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ZAP-70 Protein Promotes Tyrosine Phosphorylation of T Cell Receptor Signaling Motifs (ITAMs) in Immature CD4+8+ Thymocytes with Limiting p56lck

Ashe, Jennifer M., Wiest, David L., Abe, Ryo, Singer, Alfred·1999·The Journal of Experimental Medicine
DOI 10.1084/jem.189.7.1163PMC PMC2193013
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Abstract

As a result of interaction with epithelial cells in the thymic cortex, immature CD4+8+ (double positive, DP) thymocytes express relatively few T cell receptors (TCRs) and contain diminished numbers of coreceptor-associated p56lck (lck) PTK molecules. As a result, TCR signal transduction in DP thymocytes is significantly impaired, despite its importance for repertoire selection. We report here that, in DP thymocytes, tyrosine phosphorylation of TCR signaling motifs (ITAMs) by lck, an early event in TCR signal transduction, is dependent upon ZAP-70 protein independent of ZAP-70's kinase activity. Furthermore, the dependence on ZAP-70 protein for ITAM phosphorylation diminishes as available lck increases. Importantly, ZAP-70's role in ITAM phosphorylation in DP thymocytes is not limited to protecting phosphorylated ITAMs from dephosphorylation. Rather, this study indicates that ZAP-70 protein augments ITAM phosphorylation in DP thymocytes and so compensates in part for the relative deficiency of coreceptor-associated lck.

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Figure 1 Constitutive tyrosine phosphorylation of ζ ITAMs in DP thymocytes is dependent upon ZAP-70 protein, but not ZAP-70 kinase activity. Freshly explanted DP thymocytes were obtained from mice expressing different amounts of ZAP-70 protein (5). ST thymocytes express only a kinase-dead ZAP-70 molecule (6). Detergent (1% triton)-solubilized cell lysates were immunoprecipitated, resolved by SDS-PAGE, and immunoblotted as indicated in the figure. The extent of ζ phosphorylation in DP thymocytes correlated with the amount of ZAP-70 protein present, but not with its enzymatic capability.
Figure 1 Constitutive tyrosine phosphorylation of ζ ITAMs in DP thymocytes is dependent upon ZAP-70 protein, but not ZAP-70 kinase activity. Freshly explanted DP thymocytes were obtained from mice expressing different amounts of ZAP-70 protein (5). ST thymocytes express only a kinase-dead ZAP-70 molecule (6). Detergent (1% triton)-solubilized cell lysates were immunoprecipitated, resolved by SDS-PAGE, and immunoblotted as indicated in the figure. The extent of ζ phosphorylation in DP thymocytes correlated with the amount of ZAP-70 protein present, but not with its enzymatic capability.CC BY-NC-SA · PubMed Central
Figure 2 TCR plus CD4–signaled tyrosine phosphorylation of TCR ITAMs in DP thymocytes is dependent upon ZAP-70 both in vivo and in vitro. Freshly explanted DP thymocytes were signaled for 5 min at 37°C with medium, anti–TCR-β plus anti-CD4, or pervanadate (VO4) (13), and the cell lysates immunoprecipitated with anti-ζ. Note that with the exception of the 5 min of signaling, DP thymocytes were strictly maintained at 4°C to prevent the molecular changes that occur upon removal of DP thymocytes from their intrathymic signaling environment (2, 3, 8, 9). Detergent (1% triton) solubilized cell lysates were immunoprecipitated with anti-ζ plus protein A beads which also precipitated the anti–TCR-β and anti-CD4 mAbs used for signaling. In A, immunoprecipitates were resolved by SDS-PAGE and immunoblotted with antiphosphotyrosine to detect phospho-ζ. In B, immunoprecipitates were subjected to an immune complex kinase assay in which in vitro phosphorylation of target TCR ITAMs is indicated by phosphorylation of TCR-ζ and CD3ε chains. Enolase is an exogenously added lck substrate that is tyrosine phosphorylated by lck's enzymatic activity. Note that the immune complex kinase assay was performed in the presence of vanadate, a potent inhibitor of protein tyrosine phosphatases, to prevent tyrosine dephosphorylation. RF, running front.
Figure 2 TCR plus CD4–signaled tyrosine phosphorylation of TCR ITAMs in DP thymocytes is dependent upon ZAP-70 both in vivo and in vitro. Freshly explanted DP thymocytes were signaled for 5 min at 37°C with medium, anti–TCR-β plus anti-CD4, or pervanadate (VO4) (13), and the cell lysates immunoprecipitated with anti-ζ. Note that with the exception of the 5 min of signaling, DP thymocytes were strictly maintained at 4°C to prevent the molecular changes that occur upon removal of DP thymocytes from their intrathymic signaling environment (2, 3, 8, 9). Detergent (1% triton) solubilized cell lysates were immunoprecipitated with anti-ζ plus protein A beads which also precipitated the anti–TCR-β and anti-CD4 mAbs used for signaling. In A, immunoprecipitates were resolved by SDS-PAGE and immunoblotted with antiphosphotyrosine to detect phospho-ζ. In B, immunoprecipitates were subjected to an immune complex kinase assay in which in vitro phosphorylation of target TCR ITAMs is indicated by phosphorylation of TCR-ζ and CD3ε chains. Enolase is an exogenously added lck substrate that is tyrosine phosphorylated by lck's enzymatic activity. Note that the immune complex kinase assay was performed in the presence of vanadate, a potent inhibitor of protein tyrosine phosphatases, to prevent tyrosine dephosphorylation. RF, running front.CC BY-NC-SA · PubMed Central
Figure 3 TCR-induced ζ ITAM phosphorylation in ZAP-70−/− DP thymocytes containing different amounts of coreceptor-associated lck. Freshly explanted ZAP−/− DP thymocytes were obtained from mice differing in MHC II expression and were stimulated in vitro for 5 min. Detergent (1% triton) solubilized cell lysates were immunoprecipitated with either anti-ζ or anti-CD4, resolved by SDS-PAGE, and immunoblotted with the indicated antibodies. DP thymocytes from mice lacking MHC II expression contained increased amounts of CD4-associated lck (row c) and increased ζ phosphorylation upon TCR plus CD4 cocross-linking (row a).
Figure 3 TCR-induced ζ ITAM phosphorylation in ZAP-70−/− DP thymocytes containing different amounts of coreceptor-associated lck. Freshly explanted ZAP−/− DP thymocytes were obtained from mice differing in MHC II expression and were stimulated in vitro for 5 min. Detergent (1% triton) solubilized cell lysates were immunoprecipitated with either anti-ζ or anti-CD4, resolved by SDS-PAGE, and immunoblotted with the indicated antibodies. DP thymocytes from mice lacking MHC II expression contained increased amounts of CD4-associated lck (row c) and increased ζ phosphorylation upon TCR plus CD4 cocross-linking (row a).CC BY-NC-SA · PubMed Central
Figure 4 TCR-induced ζ phosphorylation in cultured DP thymocytes. DP thymocytes that had been cultured overnight in single cell suspension at 37°C were signaled for 5 min. DP thymocytes that have been removed from their intrathymic signaling environment and cultured overnight contain significantly increased amounts of coreceptor-associated lck (2, 3) and exhibit decreased dependence on ZAP-70 for ζ phosphorylation.
Figure 4 TCR-induced ζ phosphorylation in cultured DP thymocytes. DP thymocytes that had been cultured overnight in single cell suspension at 37°C were signaled for 5 min. DP thymocytes that have been removed from their intrathymic signaling environment and cultured overnight contain significantly increased amounts of coreceptor-associated lck (2, 3) and exhibit decreased dependence on ZAP-70 for ζ phosphorylation.CC BY-NC-SA · PubMed Central

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Research works printed but not linked: 15

  1. Van Ewijk, 1980, Distribution of H-2 microenvironments in the mouse thymus. Immunoelectron microscopic identification of I-A and H-2K bearing cells, J Histochem Cytochem, 28, 1089, 10.1177/28.10.6999083 · DOI
  2. Wiest, 1993, Regulation of T cell receptor expression in immature CD4+CD8+ thymocytes by p56lcktyrosine kinase: basis for differential signaling by CD4 and CD8 in immature thymocytes expressing both coreceptor molecules, J Exp Med, 178, 1701, 10.1084/jem.178.5.1701 · DOI
  3. Wiest, 1996, TCR activation of ZAP70 is impaired in CD4+CD8+ thymocytes as a consequence of intrathymic interactions that diminish available p56lck, Immunity, 4, 495, 10.1016/S1074-7613(00)80415-X · DOI
  4. Qian, 1996, Dominant-negative ζ-associated protein 70 inhibits T cell antigen receptor signaling, J Exp Med, 183, 611, 10.1084/jem.183.2.611 · DOI
  5. Negishi, 1995, Essential role for ZAP-70 in both positive and negative selection of thymocytes, Nature, 376, 435, 10.1038/376435a0 · DOI
  6. Wiest, 1997, A spontaneously arising mutation in the DLAARN motif of murine ZAP-70 abrogates kinase activity and arrests thymocyte development, Immunity, 6, 663, 10.1016/S1074-7613(00)80442-2 · DOI
  7. Wange, 1996, Complex complexes: signaling at the TCR, Immunity, 5, 197, 10.1016/S1074-7613(00)80315-5 · DOI
  8. Nakayama, 1990, Inhibition of T cell receptor expression and function in immature CD4+CD8+cells by CD4, Science, 249, 1558, 10.1126/science.2120773 · DOI
  9. Nakayama, 1989, Intrathymic signalling in immature CD4+CD8+thymocytes results in tyrosine phosphorylation of the T-cell receptor zeta chain, Nature, 341, 651, 10.1038/341651a0 · DOI
  10. van Oers, 1994, ZAP-70 is constitutively associated with tyrosine-phosphorylated TCR zeta in murine thymocytes and lymph node T cells, Immunity, 1, 675, 10.1016/1074-7613(94)90038-8 · DOI
  11. van Oers, 1996, Lck regulates the tyrosine phosphorylation of the T cell receptor subunits and ZAP-70 in murine thymocytes, J Exp Med, 183, 1053, 10.1084/jem.183.3.1053 · DOI
  12. Iwashima, 1994, Sequential interactions of the TCR with two distinct cytoplasmic tyrosine kinases, Science, 263, 1136, 10.1126/science.7509083 · DOI
  13. Secrist, 1993, Stimulatory effects of the protein tyrosine phosphatase inhibitor, pervanadate, on T-cell activation events, J Biol Chem, 268, 5886, 10.1016/S0021-9258(18)53403-7 · DOI
  14. Duplay, 1994, p56lckinteracts via its src homology 2 domain with the ZAP-70 kinase, J Exp Med, 179, 1163, 10.1084/jem.179.4.1163 · DOI
  15. Straus, 1996, SH2 domain function is essential for the role of the Lck tyrosine kinase in T cell receptor signal transduction, J Biol Chem, 271, 9976, 10.1074/jbc.271.17.9976 · DOI

Cited by

Metadata

Authors
Ashe, J. M., Wiest, D. L., Abe, R., & Singer, A.
Affiliations
From the *Experimental Immunology Branch, National Cancer Institute, Bethesda, Maryland 20892; ‡Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111; and the §Division of Immunobiology, Research Institute for Biological Sciences, Science University of  Tokyo, Chiba 278,  Japan
Venue
The Journal of Experimental Medicine
Publisher
Rockefeller University Press
Published
1999-04-05
Volume
189
Issue
7
Pages
1163-1168
ISSN
  • 0022-1007
  • 1540-9538
DOI
10.1084/jem.189.7.1163
PMC
PMC2193013
Cited by
26
References
0
Language
en

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