title: A specific type of membrane microdomains is involved in the maintenance and translocation of kinase active Lck to lipid rafts.
abstract: 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.
authors.affiliation: Laboratory of Immunobiology, Institute of Molecular Genetics AS CR, Prague, Czech Republic.
authors.forename: Ondřej
authors.initials: O
authors.lastname: Ballek
authors.forename: Adéla
authors.initials: A
authors.lastname: Broučková
authors.forename: Jasper
authors.initials: J
authors.lastname: Manning
authors.forename: Dominik
authors.initials: D
authors.lastname: Filipp
mesh_terms: D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D002462:Cell Membrane / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D003902:Detergents / Q000494:pharmacology; D004789:Enzyme Activation; D005260:Female; D017493:Leukocyte Common Antigens / Q000378:metabolism; D008213:Lymphocyte Activation; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000276:immunology / Q000378:metabolism*; D021962:Membrane Microdomains / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D021381:Protein Transport; D015398:Signal Transduction
chemical_list: D003902:Detergents; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D017493:Leukocyte Common Antigens; C516769:PTPRC protein, human