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.