Abstract
The Syk family tyrosine kinases play a crucial role in antigen receptor-mediated signal transduction, but their regulation and cellular targets remain incompletely defined. Following receptor engagement, phosphorylation of tyrosine residues within ZAP-70 and Syk is thought to control both kinase activity and recruitment of modulatory factors. We report here the characterization of novel mutants of ZAP-70 and Syk, in which conserved C-terminal tyrosine residues have been replaced by phenylalanines (ZAP YF-C, Syk YF-C). Both mutant kinases display a prominent gain-of-function phenotype in Jurkat T cells, as demonstrated by lymphokine promoter activation, tyrosine phosphorylation of potential targets in vivo, and elevated intracellular calcium mobilization. While the presence of p56-Lck was required for ZAP YF-C-induced signaling, Syk YF-C showed enhanced functional activity in Lck-deficient JCaM1 Jurkat cells. Our results implicate the C terminus of Syk family kinases as an important regulatory region modulating T cell activation.
Highlights
T cell activation is thought to be initiated by the interaction of the T cell antigen receptor (TCR)1 with two distinct classes of nonreceptor protein-tyrosine kinases
Significant activation of the ZAP-70 kinase mediated by its binding to ITAMs could not be demonstrated [14], but it was suggested that the recruitment of ZAP-70 into the antigen receptor complex provided a platform for “cross-talk” between the Src and ZAP-70 kinases, which leads to phosphorylation and activation of ZAP-70 [15, 16]
In order to test whether these tyrosines are important for cellular activation events mediated by ZAP-70, we altered the residues by site-directed mutagenesis (ZAP YY597/598FF, ZAP YF-C)
Summary
Vol 273, No 25, Issue of June 19, pp. 15445–15452, 1998 Printed in U.S.A. (Received for publication, February 23, 1998, and in revised form, April 13, 1998). Reconstitution experiments of antigen receptor signaling in fibroblasts [4], as well as the biochemical and functional characterization of a Jurkat cell line (JCaM1.6) that lacks Lck kinase activity [5, 6], strengthened the view that Src kinases are responsible for a very important initial step in the antigen receptor signaling cascade, namely the phosphorylation of the intracellular domains of TCR-associated proteins. This includes the homodimer, as well as the CD3 complex composed of the ␥, ␦, and ⑀ chains. Our results implicate novel regulatory mechanisms that control the cellular activity of these kinases, possibly involving inhibitory proteins
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