Accumulating data show that the tyrosine protein kinase Zap-70 plays an essential role in T cell receptor-mediated signal transduction. However, the model of action, as well as the physiologically relevant substrates of Zap-70, have not been determined. We have attempted to identify a 120-kD tyrosine-phosphorylated protein (p120) that associates with Zap-70 in activated T lymphocytes. The results of our analyses showed that p120 is largely encoded by the c-cbl protooncogene. Furthermore, the association of Zap-70 with c-Cbl was shown to be induced by T cell receptor stimulation, implying that it required posttranslational modification of one or both of these products. FynT, but not Lck, also associated with c-Cbl in activated T cells. Finally, using a heterologous system, it was demonstrated that the ability of Zap-70 to cause tyrosine phosphorylation of p120c-cbl was dependent on Lck- or FynT-mediated signals. As c-Cbl can associate with several other signaling molecules, it may couple Zap-70 to downstream effectors during T cell activation.

Recent observations suggest that the src-related tyrosine protein kinase p59fyn may be involved in antigen-induced T lymphocyte activation. As a result of alternative splicing, p59fyn exists as two isoforms that differ exclusively within a short sequence spanning the end of the Src Homology 2 (SH2) region and the beginning of the tyrosine protein kinase domain. While one p59fyn isoform (fynB) is highly expressed in brain, the alternative product (fynT) is principally found in T lymphocytes. To further understand the role of p59fyn in T cell activation and to test the hypothesis that p59fynT serves a tissue-specific function in T lymphocytes, we have examined the effects of expression of activated versions (tyrosine 528 to phenylalanine 528 mutants) of either form of p59fyn on the physiology of an antigen-specific mouse T cell hybridoma. Our results demonstrated that the two forms of fyn, expressed in equivalent amounts, efficiently enhanced antibody-induced T cell receptor (TCR)-mediated signals. In contrast, only p59fynT increased interleukin 2 production in response to antigen stimulation. This finding implies that the distinct p59fyn isoform expressed in T lymphocytes regulates the coupling of TCR stimulation by antigen/major histocompatibility complex to lymphokine production.

Accumulating data suggest that the target cells for selection events leading to establishment of the mature T cell repertoire are the functionally immature double-positive (CD4+/CD8+) thymocytes, and that the CD4 and CD8 antigens expressed on these cells play important roles in these processes. In an attempt to define the biochemical pathways implicated in these events, we have studied the effects of engagement of accessory molecules on tyrosine protein phosphorylation. The results of our experiments demonstrate that engagement of CD4 and CD8 expressed on double-positive thymocytes is coupled with a rapid tyrosine protein phosphorylation signal. Further analyses have revealed that these two surface molecules are physically associated with the internal membrane tyrosine protein kinase p56lck in immature thymocytes, and that the catalytic function of lck expressed in double-positive thymocytes is significantly enhanced upon engagement of CD4. These data provide evidence that tyrosine-specific protein phosphorylation pathways coupled to the CD4 and CD8 T cell surface antigens are functional in immature thymocytes, and therefore, formally prove that signaling functions of CD4 and CD8 molecules are operative in immature thymocytes.