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 (PIP₃) 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 PIP₃, reducing ITK membrane recruitment, inducing conformational changes at the T cell-APC contact site, and compromising phosphorylation of ITK and subsequent phosphorylation of PLCγ₁. 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 PIP₃-mediated ITK membrane recruitment from the interactions of the kinase with key components of the TCR signalosome and abrogates ITK function in T cells.