Abstract
T-cell activation requires cooperative signals generated by the T-cell antigen receptor zeta-chain complex (TCR zeta-CD3) and the costimulatory antigen CD28. CD28 interacts with three intracellular proteins-phosphatidylinositol 3-kinase (PI 3-kinase), T cell-specific protein-tyrosine kinase ITK (formerly TSK or EMT), and the complex between growth factor receptor-bound protein 2 and son of sevenless guanine nucleotide exchange protein (GRB-2-SOS). PI 3-kinase and GRB-2 bind to the CD28 phosphotyrosine-based Tyr-Met-Asn-Met motif by means of intrinsic Src-homology 2 (SH2) domains. The requirement for tyrosine phosphorylation of the Tyr-Met-Asn-Met motif for SH2 domain binding implicates an intervening protein-tyrosine kinase in the recruitment of PI 3-kinase and GRB-2 by CD28. Candidate kinases include p56Lck, p59Fyn, zeta-chain-associated 70-kDa protein (ZAP-70), and ITK. In this study, we demonstrate in coexpression studies that p56Lck and p59Fyn phosphorylate CD28 primarily at Tyr-191 of the Tyr-Met-Asn-Met motif, inducing a 3- to 8-fold increase in p85 (subunit of PI 3-kinase) and GRB-2 SH2 binding to CD28. Phosphatase digestion of CD28 eliminated binding. In contrast to Src kinases, ZAP-70 and ITK failed to induce these events. Further, ITK binding to CD28 was dependent on the presence of p56Lck and is thus likely to act downstream of p56Lck/p59Fyn in a signaling cascade. p56Lck is therefore likely to be a central switch in T-cell activation, with the dual function of regulating CD28-mediated costimulation as well as TCR-CD3-CD4 signaling.
Full text
PDF![8891](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c31d/41073/99ebc3b5708c/pnas01497-0368.png)
![8892](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c31d/41073/a88d2231bae4/pnas01497-0369.png)
![8893](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c31d/41073/bcd0890dc796/pnas01497-0370.png)
![8894](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c31d/41073/6bbc454fcdea/pnas01497-0371.png)
![8895](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c31d/41073/d2211bacab7f/pnas01497-0372.png)
Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- August A., Dupont B. Activation of src family kinase lck following CD28 crosslinking in the Jurkat leukemic cell line. Biochem Biophys Res Commun. 1994 Mar 30;199(3):1466–1473. doi: 10.1006/bbrc.1994.1396. [DOI] [PubMed] [Google Scholar]
- August A., Dupont B. CD28 of T lymphocytes associates with phosphatidylinositol 3-kinase. Int Immunol. 1994 May;6(5):769–774. doi: 10.1093/intimm/6.5.769. [DOI] [PubMed] [Google Scholar]
- August A., Gibson S., Kawakami Y., Kawakami T., Mills G. B., Dupont B. CD28 is associated with and induces the immediate tyrosine phosphorylation and activation of the Tec family kinase ITK/EMT in the human Jurkat leukemic T-cell line. Proc Natl Acad Sci U S A. 1994 Sep 27;91(20):9347–9351. doi: 10.1073/pnas.91.20.9347. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Azuma M., Ito D., Yagita H., Okumura K., Phillips J. H., Lanier L. L., Somoza C. B70 antigen is a second ligand for CTLA-4 and CD28. Nature. 1993 Nov 4;366(6450):76–79. doi: 10.1038/366076a0. [DOI] [PubMed] [Google Scholar]
- Baskar S., Ostrand-Rosenberg S., Nabavi N., Nadler L. M., Freeman G. J., Glimcher L. H. Constitutive expression of B7 restores immunogenicity of tumor cells expressing truncated major histocompatibility complex class II molecules. Proc Natl Acad Sci U S A. 1993 Jun 15;90(12):5687–5690. doi: 10.1073/pnas.90.12.5687. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Boussiotis V. A., Freeman G. J., Gribben J. G., Daley J., Gray G., Nadler L. M. Activated human B lymphocytes express three CTLA-4 counterreceptors that costimulate T-cell activation. Proc Natl Acad Sci U S A. 1993 Dec 1;90(23):11059–11063. doi: 10.1073/pnas.90.23.11059. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Carpenter C. L., Duckworth B. C., Auger K. R., Cohen B., Schaffhausen B. S., Cantley L. C. Purification and characterization of phosphoinositide 3-kinase from rat liver. J Biol Chem. 1990 Nov 15;265(32):19704–19711. [PubMed] [Google Scholar]
- Chen L., Ashe S., Brady W. A., Hellström I., Hellström K. E., Ledbetter J. A., McGowan P., Linsley P. S. Costimulation of antitumor immunity by the B7 counterreceptor for the T lymphocyte molecules CD28 and CTLA-4. Cell. 1992 Dec 24;71(7):1093–1102. doi: 10.1016/s0092-8674(05)80059-5. [DOI] [PubMed] [Google Scholar]
- Chung J., Grammer T. C., Lemon K. P., Kazlauskas A., Blenis J. PDGF- and insulin-dependent pp70S6k activation mediated by phosphatidylinositol-3-OH kinase. Nature. 1994 Jul 7;370(6484):71–75. doi: 10.1038/370071a0. [DOI] [PubMed] [Google Scholar]
- Crews C. M., Erikson R. L. Extracellular signals and reversible protein phosphorylation: what to Mek of it all. Cell. 1993 Jul 30;74(2):215–217. doi: 10.1016/0092-8674(93)90411-i. [DOI] [PubMed] [Google Scholar]
- Downward J., Graves J. D., Warne P. H., Rayter S., Cantrell D. A. Stimulation of p21ras upon T-cell activation. Nature. 1990 Aug 23;346(6286):719–723. doi: 10.1038/346719a0. [DOI] [PubMed] [Google Scholar]
- Fraser J. D., Irving B. A., Crabtree G. R., Weiss A. Regulation of interleukin-2 gene enhancer activity by the T cell accessory molecule CD28. Science. 1991 Jan 18;251(4991):313–316. doi: 10.1126/science.1846244. [DOI] [PubMed] [Google Scholar]
- Freeman G. J., Borriello F., Hodes R. J., Reiser H., Hathcock K. S., Laszlo G., McKnight A. J., Kim J., Du L., Lombard D. B. Uncovering of functional alternative CTLA-4 counter-receptor in B7-deficient mice. Science. 1993 Nov 5;262(5135):907–909. doi: 10.1126/science.7694362. [DOI] [PubMed] [Google Scholar]
- Freeman G. J., Gribben J. G., Boussiotis V. A., Ng J. W., Restivo V. A., Jr, Lombard L. A., Gray G. S., Nadler L. M. Cloning of B7-2: a CTLA-4 counter-receptor that costimulates human T cell proliferation. Science. 1993 Nov 5;262(5135):909–911. doi: 10.1126/science.7694363. [DOI] [PubMed] [Google Scholar]
- Gibson S., Leung B., Squire J. A., Hill M., Arima N., Goss P., Hogg D., Mills G. B. Identification, cloning, and characterization of a novel human T-cell-specific tyrosine kinase located at the hematopoietin complex on chromosome 5q. Blood. 1993 Sep 1;82(5):1561–1572. [PubMed] [Google Scholar]
- Guerder S., Meyerhoff J., Flavell R. The role of the T cell costimulator B7-1 in autoimmunity and the induction and maintenance of tolerance to peripheral antigen. Immunity. 1994 May;1(2):155–166. doi: 10.1016/1074-7613(94)90109-0. [DOI] [PubMed] [Google Scholar]
- Harlan D. M., Hengartner H., Huang M. L., Kang Y. H., Abe R., Moreadith R. W., Pircher H., Gray G. S., Ohashi P. S., Freeman G. J. Mice expressing both B7-1 and viral glycoprotein on pancreatic beta cells along with glycoprotein-specific transgenic T cells develop diabetes due to a breakdown of T-lymphocyte unresponsiveness. Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):3137–3141. doi: 10.1073/pnas.91.8.3137. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hathcock K. S., Laszlo G., Dickler H. B., Bradshaw J., Linsley P., Hodes R. J. Identification of an alternative CTLA-4 ligand costimulatory for T cell activation. Science. 1993 Nov 5;262(5135):905–907. doi: 10.1126/science.7694361. [DOI] [PubMed] [Google Scholar]
- Heyeck S. D., Berg L. J. Developmental regulation of a murine T-cell-specific tyrosine kinase gene, Tsk. Proc Natl Acad Sci U S A. 1993 Jan 15;90(2):669–673. doi: 10.1073/pnas.90.2.669. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hutchcroft J. E., Bierer B. E. Activation-dependent phosphorylation of the T-lymphocyte surface receptor CD28 and associated proteins. Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):3260–3264. doi: 10.1073/pnas.91.8.3260. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Jenkins M. K., Ashwell J. D., Schwartz R. H. Allogeneic non-T spleen cells restore the responsiveness of normal T cell clones stimulated with antigen and chemically modified antigen-presenting cells. J Immunol. 1988 May 15;140(10):3324–3330. [PubMed] [Google Scholar]
- June C. H., Bluestone J. A., Nadler L. M., Thompson C. B. The B7 and CD28 receptor families. Immunol Today. 1994 Jul;15(7):321–331. doi: 10.1016/0167-5699(94)90080-9. [DOI] [PubMed] [Google Scholar]
- Kolanus W., Romeo C., Seed B. T cell activation by clustered tyrosine kinases. Cell. 1993 Jul 16;74(1):171–183. doi: 10.1016/0092-8674(93)90304-9. [DOI] [PubMed] [Google Scholar]
- Koulova L., Clark E. A., Shu G., Dupont B. The CD28 ligand B7/BB1 provides costimulatory signal for alloactivation of CD4+ T cells. J Exp Med. 1991 Mar 1;173(3):759–762. doi: 10.1084/jem.173.3.759. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Li N., Batzer A., Daly R., Yajnik V., Skolnik E., Chardin P., Bar-Sagi D., Margolis B., Schlessinger J. Guanine-nucleotide-releasing factor hSos1 binds to Grb2 and links receptor tyrosine kinases to Ras signalling. Nature. 1993 May 6;363(6424):85–88. doi: 10.1038/363085a0. [DOI] [PubMed] [Google Scholar]
- Lindstein T., June C. H., Ledbetter J. A., Stella G., Thompson C. B. Regulation of lymphokine messenger RNA stability by a surface-mediated T cell activation pathway. Science. 1989 Apr 21;244(4902):339–343. doi: 10.1126/science.2540528. [DOI] [PubMed] [Google Scholar]
- Linsley P. S., Ledbetter J. A. The role of the CD28 receptor during T cell responses to antigen. Annu Rev Immunol. 1993;11:191–212. doi: 10.1146/annurev.iy.11.040193.001203. [DOI] [PubMed] [Google Scholar]
- Lu Y., Granelli-Piperno A., Bjorndahl J. M., Phillips C. A., Trevillyan J. M. CD28-induced T cell activation. Evidence for a protein-tyrosine kinase signal transduction pathway. J Immunol. 1992 Jul 1;149(1):24–29. [PubMed] [Google Scholar]
- Mizoguchi H., O'Shea J. J., Longo D. L., Loeffler C. M., McVicar D. W., Ochoa A. C. Alterations in signal transduction molecules in T lymphocytes from tumor-bearing mice. Science. 1992 Dec 11;258(5089):1795–1798. doi: 10.1126/science.1465616. [DOI] [PubMed] [Google Scholar]
- Nakanishi H., Brewer K. A., Exton J. H. Activation of the zeta isozyme of protein kinase C by phosphatidylinositol 3,4,5-trisphosphate. J Biol Chem. 1993 Jan 5;268(1):13–16. [PubMed] [Google Scholar]
- Nunès J. A., Collette Y., Truneh A., Olive D., Cantrell D. A. The role of p21ras in CD28 signal transduction: triggering of CD28 with antibodies, but not the ligand B7-1, activates p21ras. J Exp Med. 1994 Sep 1;180(3):1067–1076. doi: 10.1084/jem.180.3.1067. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pagès F., Ragueneau M., Rottapel R., Truneh A., Nunes J., Imbert J., Olive D. Binding of phosphatidylinositol-3-OH kinase to CD28 is required for T-cell signalling. Nature. 1994 May 26;369(6478):327–329. doi: 10.1038/369327a0. [DOI] [PubMed] [Google Scholar]
- Prasad K. V., Cai Y. C., Raab M., Duckworth B., Cantley L., Shoelson S. E., Rudd C. E. T-cell antigen CD28 interacts with the lipid kinase phosphatidylinositol 3-kinase by a cytoplasmic Tyr(P)-Met-Xaa-Met motif. Proc Natl Acad Sci U S A. 1994 Mar 29;91(7):2834–2838. doi: 10.1073/pnas.91.7.2834. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Prasad K. V., Janssen O., Kapeller R., Raab M., Cantley L. C., Rudd C. E. Src-homology 3 domain of protein kinase p59fyn mediates binding to phosphatidylinositol 3-kinase in T cells. Proc Natl Acad Sci U S A. 1993 Aug 1;90(15):7366–7370. doi: 10.1073/pnas.90.15.7366. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Prasad K. V., Kapeller R., Janssen O., Repke H., Duke-Cohan J. S., Cantley L. C., Rudd C. E. Phosphatidylinositol (PI) 3-kinase and PI 4-kinase binding to the CD4-p56lck complex: the p56lck SH3 domain binds to PI 3-kinase but not PI 4-kinase. Mol Cell Biol. 1993 Dec;13(12):7708–7717. doi: 10.1128/mcb.13.12.7708. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Raab M., Yamamoto M., Rudd C. E. The T-cell antigen CD5 acts as a receptor and substrate for the protein-tyrosine kinase p56lck. Mol Cell Biol. 1994 May;14(5):2862–2870. doi: 10.1128/mcb.14.5.2862. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Roy L. M., Swenson K. I., Walker D. H., Gabrielli B. G., Li R. S., Piwnica-Worms H., Maller J. L. Activation of p34cdc2 kinase by cyclin A. J Cell Biol. 1991 May;113(3):507–514. doi: 10.1083/jcb.113.3.507. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rudd C. E., Janssen O., Cai Y. C., da Silva A. J., Raab M., Prasad K. V. Two-step TCR zeta/CD3-CD4 and CD28 signaling in T cells: SH2/SH3 domains, protein-tyrosine and lipid kinases. Immunol Today. 1994 May;15(5):225–234. doi: 10.1016/0167-5699(94)90248-8. [DOI] [PubMed] [Google Scholar]
- Rudd C. E., Trevillyan J. M., Dasgupta J. D., Wong L. L., Schlossman S. F. The CD4 receptor is complexed in detergent lysates to a protein-tyrosine kinase (pp58) from human T lymphocytes. Proc Natl Acad Sci U S A. 1988 Jul;85(14):5190–5194. doi: 10.1073/pnas.85.14.5190. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Samelson L. E., Klausner R. D. Tyrosine kinases and tyrosine-based activation motifs. Current research on activation via the T cell antigen receptor. J Biol Chem. 1992 Dec 15;267(35):24913–24916. [PubMed] [Google Scholar]
- Schneider H., Cai Y. C., Prasad K. V., Shoelson S. E., Rudd C. E. T cell antigen CD28 binds to the GRB-2/SOS complex, regulators of p21ras. Eur J Immunol. 1995 Apr;25(4):1044–1050. doi: 10.1002/eji.1830250428. [DOI] [PubMed] [Google Scholar]
- Schwartz R. H. Costimulation of T lymphocytes: the role of CD28, CTLA-4, and B7/BB1 in interleukin-2 production and immunotherapy. Cell. 1992 Dec 24;71(7):1065–1068. doi: 10.1016/s0092-8674(05)80055-8. [DOI] [PubMed] [Google Scholar]
- Shahinian A., Pfeffer K., Lee K. P., Kündig T. M., Kishihara K., Wakeham A., Kawai K., Ohashi P. S., Thompson C. B., Mak T. W. Differential T cell costimulatory requirements in CD28-deficient mice. Science. 1993 Jul 30;261(5121):609–612. doi: 10.1126/science.7688139. [DOI] [PubMed] [Google Scholar]
- Siliciano J. D., Morrow T. A., Desiderio S. V. itk, a T-cell-specific tyrosine kinase gene inducible by interleukin 2. Proc Natl Acad Sci U S A. 1992 Dec 1;89(23):11194–11198. doi: 10.1073/pnas.89.23.11194. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Simon M. A., Dodson G. S., Rubin G. M. An SH3-SH2-SH3 protein is required for p21Ras1 activation and binds to sevenless and Sos proteins in vitro. Cell. 1993 Apr 9;73(1):169–177. doi: 10.1016/0092-8674(93)90169-q. [DOI] [PubMed] [Google Scholar]
- Su B., Jacinto E., Hibi M., Kallunki T., Karin M., Ben-Neriah Y. JNK is involved in signal integration during costimulation of T lymphocytes. Cell. 1994 Jun 3;77(5):727–736. doi: 10.1016/0092-8674(94)90056-6. [DOI] [PubMed] [Google Scholar]
- Townsend S. E., Allison J. P. Tumor rejection after direct costimulation of CD8+ T cells by B7-transfected melanoma cells. Science. 1993 Jan 15;259(5093):368–370. doi: 10.1126/science.7678351. [DOI] [PubMed] [Google Scholar]
- Truitt K. E., Hicks C. M., Imboden J. B. Stimulation of CD28 triggers an association between CD28 and phosphatidylinositol 3-kinase in Jurkat T cells. J Exp Med. 1994 Mar 1;179(3):1071–1076. doi: 10.1084/jem.179.3.1071. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Veillette A., Bookman M. A., Horak E. M., Bolen J. B. The CD4 and CD8 T cell surface antigens are associated with the internal membrane tyrosine-protein kinase p56lck. Cell. 1988 Oct 21;55(2):301–308. doi: 10.1016/0092-8674(88)90053-0. [DOI] [PubMed] [Google Scholar]
- Verwilghen J., Vandenberghe P., Wallays G., de Boer M., Anthony N., Panayi G. S., Ceuppens J. L. Simultaneous ligation of CD5 and CD28 on resting T lymphocytes induces T cell activation in the absence of T cell receptor/CD3 occupancy. J Immunol. 1993 Feb 1;150(3):835–846. [PubMed] [Google Scholar]
- Ward S. G., Westwick J., Hall N. D., Sansom D. M. Ligation of CD28 receptor by B7 induces formation of D-3 phosphoinositides in T lymphocytes independently of T cell receptor/CD3 activation. Eur J Immunol. 1993 Oct;23(10):2572–2577. doi: 10.1002/eji.1830231029. [DOI] [PubMed] [Google Scholar]
- Weiss A., Littman D. R. Signal transduction by lymphocyte antigen receptors. Cell. 1994 Jan 28;76(2):263–274. doi: 10.1016/0092-8674(94)90334-4. [DOI] [PubMed] [Google Scholar]
- de Boer M., Kasran A., Kwekkeboom J., Walter H., Vandenberghe P., Ceuppens J. L. Ligation of B7 with CD28/CTLA-4 on T cells results in CD40 ligand expression, interleukin-4 secretion and efficient help for antibody production by B cells. Eur J Immunol. 1993 Dec;23(12):3120–3125. doi: 10.1002/eji.1830231212. [DOI] [PubMed] [Google Scholar]