Microdomain-dependent Regulation of Lck and Fyn Protein-Tyrosine Kinases in T Lymphocyte Plasma Membranes

Abstract

Src family protein-tyrosine kinases are implicated in signaling via glycosylphosphatidylinositol (GPI)-anchored receptors. Both kinds of molecules reside in opposite leaflets of the same sphingolipid-enriched microdomains in the lymphocyte plasma membrane without making direct contact. Under detergent-free conditions, we isolated a GPI-enriched plasma membrane fraction, also containing transmembrane proteins, selectively associated with sphingolipid microdomains. Nonionic detergents released the transmembrane proteins, yielding core sphingolipid microdomains, limited amounts of which could also be obtained by detergent-free subcellular fractionation. Protein-tyrosine kinase activity in membranes containing both GPI-anchored and transmembrane proteins was much lower than in core sphingolipid microdomains but was strongly reactivated by nonionic detergents. The inhibitory mechanism acting on Lck and Fyn kinases in these membranes was independent of the protein-tyrosine phosphatase CD45 and was characterized as a mixed, noncompetitive one. We propose that in lymphocyte plasma membranes, Lck and Fyn kinases exhibit optimal activity when juxtaposed to the GPI-and sphingolipid-enriched core microdomains but encounter inhibitory conditions in surrounding membrane areas that are rich in glycerophospholipids and contain additional transmembrane proteins. INTRODUCTIONIn contrast to transmembrane glycoproteins, surface molecules inserted into the plasma membrane via a glycosylphosphatidylinositol (GPI) 1 membrane anchor are confined to the outer leaflet of the plasma membrane and do not directly communicate with the cell interior (Low, 1989). However, several GPI-anchored proteins have been shown to be potent signal transducers, because their cross-linking leads to increased protein-tyrosine phosphorylation, calcium fluxes, gene expression, and cell activation and/or proliferation (Robinson, 1991). GPI-anchored proteins such as neurotrophic factor receptors transduce signals by ligand-induced interactions with transmembrane receptor protein-tyrosine kinases (PTKs) (Massague, 1996), whereas other GPI-anchored molecules such as CD87 (uPAR), CD16B (Fc␥RIIIB), and CD14 (lipopolysaccharide receptor) interact with integrins (Petty and Todd, 1996) and appear to signal via integrin-dependent pathways. For most other GPI-anchored proteins, signaling is presumed to require association with sphingolipid microdomains (Romagnoli and Bron, 1997;Stulnig et al., 1997), and their coprecipitation with Src family PTKs has been documented in hematopoietic , epithelial (ShenoyScaria et al., 1992), and neuronal (Zisch et al., 1995;Kunz et al., 1996) cells. The molecular nature of this indirect association between GPI-anchored receptors and Src kinases, however, remains unresolved. Sphingolipid microdomains are thought to consist of clusters of sphingolipids that achieve a liquid-ordered state in the presence of cholesterol (Ahmed et al., 1997;Schroeder et al., 1998) and resist solubilization by nonionic detergents. Such sphingolipid microdomain...

Inhibition of Protein Palmitoylation, Raft Localization, and T Cell Signaling by 2-Bromopalmitate and Polyunsaturated Fatty Acids

“…As depicted in Fig. 8D, in untreated cells, 30 Ϯ 6% of Fyn localized to DRMs, in agreement with previous findings (48). Treatment with PUFAs markedly reduced the ability of Fyn to localize to DRMs, with only 16 Ϯ 6% of Fyn found in DRMs in 20:4 treated cells and 5 Ϯ 3% in 20:5 treated cells.…”

CD38 Signaling in T Cells Is Initiated within a Subset of Membrane Rafts Containing Lck and the CD3-ζ Subunit of the T Cell Antigen Receptor

“…The columns were then rinsed twice with 200 l of 1% Brij 98 lysis buffer Immunoprecipitation under Solubilizing Conditions-Pooled rafts and soluble sucrose gradient fractions were diluted with lysis buffer containing 1% Brij 98 ϩ 60 mM ODG to less than 20% sucrose. ODG is a gentle non-ionic detergent that is very efficient in solubilizing proteins associated with rafts (42)(43)(44)(45). Immunoprecipitation of protein assemblies was performed by incubation of these fractions with specific antibodies followed by capture of the immune complexes on protein G superparamagnetic microbeads as described above or on protein GSepharose 4 Fast Flow beads (Amersham Biosciences) as described elsewhere (30).…”

“…However, insolubility of a membrane protein in a non-ionic detergent could be due to its association with detergent-resistant lipid rafts and/or its anchoring to cytoskeletal elements. To distinguish between these two possibilities, we used ODG, which is a gentle non-ionic detergent that is very efficient in solubilizing proteins associated with glycolipid-enriched membranes, and it does not disrupt the cytoskeleton (42)(43)(44)(45). As shown in Fig.…”

CD44 in hematological neoplasias

“…Furthermore, crosslinking of CD44 results in higher expression levels of lymphocyte function-associated antigen-1 (LFA-1) and VLA-4 which are responsible for enhanced transendothelial migration of tumor cells [38]. The cytoplasmic tail cooperates with intracellular molecules as members of the Src tyrosine kinase family including c-Src, Lyn, Fyn, and Lck [42][43][44][45], Moesin-Ezrin-Radixin-Like Protein Merlin [46], T-cell lymphoma invasion and metastasis-inducing protein 1 and 2 (Tiam1/2) [47], intracellular OPN [48], as well as members of the Smad protein family [14,49,50].…”