Lck-dependent Fyn Activation Requires C Terminus-dependent Targeting of Kinase-active Lck to Lipid Rafts

Dominik Filipp,Behrouz Moemeni,Alessandra Ferzoco,Kirishanthy Kathirkamathamby,Jenny Zhang,Ondřej Ballek,Dominique Davidson,André Veillette,Michael Julius

doi:10.1074/jbc.m710372200

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.

Highlights

EXPERIMENTAL PROCEDURESCell Lines and T Cell Clones—Generation, selection, and growth of NIH 3T3 cells expressing wild type (WT) FynT (3T3-FynT) were described elsewhere [34]
Signals emanating from the antigen receptor complex in T cells [1, 2]
The inactive, autoinhibitory conformation is supported in large part by 2-week intra-molecular interactions formed between the SH2 domain and the phosphorylated C-terminal tyrosine and the Src homology 3 (SH3) domain and the linker region, which cooperatively contribute to down-regulate the kinase activity

Summary

EXPERIMENTAL PROCEDURES

Cell Lines and T Cell Clones—Generation, selection, and growth of NIH 3T3 cells expressing WT FynT (3T3-FynT) were described elsewhere [34]. The CD4ϩ T cell clone 2.5 infected with empty MigR1 vector or MigR1 containing various mutant constructs of Lck were selected and maintained as described elsewhere [35]. The same protocol was applied to generate SYF, 2.5, and BI-141 cells expressing indicated Lck mutants. Cell Lysis, Immunoprecipitations, Immunoblotting, and Densitometry— 4G10 and anti-Lck immunoblots were performed on aliquots of SYF, BI-141, and 2.5 cell infectants directly lysed in Laemmli loading buffer and boiled for 10 min. For evaluation of Lck, Fyn, Tyr(P)394 Lck, and Tyr(P)417 Fyn levels in 3T3-FynT infectants, cells were lysed in TNE buffer (50 mM Tris, pH 8, 20 mM EDTA, 200 mM Na3VO4, 50 mM NaF, 1% Nonidet P-40, 20 ␮g/ml leupeptin, 20 ␮g/ml aprotinin) and immunoprecipitated, blotted, and probed as described in detail elsewhere [33]. Supernatants were harvested after 16 h, and IL-2 content was quantified using an IL-2-specific ELISA (eBiosciences), according to the manufacturer’s recommendations

RESULTS

C Terminus of Lck Does Not

DISCUSSION