Abstract
While previous studies have implicated the tyrosine protein kinase p60fyn in antigenic activation of T lymphocytes, it is clear that signal transduction initiated through the antigen receptor requires the concerted actions of several proteins. Here, we report our finding that the activation of p60fyn following TcR cross-linking results in the tyrosine phosphorylation of two Fyn-associated proteins of 82 and 116 kDa. In the cells analyzed, p116 appears to represent one of the major substrates of T-cell antigen receptor-mediated tyrosine phosphorylation. In activated T-cells, the interaction of these proteins is specific for p60fyn since neither p56lck nor p62c-yes were found to detectably associate with p82 or p116. Furthermore, the p60fyn-p82/p116 complex could be dissociated and then reconstituted in vitro using purified recombinant Fyn. Using this technique we demonstrated that both p82 and p116 were capable of binding to the Fyn SH2 domain while p82 was to some extent capable of independent binding to the Fyn SH3 domain. An association between p60fyn and phosphoproteins possibly related to the T-cell p82 and p116 was also observed in other hematopoietic cells. Thus, the activation-induced phosphorylation of the p60fyn-associated proteins p116 and p82 and the wide distribution of potentially similar p60fyn-associated proteins in hematopoietic cells suggest that p116 and p82 may play a role as physiological substrates and/or regulators of p60fyn.
Highlights
As a first step torecombinantFynU. sing this technique we demon- ward identifying candidate T-cell antigen receptor (TcR) activation-dependent interacstrated that both p82 and p116 were capable ofbinding tions, we evaluated whether T-cell proteins could be found to to the Fyn S H 2 domain while p82 was to some extent complex with ~ 6 O f f"o~llowing TcR cross-linking
Protein tyrosine phosphorylation following stimulation of t h e T-cell antigen receptor (TcR)l is thought to be mediated by non-receptor tyrosine protein kinases
Similarexperimentsdemonstrated that antibodies to thefollowing proteins were unableto repre- Theresults of experiments described hereindicatethat cipitate phosphoproteins from SDS-dissociated immune com- cross-linking of the TcR results in the activation of Fyn and plexes of ~ 6 6 ~in" :the 110-130 kDa range, GTPase-activating phosphorylation of two majorFyn-associated proteins of 82 and protein, the p125'"" tyrosine protein kinase, the plakoglobin- 116 kDa
Summary
122 kDa from Fyn immune complexes following kinase assay and SDS dissociation (Fig. 6) Among these proteins, p60 and p116 were phosphorylated on tyrosine, whereas p122 was not, as assessed by reprecipitation witha n t i - p w Our first candidate to immunoprecipitates (Fig. 7) This observation argues that the represent p82 was the 85-kDa subunit of phosphatidylinosi- protein abundanceof p155 and/or thelevel of i t s tyrosine phostol-3 kinase, since this protein is knowtonbind to various Src phorylation may be low in the T-cell line studied. Thelevel of tyrosine phosphorylation of the Fyn-assonot shown) These results indicate that the Fyn-associated pc8ia2ted p116 can be increased by "cold kinase assay," i.e. adding is not identical to the 85-kDa a-subunit of phosphatidylinosi- unlabeled ATP to Fyn immune complexes (Fig. 7,A and C ).
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