Abstract
Exposure of cells to H2O2 mimics many of the effects of treatment of cells with extracellular ligands. Among these is the stimulation of tyrosine phosphorylation. In this study, we show that exposure of cells to H2O2 increases the catalytic activity of the lymphocyte-specific tyrosine protein kinase p56lck (Lck) and induces tyrosine phosphorylation of Lck at Tyr-394, the autophosphorylation site. Using mutant forms of Lck, we found that Tyr-394 is required for H2O2-induced activation of Lck, suggesting that phosphorylation of this site may activate Lck. In addition, H2O2 treatment induced phosphorylation at Tyr-394 in a catalytically inactive mutant of Lck in cells that do not express endogenous Lck. This demonstrates that a kinase other than Lck itself is capable of phosphorylating Lck at the so-called autophosphorylation site and raises the possibility that this as yet unidentified tyrosine protein kinase functions as an activator of Lck. Such an activating enzyme could play an important role in signal transduction in T cells.
Full text
PDF




Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Allen J. M., Forbush K. A., Perlmutter R. M. Functional dissection of the lck proximal promoter. Mol Cell Biol. 1992 Jun;12(6):2758–2768. doi: 10.1128/mcb.12.6.2758. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Amrein K. E., Sefton B. M. Mutation of a site of tyrosine phosphorylation in the lymphocyte-specific tyrosine protein kinase, p56lck, reveals its oncogenic potential in fibroblasts. Proc Natl Acad Sci U S A. 1988 Jun;85(12):4247–4251. doi: 10.1073/pnas.85.12.4247. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bergman M., Mustelin T., Oetken C., Partanen J., Flint N. A., Amrein K. E., Autero M., Burn P., Alitalo K. The human p50csk tyrosine kinase phosphorylates p56lck at Tyr-505 and down regulates its catalytic activity. EMBO J. 1992 Aug;11(8):2919–2924. doi: 10.1002/j.1460-2075.1992.tb05361.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Boulet I., Fagard R., Fischer S. Correlation between phosphorylation and kinase activity of a tyrosine protein kinase: p56lck. Biochem Biophys Res Commun. 1987 Nov 30;149(1):56–64. doi: 10.1016/0006-291x(87)91604-4. [DOI] [PubMed] [Google Scholar]
- Caron L., Abraham N., Pawson T., Veillette A. Structural requirements for enhancement of T-cell responsiveness by the lymphocyte-specific tyrosine protein kinase p56lck. Mol Cell Biol. 1992 Jun;12(6):2720–2729. doi: 10.1128/mcb.12.6.2720. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Casnellie J. E., Harrison M. L., Hellstrom K. E., Krebs E. G. A lymphoma protein with an in vitro site of tyrosine phosphorylation homologous to that in pp60src. J Biol Chem. 1982 Dec 10;257(23):13877–13879. [PubMed] [Google Scholar]
- Collins T. L., Burakoff S. J. Tyrosine kinase activity of CD4-associated p56lck may not be required for CD4-dependent T-cell activation. Proc Natl Acad Sci U S A. 1993 Dec 15;90(24):11885–11889. doi: 10.1073/pnas.90.24.11885. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cooper J. A. Estimation of phosphorylation stoichiometry by separation of phosphorylated isoforms. Methods Enzymol. 1991;201:251–261. doi: 10.1016/0076-6879(91)01023-u. [DOI] [PubMed] [Google Scholar]
- Cooper J. A., MacAuley A. Potential positive and negative autoregulation of p60c-src by intermolecular autophosphorylation. Proc Natl Acad Sci U S A. 1988 Jun;85(12):4232–4236. doi: 10.1073/pnas.85.12.4232. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Couture C., Baier G., Oetken C., Williams S., Telford D., Marie-Cardine A., Baier-Bitterlich G., Fischer S., Burn P., Altman A. Activation of p56lck by p72syk through physical association and N-terminal tyrosine phosphorylation. Mol Cell Biol. 1994 Aug;14(8):5249–5258. doi: 10.1128/mcb.14.8.5249. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Devary Y., Gottlieb R. A., Smeal T., Karin M. The mammalian ultraviolet response is triggered by activation of Src tyrosine kinases. Cell. 1992 Dec 24;71(7):1081–1091. doi: 10.1016/s0092-8674(05)80058-3. [DOI] [PubMed] [Google Scholar]
- 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]
- Heffetz D., Bushkin I., Dror R., Zick Y. The insulinomimetic agents H2O2 and vanadate stimulate protein tyrosine phosphorylation in intact cells. J Biol Chem. 1990 Feb 15;265(5):2896–2902. [PubMed] [Google Scholar]
- Hunter T., Sefton B. M. Transforming gene product of Rous sarcoma virus phosphorylates tyrosine. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1311–1315. doi: 10.1073/pnas.77.3.1311. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hurley T. R., Amrein K. E., Sefton B. M. Creation and characterization of temperature-sensitive mutants of the lck tyrosine protein kinase. J Virol. 1992 Dec;66(12):7406–7413. doi: 10.1128/jvi.66.12.7406-7413.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Karnitz L., Sutor S. L., Torigoe T., Reed J. C., Bell M. P., McKean D. J., Leibson P. J., Abraham R. T. Effects of p56lck deficiency on the growth and cytolytic effector function of an interleukin-2-dependent cytotoxic T-cell line. Mol Cell Biol. 1992 Oct;12(10):4521–4530. doi: 10.1128/mcb.12.10.4521. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kmiecik T. E., Johnson P. J., Shalloway D. Regulation by the autophosphorylation site in overexpressed pp60c-src. Mol Cell Biol. 1988 Oct;8(10):4541–4546. doi: 10.1128/mcb.8.10.4541. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kmiecik T. E., Shalloway D. Activation and suppression of pp60c-src transforming ability by mutation of its primary sites of tyrosine phosphorylation. Cell. 1987 Apr 10;49(1):65–73. doi: 10.1016/0092-8674(87)90756-2. [DOI] [PubMed] [Google Scholar]
- Marth J. D., Cooper J. A., King C. S., Ziegler S. F., Tinker D. A., Overell R. W., Krebs E. G., Perlmutter R. M. Neoplastic transformation induced by an activated lymphocyte-specific protein tyrosine kinase (pp56lck). Mol Cell Biol. 1988 Feb;8(2):540–550. doi: 10.1128/mcb.8.2.540. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Miller A. D., Rosman G. J. Improved retroviral vectors for gene transfer and expression. Biotechniques. 1989 Oct;7(9):980-2, 984-6, 989-90. [PMC free article] [PubMed] [Google Scholar]
- Molina T. J., Kishihara K., Siderovski D. P., van Ewijk W., Narendran A., Timms E., Wakeham A., Paige C. J., Hartmann K. U., Veillette A. Profound block in thymocyte development in mice lacking p56lck. Nature. 1992 May 14;357(6374):161–164. doi: 10.1038/357161a0. [DOI] [PubMed] [Google Scholar]
- Morikawa S., Tatsumi E., Baba M., Harada T., Yasuhira K. Two E-rosette-forming lymphoid cell lines. Int J Cancer. 1978 Feb 15;21(2):166–170. doi: 10.1002/ijc.2910210207. [DOI] [PubMed] [Google Scholar]
- Muller A. J., Young J. C., Pendergast A. M., Pondel M., Landau N. R., Littman D. R., Witte O. N. BCR first exon sequences specifically activate the BCR/ABL tyrosine kinase oncogene of Philadelphia chromosome-positive human leukemias. Mol Cell Biol. 1991 Apr;11(4):1785–1792. doi: 10.1128/mcb.11.4.1785. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mustelin T., Coggeshall K. M., Altman A. Rapid activation of the T-cell tyrosine protein kinase pp56lck by the CD45 phosphotyrosine phosphatase. Proc Natl Acad Sci U S A. 1989 Aug;86(16):6302–6306. doi: 10.1073/pnas.86.16.6302. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nakamura K., Hori T., Sato N., Sugie K., Kawakami T., Yodoi J. Redox regulation of a src family protein tyrosine kinase p56lck in T cells. Oncogene. 1993 Nov;8(11):3133–3139. [PubMed] [Google Scholar]
- O'Shea J. J., McVicar D. W., Bailey T. L., Burns C., Smyth M. J. Activation of human peripheral blood T lymphocytes by pharmacological induction of protein-tyrosine phosphorylation. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10306–10310. doi: 10.1073/pnas.89.21.10306. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Ostergaard H. L., Trowbridge I. S. Coclustering CD45 with CD4 or CD8 alters the phosphorylation and kinase activity of p56lck. J Exp Med. 1990 Jul 1;172(1):347–350. doi: 10.1084/jem.172.1.347. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Reynolds P. J., Hurley T. R., Sefton B. M. Functional analysis of the SH2 and SH3 domains of the lck tyrosine protein kinase. Oncogene. 1992 Oct;7(10):1949–1955. [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]
- Schieven G. L., Kirihara J. M., Burg D. L., Geahlen R. L., Ledbetter J. A. p72syk tyrosine kinase is activated by oxidizing conditions that induce lymphocyte tyrosine phosphorylation and Ca2+ signals. J Biol Chem. 1993 Aug 5;268(22):16688–16692. [PubMed] [Google Scholar]
- Schieven G. L., Kirihara J. M., Myers D. E., Ledbetter J. A., Uckun F. M. Reactive oxygen intermediates activate NF-kappa B in a tyrosine kinase-dependent mechanism and in combination with vanadate activate the p56lck and p59fyn tyrosine kinases in human lymphocytes. Blood. 1993 Aug 15;82(4):1212–1220. [PubMed] [Google Scholar]
- Schreck R., Rieber P., Baeuerle P. A. Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-kappa B transcription factor and HIV-1. EMBO J. 1991 Aug;10(8):2247–2258. doi: 10.1002/j.1460-2075.1991.tb07761.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Secrist J. P., Burns L. A., Karnitz L., Koretzky G. A., Abraham R. T. Stimulatory effects of the protein tyrosine phosphatase inhibitor, pervanadate, on T-cell activation events. J Biol Chem. 1993 Mar 15;268(8):5886–5893. [PubMed] [Google Scholar]
- Sefton B. M., Beemon K., Hunter T. Comparison of the expression of the src gene of Rous sarcoma virus in vitro and in vivo. J Virol. 1978 Dec;28(3):957–971. doi: 10.1128/jvi.28.3.957-971.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sieh M., Bolen J. B., Weiss A. CD45 specifically modulates binding of Lck to a phosphopeptide encompassing the negative regulatory tyrosine of Lck. EMBO J. 1993 Jan;12(1):315–321. doi: 10.1002/j.1460-2075.1993.tb05659.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stevenson M. A., Pollock S. S., Coleman C. N., Calderwood S. K. X-irradiation, phorbol esters, and H2O2 stimulate mitogen-activated protein kinase activity in NIH-3T3 cells through the formation of reactive oxygen intermediates. Cancer Res. 1994 Jan 1;54(1):12–15. [PubMed] [Google Scholar]
- Taddie J. A., Hurley T. R., Hardwick B. S., Sefton B. M. Activation of B- and T-cells by the cytoplasmic domains of the B-cell antigen receptor proteins Ig-alpha and Ig-beta. J Biol Chem. 1994 May 6;269(18):13529–13535. [PubMed] [Google Scholar]
- Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [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]
- Veillette A., Fournel M. The CD4 associated tyrosine protein kinase p56lck is positively regulated through its site of autophosphorylation. Oncogene. 1990 Oct;5(10):1455–1462. [PubMed] [Google Scholar]
- Voronova A. F., Buss J. E., Patschinsky T., Hunter T., Sefton B. M. Characterization of the protein apparently responsible for the elevated tyrosine protein kinase activity in LSTRA cells. Mol Cell Biol. 1984 Dec;4(12):2705–2713. doi: 10.1128/mcb.4.12.2705. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Voronova A. F., Sefton B. M. Expression of a new tyrosine protein kinase is stimulated by retrovirus promoter insertion. Nature. 1986 Feb 20;319(6055):682–685. doi: 10.1038/319682a0. [DOI] [PubMed] [Google Scholar]