 and SH3 domain-containing protein GRB2 links receptor tyrosine kinases to ras signaling. Cell. 1992 Aug 7;70(3):431–442. doi: 10.1016/0092-8674(92)90167-b. [DOI] [PubMed] [Google Scholar]
  41. Margolis B., Hu P., Katzav S., Li W., Oliver J. M., Ullrich A., Weiss A., Schlessinger J. Tyrosine phosphorylation of vav proto-oncogene product containing SH2 domain and transcription factor motifs. Nature. 1992 Mar 5;356(6364):71–74. doi: 10.1038/356071a0. [DOI] [PubMed] [Google Scholar]
  42. Mimori T., Akizuki M., Yamagata H., Inada S., Yoshida S., Homma M. Characterization of a high molecular weight acidic nuclear protein recognized by autoantibodies in sera from patients with polymyositis-scleroderma overlap. J Clin Invest. 1981 Sep;68(3):611–620. doi: 10.1172/JCI110295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Miura O., Miura Y., Nakamura N., Quelle F. W., Witthuhn B. A., Ihle J. N., Aoki N. Induction of tyrosine phosphorylation of Vav and expression of Pim-1 correlates with Jak2-mediated growth signaling from the erythropoietin receptor. Blood. 1994 Dec 15;84(12):4135–4141. [PubMed] [Google Scholar]
  44. Musacchio A., Gibson T., Rice P., Thompson J., Saraste M. The PH domain: a common piece in the structural patchwork of signalling proteins. Trends Biochem Sci. 1993 Sep;18(9):343–348. doi: 10.1016/0968-0004(93)90071-t. [DOI] [PubMed] [Google Scholar]
  45. Niu H., Jacob S. T. Enhancer 1 binding factor (E1BF), a Ku-related protein, is a growth-regulated RNA polymerase I transcription factor: association of a repressor activity with purified E1BF from serum-deprived cells. Proc Natl Acad Sci U S A. 1994 Sep 13;91(19):9101–9105. doi: 10.1073/pnas.91.19.9101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Pallard C., Gouilleux F., Bénit L., Cocault L., Souyri M., Levy D., Groner B., Gisselbrecht S., Dusanter-Fourt I. Thrombopoietin activates a STAT5-like factor in hematopoietic cells. EMBO J. 1995 Jun 15;14(12):2847–2856. doi: 10.1002/j.1460-2075.1995.tb07284.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Platanias L. C., Sweet M. E. Interferon alpha induces rapid tyrosine phosphorylation of the vav proto-oncogene product in hematopoietic cells. J Biol Chem. 1994 Feb 4;269(5):3143–3146. [PubMed] [Google Scholar]
  48. Puil L., Pawson T. Vagaries of vav. Curr Biol. 1992 May;2(5):275–277. doi: 10.1016/0960-9822(92)90396-r. [DOI] [PubMed] [Google Scholar]
  49. Ramos-Morales F., Druker B. J., Fischer S. Vav binds to several SH2/SH3 containing proteins in activated lymphocytes. Oncogene. 1994 Jul;9(7):1917–1923. [PubMed] [Google Scholar]
  50. Ramos-Morales F., Romero F., Schweighoffer F., Bismuth G., Camonis J., Tortolero M., Fischer S. The proline-rich region of Vav binds to Grb2 and Grb3-3. Oncogene. 1995 Oct 19;11(8):1665–1669. [PubMed] [Google Scholar]
  51. Reeves W. H. Use of monoclonal antibodies for the characterization of novel DNA-binding proteins recognized by human autoimmune sera. J Exp Med. 1985 Jan 1;161(1):18–39. doi: 10.1084/jem.161.1.18. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Roberts M. R., Miskimins W. K., Ruddle F. H. Nuclear proteins TREF1 and TREF2 bind to the transcriptional control element of the transferrin receptor gene and appear to be associated as a heterodimer. Cell Regul. 1989 Nov;1(1):151–164. doi: 10.1091/mbc.1.1.151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. 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]
  54. Stuiver M. H., Coenjaerts F. E., van der Vliet P. C. The autoantigen Ku is indistinguishable from NF IV, a protein forming multimeric protein-DNA complexes. J Exp Med. 1990 Oct 1;172(4):1049–1054. doi: 10.1084/jem.172.4.1049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Taccioli G. E., Gottlieb T. M., Blunt T., Priestley A., Demengeot J., Mizuta R., Lehmann A. R., Alt F. W., Jackson S. P., Jeggo P. A. Ku80: product of the XRCC5 gene and its role in DNA repair and V(D)J recombination. Science. 1994 Sep 2;265(5177):1442–1445. doi: 10.1126/science.8073286. [DOI] [PubMed] [Google Scholar]
  56. Tarakhovsky A., Turner M., Schaal S., Mee P. J., Duddy L. P., Rajewsky K., Tybulewicz V. L. Defective antigen receptor-mediated proliferation of B and T cells in the absence of Vav. Nature. 1995 Mar 30;374(6521):467–470. doi: 10.1038/374467a0. [DOI] [PubMed] [Google Scholar]
  57. Van Aelst L., Barr M., Marcus S., Polverino A., Wigler M. Complex formation between RAS and RAF and other protein kinases. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):6213–6217. doi: 10.1073/pnas.90.13.6213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Vojtek A. B., Hollenberg S. M., Cooper J. A. Mammalian Ras interacts directly with the serine/threonine kinase Raf. Cell. 1993 Jul 16;74(1):205–214. doi: 10.1016/0092-8674(93)90307-c. [DOI] [PubMed] [Google Scholar]
  59. Yaneva M., Ochs R., McRorie D. K., Zweig S., Busch H. Purification of an 86-70 kDa nuclear DNA-associated protein complex. Biochim Biophys Acta. 1985 Jul 26;841(1):22–29. doi: 10.1016/0304-4165(85)90270-3. [DOI] [PubMed] [Google Scholar]
  60. Ye Z. S., Baltimore D. Binding of Vav to Grb2 through dimerization of Src homology 3 domains. Proc Natl Acad Sci U S A. 1994 Dec 20;91(26):12629–12633. doi: 10.1073/pnas.91.26.12629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Zhang R., Alt F. W., Davidson L., Orkin S. H., Swat W. Defective signalling through the T- and B-cell antigen receptors in lymphoid cells lacking the vav proto-oncogene. Nature. 1995 Mar 30;374(6521):470–473. doi: 10.1038/374470a0. [DOI] [PubMed] [Google Scholar]
  62. Zmuidzinas A., Fischer K. D., Lira S. A., Forrester L., Bryant S., Bernstein A., Barbacid M. The vav proto-oncogene is required early in embryogenesis but not for hematopoietic development in vitro. EMBO J. 1995 Jan 3;14(1):1–11. doi: 10.1002/j.1460-2075.1995.tb06969.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. de Vries E., van Driel W., Bergsma W. G., Arnberg A. C., van der Vliet P. C. HeLa nuclear protein recognizing DNA termini and translocating on DNA forming a regular DNA-multimeric protein complex. J Mol Biol. 1989 Jul 5;208(1):65–78. doi: 10.1016/0022-2836(89)90088-0. [DOI] [PubMed] [Google Scholar]

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