Abstract
A proximal and critical biochemical event upon T cell antigen receptor (TCR) stimulation is the activation of a protein tyrosine kinase (PTK) pathway. ZAP-70, a PTK of the p72syk family, associates with phosphorylated TCR subunits upon TCR stimulation. Here we report that the tandem SH2 domains of ZAP-70, expressed as a fusion protein, bind to tyrosine-phosphorylated CD3 epsilon and TCR zeta from activated Jurkat T cell lysates. The single N- and C-terminal SH2 domains of ZAP-70, expressed separately, do not bind these TCR subunits. In comparison to fusion proteins containing SH2 domains from other proteins, the tandem SH2 domains of ZAP-70 demonstrate a remarkably restricted repertoire of protein binding, binding only TCR zeta and CD3 epsilon. ZAP-70 is also recovered in the binding assay, but this is likely to be a consequence of its interaction with multiple SH2 binding sites on the zeta-zeta and CD3 epsilon-containing dimers.
Highlights
A proximal and critical biochemical event upTocnell sion of kinase-dead p66Yn has an inhibitory effect on T cell antigen receptor (TCR) acantigen receptor(TCR) stimulationis the activationof a tivation, and knock-out experiments confirm that, at least in protein tyrosine kina(sFeTK) pathway
TCR engagement results inbinding fusion proteins containinSgH2 domains from otheprroteins, thetandem S H 2 domains of ZAP-70demonstrate a remarkablyrestrictedrepertoire of proteinbinding, binding onlyTCW and CD3r.ZAP-70 is recovered in the binding assay, but this likely tobe a consequence of its interaction with multiplSeH2 binding sites on the of ZAF"70 to CD3e [16].The cDNA encoding this protein was cloned,and sequenceanalysis revealed that ZAP-70is aprotein tyrosine kinase of the p72Qkfamily [17]
As has been demonstrated previously, activation induced by OKT3 ligation of the TCR caused phosphorylation and coprecipitation of ZAP-70 with CD3, as well as stimulating tyrosine phosphorylation of CD36 [16]
Summary
Construction of pGEX-3X-SH2 ExpressionP lasmidsA cassette encoding the tandem SH2 domains ofZAP-70 (residues 1-263) and a C-terminal epitopic myc tag was constructed by amplifying the corresponding region of the ZAP-70 cDNA(a gift of Dr A. University of California, San Francisco) using the polymerase chain reaction. Plasmids encodingGST fusion proteins of the SH2 domain of blk, fyn, and lyn have been described elsewhere.. Ratnofsky (BASF).A plasmid encoding a GST fusion to the N-terminal SH2 domain of Ras-GAP was a gift of Dr B. JMlOl Escherichia coli transformed with plasmids encodingGST fusion proteins of the SH2 domains (both the single and tandem domains) of the phosphatidylinositol3-kinase p85 protein and PLCy, were gifts of Dr T. Purification of GSTSH2 Fusion Proteins and SH2 Binding Assay "GST-SH2 fusion proteins were purified from transformed E. coli essentially as described elsewhere: except that 1% Triton X-100 was added to the lysis buffer. Weiss (University of California, San Francisco).In some experiments pervanadate was used for T cell stimulation. Gel electrophoresis and immunoblotting were carried out as described [16]
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