A conserved motif in the ITK PH-domain is required for phosphoinositide binding and TCR signaling but dispensable for adaptor protein interactions.

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.

In vivo significance of ITK-SLP-76 interaction in cytokine production.

In vitro data have suggested that activation of the inducible T-cell kinase (ITK) requires an interaction with the adaptor protein SLP-76. One means for this interaction involves binding of the ITK SH3 domain to the polyproline-rich (PR) region of SLP-76. However, the biological significance of this association in live cells and the consequences of its disruption have not been demonstrated. Here, we utilized a polyarginine-rich, cell-permeable peptide that represents the portion of the SLP-76 PR region that interacts with the ITK SH3 domain as a competitive inhibitor to disrupt the association between ITK and SLP-76 in live cells. We demonstrate that treatment of cells with this peptide, by either in vitro incubation or intraperitoneal injection of the peptide in mice, inhibits the T-cell receptor (TCR)-induced association between ITK and SLP-76, recruitment and transphosphorylation of ITK, actin polarization at the T-cell contact site, and expression of Th2 cytokines. The inhibition is specific, as indicated by lack of effects by the polyarginine vehicle alone or a scrambled sequence of the cargo peptide. In view of the role of ITK as a regulator of Th2 cytokine expression, the data underscore the significance of ITK as a target for pharmacological intervention.

Positive regulation of Itk PH domain function by soluble IP4.

Pleckstrin homology (PH) domain-mediated protein recruitment to cellular membranes is of paramount importance for signal transduction. The recruitment of many PH domains is controlled through production and turnover of their membrane ligand, phosphatidylinositol 3,4,5-trisphosphate (PIP3). We show that phosphorylation of the second messenger inositol 1,4,5-trisphosphate (IP3) into inositol 1,3,4,5-tetrakisphosphate (IP4) establishes another mode of PH domain regulation through a soluble ligand. At physiological concentrations, IP4 promoted PH domain binding to PIP3. In primary mouse CD4+CD8+ thymocytes, this was required for full activation of the protein tyrosine kinase Itk after T cell receptor engagement. Our data suggest that IP4 establishes a feedback loop of phospholipase C-gamma1 activation through Itk that is essential for T cell development.

Bacterial superantigens bypass Lck-dependent T cell receptor signaling by activating a Galpha11-dependent, PLC-beta-mediated pathway.

The paradigm to explain antigen-dependent T cell receptor (TCR) signaling is based on the activation of the CD4 or CD8 coreceptor-associated kinase Lck. It is widely assumed that this paradigm is also applicable to signaling by bacterial superantigens. However, these bacterial toxins can activate human T cells lacking Lck, suggesting the existence of an additional pathway of TCR signaling. Here we showed that this alternative pathway operates in the absence of Lck-dependent tyrosine-phosphorylation events and was initiated by the TCR-dependent activation of raft-enriched heterotrimeric Galpha11 proteins. This event, in turn, activated a phospholipase C-beta and protein kinase C-mediated cascade that turned on the mitogen-activated protein kinases ERK-1 and ERK-2, triggered Ca(2+) influx, and translocated the transcription factors NF-AT and NF-kappaB to the nucleus, ultimately inducing the production of interleukin-2 in Lck-deficient T cells. The triggering of this alternative pathway by superantigens suggests that these toxins use a G protein-coupled receptor as a coreceptor on T cells.