A specific type of membrane microdomains is involved in the maintenance and translocation of kinase active Lck to lipid rafts

Lck is the principal signal-generating tyrosine kinase of the T cell activation mechanism. We have previously demonstrated that induced Lck activation outside of lipid rafts (LR) results in the rapid translocation of a fraction of Lck to LR. While this translocation predicates the subsequent production of IL-2, the mechanism underpinning this process is unknown. Here, we describe the main attributes of this translocating pool of Lck. Using fractionation of Brij58 lysates, derived from primary naive non-activated CD4+ T cells, we show that a significant portion of Lck is associated with high molecular weight complexes representing a special type of detergent-resistant membranes (DRMs) of relatively high density and sensitivity to laurylmaltoside, thus called heavy DRMs. TcR/CD4 coaggregation-mediated activation resulted in the redistribution of more than 50% of heavy DRM-associated Lck to LR in a microtubular network-dependent fashion. Remarkably, in non-activated CD4+ T-cells, only heavy DRM-associated Lck is phosphorylated on its activatory tyrosine 394 and this pool of Lck is found to be membrane confined with CD45 phosphatase. These data are the first to illustrate a lipid microdomain-based mechanism concentrating the preactivated pool of cellular Lck and supporting its high stoichiometry of colocalization with CD45 in CD4+ T cells. They also provide a new structural framework to assess the mechanism underpinning the compartmentalization of critical signaling elements and regulation of spatio-temporal delivery of Lck function during the T cell proximal signaling.

Graphical abstractFigure optionsDownload full-size imageDownload as PowerPoint slideHighlights► In resting CD4+ T cells the pool of active pY394Lck represents ∼2% of the total Lck. ► Preactivated pY394Lck is associated with a special type of microdomains, heavy DRMs. ► Heavy DRM-associated pY394Lck is colocalized with CD45 via membrane confinement. ► T cell activation leads to redistribution of pY394Lck from heavy to light DRMs. ► Data describe a lipid microdomain-based mechanism segregating the pool of pY394Lck.