Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1997 Nov 1;327(Pt 3):867–876. doi: 10.1042/bj3270867

CD45 and RPTPalpha display different protein tyrosine phosphatase activities in T lymphocytes.

D H Ng 1, M D Jabali 1, A Maiti 1, P Borodchak 1, K W Harder 1, T Brocker 1, B Malissen 1, F R Jirik 1, P Johnson 1
PMCID: PMC1218869  PMID: 9581568

Abstract

To examine the substrate specificity and function of two receptor protein tyrosine phosphatases, CD45 and RPTPalpha, RPTPalpha was expressed in a CD45(-), T-cell receptor (TCR)+, BW5147 T-lymphoma cell. High levels of expression of RPTPalpha did not fully restore either proximal or distal TCR-mediated signalling events. RPTPalpha was unable to reconstitute the phosphorylation of CD3zeta and did not increase the expression of the activation marker, CD69, on stimulation with TCR/CD3. RPTPalpha did not significantly alter the phosphorylation state or kinase activity of two CD45 substrates, p56(lck) or p59(fyn), suggesting that RPTPalpha does not have the same specificity or function as CD45 in T-cells. Further comparison of the two phosphatases indicated that immunoprecipitated RPTPalpha was approx. one-seventh to one-tenth as active as CD45 when tested against artificial substrates. This difference in activity was also observed in vitro with purified recombinant enzymes at physiological pH. Additional analysis with Src family phosphopeptides and recombinant p56(lck) as substrates indicated that CD45 was consistently more active than RPTPalpha, having both higher Vmax and lower Km values. Thus CD45 is intrinsically a much more active phosphatase than RPTPalpha, which provides one reason why RPTPalpha cannot effectively dephosphorylate p56(lck) and substitute for CD45 in T-cells. This work establishes that these two related protein tyrosine phosphatases are not interchangeable in T-cells and that this is due, at least in part, to quantitative differences in phosphatase activity.

Full Text

The Full Text of this article is available as a PDF (509.3 KB).

Selected References

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

  1. Barford D., Jia Z., Tonks N. K. Protein tyrosine phosphatases take off. Nat Struct Biol. 1995 Dec;2(12):1043–1053. doi: 10.1038/nsb1295-1043. [DOI] [PubMed] [Google Scholar]
  2. Cahir McFarland E. D., Hurley T. R., Pingel J. T., Sefton B. M., Shaw A., Thomas M. L. Correlation between Src family member regulation by the protein-tyrosine-phosphatase CD45 and transmembrane signaling through the T-cell receptor. Proc Natl Acad Sci U S A. 1993 Feb 15;90(4):1402–1406. doi: 10.1073/pnas.90.4.1402. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Charbonneau H., Tonks N. K., Kumar S., Diltz C. D., Harrylock M., Cool D. E., Krebs E. G., Fischer E. H., Walsh K. A. Human placenta protein-tyrosine-phosphatase: amino acid sequence and relationship to a family of receptor-like proteins. Proc Natl Acad Sci U S A. 1989 Jul;86(14):5252–5256. doi: 10.1073/pnas.86.14.5252. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cooke M. P., Perlmutter R. M. Expression of a novel form of the fyn proto-oncogene in hematopoietic cells. New Biol. 1989 Oct;1(1):66–74. [PubMed] [Google Scholar]
  5. Daum G., Zander N. F., Morse B., Hurwitz D., Schlessinger J., Fischer E. H. Characterization of a human recombinant receptor-linked protein tyrosine phosphatase. J Biol Chem. 1991 Jul 5;266(19):12211–12215. [PubMed] [Google Scholar]
  6. Davidson D., Chow L. M., Fournel M., Veillette A. Differential regulation of T cell antigen responsiveness by isoforms of the src-related tyrosine protein kinase p59fyn. J Exp Med. 1992 Jun 1;175(6):1483–1492. doi: 10.1084/jem.175.6.1483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Desai D. M., Sap J., Schlessinger J., Weiss A. Ligand-mediated negative regulation of a chimeric transmembrane receptor tyrosine phosphatase. Cell. 1993 May 7;73(3):541–554. doi: 10.1016/0092-8674(93)90141-c. [DOI] [PubMed] [Google Scholar]
  8. Desai D. M., Sap J., Silvennoinen O., Schlessinger J., Weiss A. The catalytic activity of the CD45 membrane-proximal phosphatase domain is required for TCR signaling and regulation. EMBO J. 1994 Sep 1;13(17):4002–4010. doi: 10.1002/j.1460-2075.1994.tb06716.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Furukawa T., Itoh M., Krueger N. X., Streuli M., Saito H. Specific interaction of the CD45 protein-tyrosine phosphatase with tyrosine-phosphorylated CD3 zeta chain. Proc Natl Acad Sci U S A. 1994 Nov 8;91(23):10928–10932. doi: 10.1073/pnas.91.23.10928. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hara T., Jung L. K., Bjorndahl J. M., Fu S. M. Human T cell activation. III. Rapid induction of a phosphorylated 28 kD/32 kD disulfide-linked early activation antigen (EA 1) by 12-o-tetradecanoyl phorbol-13-acetate, mitogens, and antigens. J Exp Med. 1986 Dec 1;164(6):1988–2005. doi: 10.1084/jem.164.6.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hovis R. R., Donovan J. A., Musci M. A., Motto D. G., Goldman F. D., Ross S. E., Koretzky G. A. Rescue of signaling by a chimeric protein containing the cytoplasmic domain of CD45. Science. 1993 Apr 23;260(5107):544–546. doi: 10.1126/science.8475387. [DOI] [PubMed] [Google Scholar]
  12. Hunter T. Protein kinases and phosphatases: the yin and yang of protein phosphorylation and signaling. Cell. 1995 Jan 27;80(2):225–236. doi: 10.1016/0092-8674(95)90405-0. [DOI] [PubMed] [Google Scholar]
  13. Hurley T. R., Hyman R., Sefton B. M. Differential effects of expression of the CD45 tyrosine protein phosphatase on the tyrosine phosphorylation of the lck, fyn, and c-src tyrosine protein kinases. Mol Cell Biol. 1993 Mar;13(3):1651–1656. doi: 10.1128/mcb.13.3.1651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hyman R., Trowbridge I. Two complementation classes of T200 (Ly-5) glycoprotein-negative mutants. Immunogenetics. 1981 Mar 1;12(5-6):511–523. doi: 10.1007/BF01561692. [DOI] [PubMed] [Google Scholar]
  15. Itoh M., Streuli M., Krueger N. X., Saito H. Purification and characterization of the catalytic domains of the human receptor-linked protein tyrosine phosphatases HPTP beta, leukocyte common antigen (LCA), and leukocyte common antigen-related molecule (LAR). J Biol Chem. 1992 Jun 15;267(17):12356–12363. [PubMed] [Google Scholar]
  16. Iwashima M., Irving B. A., van Oers N. S., Chan A. C., Weiss A. Sequential interactions of the TCR with two distinct cytoplasmic tyrosine kinases. Science. 1994 Feb 25;263(5150):1136–1139. doi: 10.1126/science.7509083. [DOI] [PubMed] [Google Scholar]
  17. Jirik F. R., Janzen N. M., Melhado I. G., Harder K. W. Cloning and chromosomal assignment of a widely expressed human receptor-like protein-tyrosine phosphatase. FEBS Lett. 1990 Oct 29;273(1-2):239–242. doi: 10.1016/0014-5793(90)81094-5. [DOI] [PubMed] [Google Scholar]
  18. Johnson P., Ostergaard H. L., Wasden C., Trowbridge I. S. Mutational analysis of CD45. A leukocyte-specific protein tyrosine phosphatase. J Biol Chem. 1992 Apr 25;267(12):8035–8041. [PubMed] [Google Scholar]
  19. Kaplan R., Morse B., Huebner K., Croce C., Howk R., Ravera M., Ricca G., Jaye M., Schlessinger J. Cloning of three human tyrosine phosphatases reveals a multigene family of receptor-linked protein-tyrosine-phosphatases expressed in brain. Proc Natl Acad Sci U S A. 1990 Sep;87(18):7000–7004. doi: 10.1073/pnas.87.18.7000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Karasuyama H., Kudo A., Melchers F. The proteins encoded by the VpreB and lambda 5 pre-B cell-specific genes can associate with each other and with mu heavy chain. J Exp Med. 1990 Sep 1;172(3):969–972. doi: 10.1084/jem.172.3.969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Koretzky G. A., Picus J., Schultz T., Weiss A. Tyrosine phosphatase CD45 is required for T-cell antigen receptor and CD2-mediated activation of a protein tyrosine kinase and interleukin 2 production. Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2037–2041. doi: 10.1073/pnas.88.6.2037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Krueger N. X., Streuli M., Saito H. Structural diversity and evolution of human receptor-like protein tyrosine phosphatases. EMBO J. 1990 Oct;9(10):3241–3252. doi: 10.1002/j.1460-2075.1990.tb07523.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Leitenberg D., Constant S., Lu D. D., Smith B. R., Bottomly K. CD4 and CD45 regulate qualitatively distinct patterns of calcium mobilization in individual CD4+ T cells. Eur J Immunol. 1995 Sep;25(9):2445–2451. doi: 10.1002/eji.1830250906. [DOI] [PubMed] [Google Scholar]
  24. Matthews R. J., Cahir E. D., Thomas M. L. Identification of an additional member of the protein-tyrosine-phosphatase family: evidence for alternative splicing in the tyrosine phosphatase domain. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4444–4448. doi: 10.1073/pnas.87.12.4444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Motto D. G., Musci M. A., Koretzky G. A. Surface expression of a heterologous phosphatase complements CD45 deficiency in a T cell clone. J Exp Med. 1994 Oct 1;180(4):1359–1366. doi: 10.1084/jem.180.4.1359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Mustelin T., Altman A. Dephosphorylation and activation of the T cell tyrosine kinase pp56lck by the leukocyte common antigen (CD45). Oncogene. 1990 Jun;5(6):809–813. [PubMed] [Google Scholar]
  27. Mustelin T., Pessa-Morikawa T., Autero M., Gassmann M., Andersson L. C., Gahmberg C. G., Burn P. Regulation of the p59fyn protein tyrosine kinase by the CD45 phosphotyrosine phosphatase. Eur J Immunol. 1992 May;22(5):1173–1178. doi: 10.1002/eji.1830220510. [DOI] [PubMed] [Google Scholar]
  28. Mustelin T., Williams S., Tailor P., Couture C., Zenner G., Burn P., Ashwell J. D., Altman A. Regulation of the p70zap tyrosine protein kinase in T cells by the CD45 phosphotyrosine phosphatase. Eur J Immunol. 1995 Apr;25(4):942–946. doi: 10.1002/eji.1830250413. [DOI] [PubMed] [Google Scholar]
  29. Ng D. H., Harder K. W., Clark-Lewis I., Jirik F., Johnson P. Non-radioactive method to measure CD45 protein tyrosine phosphatase activity isolated directly from cells. J Immunol Methods. 1995 Feb 27;179(2):177–185. doi: 10.1016/0022-1759(94)00281-z. [DOI] [PubMed] [Google Scholar]
  30. Ng D. H., Maiti A., Johnson P. Point mutation in the second phosphatase domain of CD45 abrogates tyrosine phosphatase activity. Biochem Biophys Res Commun. 1995 Jan 5;206(1):302–309. doi: 10.1006/bbrc.1995.1042. [DOI] [PubMed] [Google Scholar]
  31. Ng D. H., Watts J. D., Aebersold R., Johnson P. Demonstration of a direct interaction between p56lck and the cytoplasmic domain of CD45 in vitro. J Biol Chem. 1996 Jan 19;271(3):1295–1300. doi: 10.1074/jbc.271.3.1295. [DOI] [PubMed] [Google Scholar]
  32. Niklinska B. B., Hou D., June C., Weissman A. M., Ashwell J. D. CD45 tyrosine phosphatase activity and membrane anchoring are required for T-cell antigen receptor signaling. Mol Cell Biol. 1994 Dec;14(12):8078–8084. doi: 10.1128/mcb.14.12.8078. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Novak T. J., Farber D., Leitenberg D., Hong S. C., Johnson P., Bottomly K. Isoforms of the transmembrane tyrosine phosphatase CD45 differentially affect T cell recognition. Immunity. 1994 May;1(2):109–119. doi: 10.1016/1074-7613(94)90104-x. [DOI] [PubMed] [Google Scholar]
  34. Ostergaard H. L., Shackelford D. A., Hurley T. R., Johnson P., Hyman R., Sefton B. M., Trowbridge I. S. Expression of CD45 alters phosphorylation of the lck-encoded tyrosine protein kinase in murine lymphoma T-cell lines. Proc Natl Acad Sci U S A. 1989 Nov;86(22):8959–8963. doi: 10.1073/pnas.86.22.8959. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Pulido R., Serra-Pagès C., Tang M., Streuli M. The LAR/PTP delta/PTP sigma subfamily of transmembrane protein-tyrosine-phosphatases: multiple human LAR, PTP delta, and PTP sigma isoforms are expressed in a tissue-specific manner and associate with the LAR-interacting protein LIP.1. Proc Natl Acad Sci U S A. 1995 Dec 5;92(25):11686–11690. doi: 10.1073/pnas.92.25.11686. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sap J., D'Eustachio P., Givol D., Schlessinger J. Cloning and expression of a widely expressed receptor tyrosine phosphatase. Proc Natl Acad Sci U S A. 1990 Aug;87(16):6112–6116. doi: 10.1073/pnas.87.16.6112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Shiroo M., Goff L., Biffen M., Shivnan E., Alexander D. CD45 tyrosine phosphatase-activated p59fyn couples the T cell antigen receptor to pathways of diacylglycerol production, protein kinase C activation and calcium influx. EMBO J. 1992 Dec;11(13):4887–4897. doi: 10.1002/j.1460-2075.1992.tb05595.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Stone R. L., Dixon J. E. Protein-tyrosine phosphatases. J Biol Chem. 1994 Dec 16;269(50):31323–31326. [PubMed] [Google Scholar]
  39. Streuli M., Krueger N. X., Thai T., Tang M., Saito H. Distinct functional roles of the two intracellular phosphatase like domains of the receptor-linked protein tyrosine phosphatases LCA and LAR. EMBO J. 1990 Aug;9(8):2399–2407. doi: 10.1002/j.1460-2075.1990.tb07415.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Streuli M., Krueger N. X., Tsai A. Y., Saito H. A family of receptor-linked protein tyrosine phosphatases in humans and Drosophila. Proc Natl Acad Sci U S A. 1989 Nov;86(22):8698–8702. doi: 10.1073/pnas.86.22.8698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Sun H., Tonks N. K. The coordinated action of protein tyrosine phosphatases and kinases in cell signaling. Trends Biochem Sci. 1994 Nov;19(11):480–485. doi: 10.1016/0968-0004(94)90134-1. [DOI] [PubMed] [Google Scholar]
  42. Tan X., Stover D. R., Walsh K. A. Demonstration of protein tyrosine phosphatase activity in the second of two homologous domains of CD45. J Biol Chem. 1993 Apr 5;268(10):6835–6838. [PubMed] [Google Scholar]
  43. Testi R., Phillips J. H., Lanier L. L. Leu 23 induction as an early marker of functional CD3/T cell antigen receptor triggering. Requirement for receptor cross-linking, prolonged elevation of intracellular [Ca++] and stimulation of protein kinase C. J Immunol. 1989 Mar 15;142(6):1854–1860. [PubMed] [Google Scholar]
  44. Tonks N. K., Diltz C. D., Fischer E. H. Characterization of the major protein-tyrosine-phosphatases of human placenta. J Biol Chem. 1988 May 15;263(14):6731–6737. [PubMed] [Google Scholar]
  45. Tonks N. K., Diltz C. D., Fischer E. H. Purification of the major protein-tyrosine-phosphatases of human placenta. J Biol Chem. 1988 May 15;263(14):6722–6730. [PubMed] [Google Scholar]
  46. Trowbridge I. S., Thomas M. L. CD45: an emerging role as a protein tyrosine phosphatase required for lymphocyte activation and development. Annu Rev Immunol. 1994;12:85–116. doi: 10.1146/annurev.iy.12.040194.000505. [DOI] [PubMed] [Google Scholar]
  47. Volarević S., Niklinska B. B., Burns C. M., June C. H., Weissman A. M., Ashwell J. D. Regulation of TCR signaling by CD45 lacking transmembrane and extracellular domains. Science. 1993 Apr 23;260(5107):541–544. doi: 10.1126/science.8475386. [DOI] [PubMed] [Google Scholar]
  48. Walton K. M., Dixon J. E. Protein tyrosine phosphatases. Annu Rev Biochem. 1993;62:101–120. doi: 10.1146/annurev.bi.62.070193.000533. [DOI] [PubMed] [Google Scholar]
  49. Wang Y., Pallen C. J. The receptor-like protein tyrosine phosphatase HPTP alpha has two active catalytic domains with distinct substrate specificities. EMBO J. 1991 Nov;10(11):3231–3237. doi: 10.1002/j.1460-2075.1991.tb04886.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Wegener A. M., Letourneur F., Hoeveler A., Brocker T., Luton F., Malissen B. The T cell receptor/CD3 complex is composed of at least two autonomous transduction modules. Cell. 1992 Jan 10;68(1):83–95. doi: 10.1016/0092-8674(92)90208-t. [DOI] [PubMed] [Google Scholar]
  51. Zheng X. M., Wang Y., Pallen C. J. Cell transformation and activation of pp60c-src by overexpression of a protein tyrosine phosphatase. Nature. 1992 Sep 24;359(6393):336–339. doi: 10.1038/359336a0. [DOI] [PubMed] [Google Scholar]
  52. den Hertog J., Pals C. E., Peppelenbosch M. P., Tertoolen L. G., de Laat S. W., Kruijer W. Receptor protein tyrosine phosphatase alpha activates pp60c-src and is involved in neuronal differentiation. EMBO J. 1993 Oct;12(10):3789–3798. doi: 10.1002/j.1460-2075.1993.tb06057.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. van Oers N. S., Teh S. J., Irving B. A., Tiong J., Weiss A., Teh H. S. Production and characterization of monoclonal antibodies specific for the murine T cell receptor zeta chain. J Immunol Methods. 1994 Apr 15;170(2):261–268. doi: 10.1016/0022-1759(94)90401-4. [DOI] [PubMed] [Google Scholar]

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

RESOURCES