Itk Phosphorylation Sites Are Required for Functional Activity in Primary T Cells

Heather M Wilcox,Leslie J Berg

doi:10.1074/jbc.m304811200

Abstract

The Tec family kinase Itk plays a critical role in signal transduction downstream of the T cell antigen receptor and has been implicated in the activation of phospholipase C-gamma1, a key regulator of calcium mobilization and extracellular signal-regulated kinase (ERK) activation. We have shown previously that Itk is regulated by an activating transphosphorylation event in which Tyr-511 in the kinase domain is phosphorylated by Lck (Heyeck, S. D., Wilcox, H. M., Bunnell, S. C., and Berg, L. J. (1997) J. Biol. Chem. 272, 25401-25408). In this study, we present evidence for another mode of regulation for Itk, the autophosphorylation of Tyr-180 in the Src homology 3 (SH3) domain. To investigate the role of Itk trans- and autophosphorylation in T cell signaling, a retroviral transduction system was used to introduce different versions of Itk into Itk-deficient primary T cells. We report that Itk mutated at either the trans- or the autophosphorylation site is unable to fully restore cytokine production and ERK activation in the Itk-deficient cells; Itk-Y511F is severely defective, whereas Itk-Y180F has partial activity. Because phosphorylation at Tyr-180 is predicted to interfere with ligand binding by the SH3 domain, an SH3 point mutant that cannot bind ligand was also examined and found to be unable to restore function to the Itk-/- cells. These data provide new insights into the complex regulation of Itk in primary T cells.

Highlights

Engagement of the T cell antigen receptor (TCR)1 results in the rapid recruitment and activation of protein tyrosine kinases of the Src, Syk, and Tec families
Our data strongly support a model in which transphosphorylation of the activation loop tyrosine precedes Itk autophosphorylation in the Src homology 3 (SH3) domain
This model is based on studies of recombinant proteins expressed in insect cells, which indicate that the Src-family kinase, Lck, provides the initial activating phosphorylation event at Tyr511, stimulating Itk to autophosphorylate at Tyr-180

Summary

Introduction

Engagement of the T cell antigen receptor (TCR)1 results in the rapid recruitment and activation of protein tyrosine kinases of the Src, Syk, and Tec families. For this gene transfer system to be useful, it was necessary to ensure the following: 1) that the cultured cell lines maintain the functional defects seen with freshly isolated Itk and ItkϪ/Ϫ T cells; and 2) that reconstitution of the cells with retrovirally introduced wild type Itk restores IL-2 production.

Results

Conclusion