A Conserved Motif in the ITK PH-Domain Is Required for Phosphoinositide Binding and TCR Signaling but Dispensable for Adaptor Protein Interactions

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.

Phosphoinositides and intracellular calcium signaling: novel insights into phosphoinositides and calcium coupling as negative regulators of cellular signaling

“…PH domains are small protein modules found in major regulators of vesicle biogenesis and are thus involved in intracellular trafficking; the proteins carrying these domains include pleckstrin, the AKT/RAC family serine/threonine kinases, Btk/Itk/Tec subfamily nonreceptor tyrosine kinases, NrKA/NrKB protein kinases, the breakpoint cluster region protein (BCR), the Rho/Rac guanine nucleotide exchange factor FGDs, insulin receptor substrates (IRSs), PLCs, the Rho family of guanosine triphosphatases (GTPases), the Ras GTPase-activating protein (RasGAP), oxysterol-binding proteins, ceramide kinase, G protein receptor kinases, Src homology 2B (SH2B) adapter proteins involved in tyrosine receptor kinase family member binding, KIF1A, and ADP-ribosylation factors (ARFs) 12 , 13 , 15 , 32 – 36 . The PH domain comprises approximately 120 amino acids and can bind PIPs, which mediate intracellular signaling, trafficking, and regulation 37 .…”

“…These second messengers are transiently generated by cell surface receptor-stimulated PI3K activation following insulin, growth factor, or cytokine stimulation 3 . Therefore, the PH domains of kinases downstream of PI3K, such as AKT 32 , BTK 11 , 12 , ITK 13 , and GRP1 14 , recognize either PI(3,4)P 2 , PI(3,4,5)P 3 or both with remarkable specificity and affinity. This precise recognition of the PI3K products PI(3,4)P 2 and PI(3,4,5)P 3 by PH domains is crucial for signaling-dependent membrane recruitment of kinases such as AKT, BTK, ITK, and GRP1 in response to hormone and cytokine stimulation 40 .…”

“…Among PIP-binding domains, PH domains are effectors that recognize the lipid second messengers PI(3,4)P 2 or PI(3,4,5)P 3 , which are transiently generated by phosphatidylinositol (PI) 3-kinase (PI3K) 1 , 10 in response to insulin, growth factors, and cytokines. In addition, insulin- or growth factor-stimulated PI3K products are critical for signal-dependent membrane recruitment of the PH domain-carrying several kinases, such as protein kinase B (AKT), Bruton’s tyrosine kinase (BTK) 11 , 12 , interleukin-2-inducible T-cell kinase (ITK) 13 , and general receptor for phosphoinositide 1 (GRP1) 14 , in response to hormone and cytokine stimulation. Mutations in the PH domain that disrupt PI(3,4)P 2 or PI(3,4,5)P 3 binding have been reported to cause severe signaling defects such as those causing X-linked agammaglobulinemia in humans and X-linked immunodeficiency in mice 11 , 15 .…”

Partial defects of T-cell development associated with poor T-cell function

“…1). In particular, ITK pleckstrin homology domain binds to phosphatidylinositol monophosphates, allowing ITK recruitment to the cell membrane 86 . Following TCR/CD3 cross-linking, ITK activation increases PLC-γ1 activation and Ca 2+ influx 87–89 .…”