title: Lck-dependent Fyn activation requires C terminus-dependent targeting of kinase-active Lck to lipid rafts.
abstract: Mechanisms regulating the activation and delivery of function of Lck and Fyn are central to the generation of the most proximal signaling events emanating from the T cell antigen receptor (TcR) complex. Recent results demonstrate that lipid rafts (LR) segregate Lck and Fyn and play a fundamental role in the temporal and spatial coordination of their activation. Specifically, TcR-CD4 co-aggregation-induced Lck activation outside LR results in Lck translocation to LR where the activation of LR-resident Fyn ensues. Here we report a structure-function analysis toward characterizing the mechanism supporting Lck partitioning to LR and its capacity to activate co-localized Fyn. Using NIH 3T3 cells ectopically expressing FynT, we demonstrate that only LR-associated, kinase-active (Y505F)Lck reciprocally co-immunoprecipitates with and activates Fyn. Mutational analyses revealed a profound reduction in the formation of Lck-Fyn complexes and Fyn activation, using kinase domain mutants K273R and Y394F of (Y505F)Lck, both of which have profoundly compromised kinase activity. The only kinase-active Lck mutants tested that revealed impaired physical and enzymatic engagement with Fyn were those involving truncation of the C-terminal sequence YQPQP. Remarkably, sequential truncation of YQPQP resulted in an increasing reduction of kinase-active Lck partitioning to LR, in both fibroblasts and T cells. This in turn correlated with an ablation of the capacity of these truncates to enhance TcR-mediated interleukin-2 production. Thus, Lck-dependent Fyn activation is predicated by proximity-mediated transphosphorylation of the Fyn kinase domain, and targeting kinase-active Lck to LR is dependent on the C-terminal sequence QPQP.
authors.affiliation: Sunnybrook Research Institute and the Department of Immunology, University of Toronto, Toronto, Ontario M4N 3M5, Canada.
authors.forename: Dominik
authors.initials: D
authors.lastname: Filipp
authors.forename: Behrouz
authors.initials: B
authors.lastname: Moemeni
authors.forename: Alessandra
authors.initials: A
authors.lastname: Ferzoco
authors.forename: Kirishanthy
authors.initials: K
authors.lastname: Kathirkamathamby
authors.forename: Jenny
authors.initials: J
authors.lastname: Zhang
authors.forename: Ondrej
authors.initials: O
authors.lastname: Ballek
authors.forename: Dominique
authors.initials: D
authors.lastname: Davidson
authors.forename: André
authors.initials: A
authors.lastname: Veillette
authors.forename: Michael
authors.initials: M
authors.lastname: Julius
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D015704:CD4 Antigens / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation / Q000235:genetics / Q000502:physiology; D007376:Interleukin-2 / Q000096:biosynthesis; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000235:genetics / Q000378:metabolism*; D021962:Membrane Microdomains / Q000201:enzymology* / Q000235:genetics; D051379:Mice; D020125:Mutation, Missense; D041681:NIH 3T3 Cells; D021381:Protein Transport / Q000502:physiology; D051076:Proto-Oncogene Proteins c-fyn / Q000235:genetics / Q000378:metabolism*; D011948:Receptors, Antigen, T-Cell / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D015398:Signal Transduction / Q000502:physiology*; D013601:T-Lymphocytes / Q000201:enzymology*
chemical_list: D015704:CD4 Antigens; D007376:Interleukin-2; D011948:Receptors, Antigen, T-Cell; C494960:Fyn protein, mouse; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D051076:Proto-Oncogene Proteins c-fyn