title: Substrate recognition of PLCγ1 via a specific docking surface on Itk.
abstract: Itk (interleukin-2 inducible T cell kinase) is a non-receptor protein tyrosine kinase expressed primarily in T cells. Itk catalyzes phosphorylation on tyrosine residues within a number of its natural substrates, including the well-characterized Y783 of PLCγ1. However, the molecular mechanisms Itk exploits to recognize its substrates are not completely understood. We have previously identified a specific docking interaction between the kinase domain of Itk and the C-terminal Src homology 2 (SH2C) domain of PLCγ1 that promotes substrate specificity for this enzyme/substrate pair. In the current study, we identify and map the interaction surface on the Itk kinase domain as an acidic patch centered on the G helix. Mutation of the residues on and adjacent to the G helix within the Itk kinase domain impairs the catalytic efficacy of PLCγ1 substrate phosphorylation by specifically altering the protein-protein interaction interface and not the inherent catalytic activity of Itk. NMR titration experiments using a Btk (Bruton's tyrosine kinase) kinase domain as a surrogate for the Itk kinase domain provide further support for an Itk/PLCγ1 SH2C interaction surrounding the G helix of the kinase domain. The work presented here provides structural insight into how the Itk kinase uses the G helix to single out Y783 of PLCγ1 for specific phosphorylation. Comparing these results to other well-characterized kinase/substrate systems suggests that the G helix is a general structural feature used by kinases for substrate recognition during signaling.
authors.affiliation: Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, USA.
authors.forename: Qian
authors.initials: Q
authors.lastname: Xie
authors.forename: Raji E
authors.initials: RE
authors.lastname: Joseph
authors.forename: D Bruce
authors.initials: DB
authors.lastname: Fulton
authors.forename: Amy H
authors.initials: AH
authors.lastname: Andreotti
mesh_terms: D000077329:Agammaglobulinaemia Tyrosine Kinase; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D055162:Biocatalysis; D015153:Blotting, Western; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D051966:Phospholipase C gamma / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018909:src Homology Domains / Q000235:genetics
chemical_list: D010455:Peptides; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D000077329:Agammaglobulinaemia Tyrosine Kinase; C000625951:Btk protein, mouse; C080813:emt protein-tyrosine kinase; D051966:Phospholipase C gamma