Serine 6 of Lck Tyrosine Kinase: A Critical Site for Lck Myristoylation, Membrane Localization, and Function in T Lymphocytes

Abstract

Lck is a member of the Src family kinases expressed predominantly in T cells, and plays a pivotal role in TCR-mediated signal transduction. Myristoylation of glysine 2 in the N-terminal Src homology 4 (SH4) domain of Lck is essential for membrane localization and function. In this study, we examined a site within the SH4 domain of Lck regulating myristoylation, membrane localization, and function of Lck. A Lck mutant in which serine 6 (Ser6) was substituted by an alanine was almost completely cytosolic in COS-7 cells, and this change of localization was associated with a drastic inhibition of myristoylation in this mutant. To assess the role of Ser6 of Lck in T cell function, we established stable transfectants expressing various Lck mutants using Lck-negative JCaM1 cells. The Lck mutant of Ser6 to alanine, most of which did not target to the plasma membrane, was not able to reconstitute TCR-mediated signaling events in JCaM1 cells, as analyzed by tyrosine phosphorylation of intracellular proteins and CD69 expression. These results demonstrate that Ser6 is a critical factor for Lck myristoylation, membrane localization, and function in T cells, presumably because the residue is important for N-myristoyl transferase recognition.

Cloning, characterization, and expression pattern of Atlantic halibut (Hippoglossus hippoglossus L.) CD4-2, Lck, and ZAP-70

“…Other feature of the unique domain of Lck is the presence of a GCxCS motif. This motif is believed to be important for membrane localization of Lck in a CD4/CD8 independent manner, due to palmiotylation of C 3 and C 5 dependent on the myristoylation of G 2 [23][24][25]. This motif was identified within The SH2 domain of Lck is required for efficient TCR-mediated signaling in higher vertebrates [67], as it is found to interact with ZAP-70 following T-cell stimulation [68].…”

Lowering Glycosphingolipid Levels in CD4+ T Cells Attenuates T Cell Receptor Signaling, Cytokine Production, and Differentiation to the Th17 Lineage

“…Lck is normally acylated at the N terminus through myristoylation and S-palmitoylation, which mediate its localization to lipid rafts. Lck-deficient T cells or cells harboring acylation-defective Lck mutants that retain enzymatic activity but are incapable of translocating into lipid rafts are unable to reproduce the early signaling events triggered by engagement of the TCR (15,17,22). In addition, an Lck chimeric protein with a non-lipid raft-localizing transmembrane domain also fails to reconstitute early TCR signaling events unless it is brought into proximity of the TCR-CD3 complex by antibody-mediated cross-linking (7).…”

“…These findings corroborate those noted above with Genz-122346 that lowering the GSL levels reduces T cell activation. 17 Lineage-Naive T cells are able to differentiate into distinct subsets of effector cells depending on the activation signal and milieu of cytokines. The observation of a dampened TCR signaling activity and T cell activation in immune cells harboring lower amounts of GSLs prompted us to probe if this also impacted their ability to differentiate.…”

“…For example, it has been reported that although the TCR-CD3 complex is only weakly associated with lipid rafts in resting T cells, this association is significantly increased upon their activation (14). It is likely that this increase in association of TCR-CD3 with the lipid rafts helps to bring the complex into the proximity of raftresident protein-tyrosine kinases of the Src family, such as Lck (15)(16)(17). This immediacy facilitates the phosphorylation of key TCR proximal signaling molecules, such as Zap70 and LAT (adaptor protein linker for activation of T cells).…”

Genetically Encoded Förster Resonance Energy Transfer Sensors for the Conformation of the Src Family Kinase Lck

“…Constructs CLckY-2 and CLckY-3 contain the ECFP at the linker regions between the SH3 and SH2 domains and the SH2 and catalytic domains, respectively. The N terminus is critical for membrane targeting of Lck via myristoylation (28) and palmitoylation (29) and thus was left untouched in all constructs (Fig. 1A).…”