|In Vivo Consequences of Disrupting SH3-Mediated Interactions of the Inducible T-Cell Kinase.|
Levytskyy, RM; Hirve, N; Guimond, DM; Min, L; Andreotti, AH; Tsoukas, CD
Journal of signal transduction
ITK-SH3-mediated interactions, both with exogenous ligands and via intermolecular self-association with ITK-SH2, have been shown to be important for regulation of ITK activity. The biological significance of these competing SH3 interactions is not completely understood. A mutant of ITK where substitution of the SH3 domain with that of the related kinase BTK (ITK-BTK((SH3))) was used to disrupt intermolecular self-association of ITK while maintaining canonical binding to exogenous ligands such as SLP-76. ITK-BTK((SH3)) displays reduced association with SLP-76 leading to inefficient transphosphorylation, reduced phosphorylation of PLCγ1, and diminished Th(2) cytokine production. In contrast, ITK-BTK((SH3)) displays no defect in its localization to the T-cell-APC contact site. Another mutation, Y511F, in the activation loop of ITK, impairs ITK activation. T cells expressing ITK-Y511F display defective phosphorylation of ITK and its downstream target PLCγ1, as well as significant inhibition of Th(2) cytokines. In contrast, the inducible localization of ITK-Y511F to the T cell-APC contact site and its association with SLP-76 are not affected. The presented data lend further support to the hypothesis that precise interactions between ITK and its signaling partners are required to support ITK signaling downstream of the TCR.
|A conserved motif in the ITK PH-domain is required for phosphoinositide binding and TCR signaling but dispensable for adaptor protein interactions.|
Hirve, N; Levytskyy, RM; Rigaud, S; Guimond, DM; Zal, T; Sauer, K; Tsoukas, CD
Binding of the membrane phospholipid phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) to the Pleckstrin Homology (PH) domain of the Tec family protein tyrosine kinase, Inducible T cell Kinase (ITK), is critical for the recruitment of the kinase to the plasma membrane and its co-localization with the TCR-CD3 molecular complex. Three aromatic residues, termed the FYF motif, located in the inner walls of the phospholipid-binding pocket of the ITK PH domain, are conserved in the PH domains of all Tec kinases, but not in other PH-domain containing proteins, suggesting an important function of the FYF motif in the Tec kinase family. However, the biological significance of the FYF amino acid motif in the ITK-PH domain is unknown. To elucidate it, we have tested the effects of a FYF triple mutant (F26S, Y90F, F92S), henceforth termed FYF-ITK mutant, on ITK function. We found that FYF triple mutation inhibits the TCR-induced production of IL-4 by impairing ITK binding to PIP(3), reducing ITK membrane recruitment, inducing conformational changes at the T cell-APC contact site, and compromising phosphorylation of ITK and subsequent phosphorylation of PLCγ(1). Interestingly, however, the FYF motif is dispensable for the interaction of ITK with two of its signaling partners, SLP-76 and LAT. Thus, the FYF mutation uncouples PIP(3)-mediated ITK membrane recruitment from the interactions of the kinase with key components of the TCR signalosome and abrogates ITK function in T cells.
|The EMT/ITK/TSK (EMT) tyrosine kinase is activated during TCR signaling: LCK is required for optimal activation of EMT.|
Gibson, S, et al.
J. Immunol., 156: 2716-22 (1996)
Functional T lymphocyte activation requires concurrent stimulation of the TCR complex and an accessory molecule, most frequently CD28. We have previously demonstrated that the TEC family tyrosine kinase EMT/ITK/TSK (EMT) is activated following cross-linking of CD28. We demonstrate herein that cross-linking of the CD3 component of the TCR complex also leads to EMT activation as indicated by a rapid and transient increase in EMT tyrosine phosphorylation and kinase activity in anti-EMT immunoprecipitates. However, although concurrent cross-linking of the TCR and CD28 results in a marked increase in production of the T cell growth factor IL-2, it does not result in a significant alteration in the magnitude or duration of EMT activation. Somatic cell mutants of the Jurkat T cell line, which lack the SRC family kinase LCK (JCaM1.6), fail to produce IL-2 when stimulated through the TCR complex. EMT activation, as evidenced by increased EMT tyrosine phosphorylation and EMT-associated kinase activity, was also greatly reduced following stimulation of the TCR in the JCaM1.6 Jurkat T cell mutants that lack LCK. In support of a role for LCK in EMT activation, reconstitution of the LCK-negative Jurkat T cell line by enforced expression of LCK restored TCR-mediated EMT activation. Taken together, the data indicate that the EMT tyrosine kinase is activated following cross-linking of the TCR, a process in which LCK likely plays an important role.
|Structure and expression of novel protein-tyrosine kinases, Emb and Emt, in hematopoietic cells.|
Yamada, N, et al.
Biochem. Biophys. Res. Commun., 192: 231-40 (1993)
Two novel tyrosine kinase cDNAs were obtained from murine mast cells. These kinases, Emb and Emt, constitute a novel tyrosine kinase subfamily which may also include Tec, a kinase preferentially expressed in liver, and Dsrc28, a fruit fly kinase. Both lack hydrophobic stretches characteristic of the transmembrane domains found in growth factor receptor tyrosine kinases and carboxyl-terminal, negative regulatory tyrosine residue found in Src family kinases. In addition to the Src homology region 2 (SH2) and SH3 domains characteristic of the Src family kinases and other signaling molecules, Emb and Emt share a similar amino-terminal domain comprised mainly of two repeat segments. The emb 2.7-kb transcript was expressed in mast cells, myeloid cells and B lymphocytes while the emt 4.6-kb mRNA in mast cells, myeloid cells and T lymphocytes. The evidence for in vitro tyrosine kinase activity of Emb and Emt proteins is also provided.