Abstract
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.
Highlights
The Tec family of non-receptor protein tyrosine kinases is important for hematopoietic cell development and function [1]
The possibility that the signal seen with FYF-Inducible T cell Kinase (ITK) mutant may not be a reflection of enzymatic activity, but rather due to a co-precipitated Src kinase in HEK-293T cells that might have phosphorylated the FYF mutant on Y511 or Y180 is very unlikely because if this were the case, we would have seen a signal in the K390R mutant
One possible explanation is that even though the T cells used in these experiments lacked ITK, other Tec kinases (e.g. TEC, RLK) are present, and they may be contributing to the production of IL-4 in the culture supernatants [12]
Summary
The Tec family of non-receptor protein tyrosine kinases is important for hematopoietic cell development and function [1]. The Tec family member, Inducible T cell Kinase (ITK), regulates TCR-induced signaling events including intracellular Ca++ mobilization, actin polymerization, and the transcriptional activation of Th2 cytokine genes ITK is organized in structural domains that play critical roles in the regulation of its functions [3]. The most N-terminally located domain of ITK, the Pleckstrin Homology (PH) domain, mediates TCR-induced recruitment and localization of the kinase to the plasma membrane [4,5]. Production and turnover of the membrane phospholipid phosphatidylinositol 3,4,5-trisphosphate (PIP3) and its interaction with the PH domain are critical for ITK recruitment to the plasma membrane and its co-localization with the TCR-CD3 molecular complex [4,5,6]. We recently demonstrated that the soluble ligand inositol 1,3,4,5 tetrakisphosphate (IP4) plays a critical role in regulating ITK-PH domain interactions with the plasma membrane [5]
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