The Inducible T cell kinase (Itk), a member of Tec family of non receptor kinases, is a key component of the T cell receptor (TCR) signaling pathway. Functional activation of Itk requires multiple events such as membrane recruitment, phosphorylation by Lck, and binding to the SLP76/LAT adaptor complex in a yet unknown order. Activated Itk phosphorylates phospholipase C _1 (PLC _1) leading to elevated intracellular calcium and increased expression of T helper type 2 (Th2) cytokine genes. The Pleckstrin homology (PH) domain of Itk interacts reversibly with membrane phospholipids thereby recruiting the kinase to the membrane. Three aromatic residues termed the FYF motif, located on the inner walls of the phospholipid-binding pocket, are conserved in the PH domains of all Tec kinases. To study the biological significance of these residues in the Itk PH domain, a FYF triple mutant was created and tested for its effects on Itk recruitment and other downstream signaling events. FYF-Itk mutant displayed defective membrane localization, reduced phosphorylation upon TCR stimulation and almost complete abrogation of phospholipid binding. This mutant also failed to phosphorylate PLC _1 and as a result had decreased Th2 cytokine production. Interestingly, the TCR inducible association of mutant Itk with SLP76/LAT adaptor complex remained unaffected. Based on this and previously published reports, a model of Itk activation is proposed suggesting separate mechanisms for membrane phospholipid binding and association of Itk in signaling complex.
