title: 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 (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.
authors.affiliation: Molecular Biology Institute and Center for Microbial Sciences, Department of Biology, San Diego State University, San Diego, California, USA.
authors.forename: Nupura
authors.initials: N
authors.lastname: Hirve
authors.forename: Roman M
authors.initials: RM
authors.lastname: Levytskyy
authors.forename: Stephanie
authors.initials: S
authors.lastname: Rigaud
authors.forename: David M
authors.initials: DM
authors.lastname: Guimond
authors.forename: Tomasz
authors.initials: T
authors.lastname: Zal
authors.forename: Karsten
authors.initials: K
authors.lastname: Sauer
authors.forename: Constantine D
authors.initials: CD
authors.lastname: Tsoukas
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism; D017124:Conserved Sequence; D057809:HEK293 Cells; D006801:Humans; D015847:Interleukin-4 / Q000096:biosynthesis; D019169:Jurkat Cells; D051379:Mice; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D051966:Phospholipase C gamma / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000737:chemistry* / Q000378:metabolism*; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D013601:T-Lymphocytes / Q000378:metabolism; D014162:Transfection
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D050505:Mutant Proteins; D018129:Phosphatidylinositol Phosphates; D011948:Receptors, Antigen, T-Cell; C060974:phosphatidylinositol 3,4,5-triphosphate; D015847:Interleukin-4; D011505:Protein-Tyrosine Kinases; C080813:emt protein-tyrosine kinase; D051966:Phospholipase C gamma