A remote substrate docking mechanism for the tec family tyrosine kinases.
During T cell signaling, Itk selectively phosphorylates a tyrosine within its own SH3 domain and a tyrosine within PLCgamma1. We find that the remote SH2 domain in each of these substrates is required to achieve efficient tyrosine phosphorylation by Itk and extend this observation to two other Tec family kinases, Btk and Tec. Additionally, we detect a stable interaction between the substrate SH2 domains and the kinase domain of Itk and find that addition of specific, exogenous SH2 domains to the in vitro kinase assay competes directly with substrate phosphorylation. On the basis of these results, we show that the kinetic parameters of a generic peptide substrate of Itk are significantly improved via fusion of the peptide substrate to the SH2 domain of PLCgamma1. This work is the first characterization of a substrate docking mechanism for the Tec kinases and provides evidence of a novel, phosphotyrosine-independent regulatory role for the ubiquitous SH2 domain.
Joseph
Raji E
RE
Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, Iowa 50011, USA.
Min
Lie
L
Xu
Ruo
R
Musselman
Eli D
ED
Andreotti
Amy H
AH
D000595:Amino Acid Sequence; D001667:Binding, Competitive; D002384:Catalysis; D020134:Catalytic Domain; D017124:Conserved Sequence / Q000502:physiology; D008969:Molecular Sequence Data; D051966:Phospholipase C gamma / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000737:chemistry / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000737:chemistry* / Q000378:metabolism* / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology; D013379:Substrate Specificity; D018909:src Homology Domains / Q000502:physiology
D019000:Phosphotyrosine; C089848:Tec protein-tyrosine kinase; D011505:Protein-Tyrosine Kinases; C080813:emt protein-tyrosine kinase; D051966:Phospholipase C gamma