title: Lck phosphorylates the activation loop tyrosine of the Itk kinase domain and activates Itk kinase activity.
abstract: The Tec family tyrosine kinase Itk has been implicated in T cell receptor (TCR) signaling, yet its precise role and mechanism of activation remain undefined. To investigate these issues, we examined the biochemical response of Itk to TCR stimulation. We found that Itk is tyrosine-phosphorylated after TCR cross-linking and that this phosphorylation depends on the presence of functional Lck. To determine if this Lck dependence results from direct phosphorylation of Itk by Lck, we generated recombinant Itk and Lck using a baculovirus expression system and used these proteins in subsequent biochemical analyses. We found that Lck phosphorylates Itk upon co-expression in insect cells and, further, that this phosphorylation of Itk results in increased Itk in vitro kinase activity. The major site of Lck phosphorylation on Itk was mapped to the conserved tyrosine (Tyr511) in the activation loop of the Itk kinase domain. Substitution of this tyrosine with phenylalanine abolishes Itk kinase activity in insect cells, indicating that phosphorylation at this site plays a critical role in regulating Itk function.
authors.affiliation: Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
authors.forename: S D
authors.initials: SD
authors.lastname: Heyeck
authors.forename: H M
authors.initials: HM
authors.lastname: Wilcox
authors.forename: S C
authors.initials: SC
authors.lastname: Bunnell
authors.forename: L J
authors.initials: LJ
authors.lastname: Berg
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D017124:Conserved Sequence; D004789:Enzyme Activation; D006801:Humans; D019169:Jurkat Cells; D007700:Kinetics; D008213:Lymphocyte Activation; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry; D010748:Phosphopeptides / Q000737:chemistry; D010766:Phosphorylation; D019000:Phosphotyrosine; D017354:Point Mutation; D011505:Protein-Tyrosine Kinases / Q000737:chemistry* / Q000378:metabolism*; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D018411:Spodoptera; D013601:T-Lymphocytes; D014162:Transfection; D014443:Tyrosine; D014638:Vanadates / Q000494:pharmacology; D019061:src-Family Kinases / Q000378:metabolism*
chemical_list: D010446:Peptide Fragments; D010748:Phosphopeptides; D011948:Receptors, Antigen, T-Cell; D011994:Recombinant Proteins; C076715:pervanadate; D019000:Phosphotyrosine; D014638:Vanadates; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); C080813:emt protein-tyrosine kinase; D019061:src-Family Kinases