Biol Res 35: 127-131, 2002

Lipid rafts: cell surface platforms for T cell signaling

TONY MAGEE^1#, NIINA PIRINEN¹, JEREMY ADLER², STAMATIS N. PAGAKIS² AND INGELA PARMRYD¹

AZ and 2, National Institute for Medical Research, Mill Hill, London, NW71AA, UK.

ABSTRACT

The Src family tyrosine kinase Lck is essential for T cell development and T cell receptor (TCR)* signaling. Lck is post-translationally fatty acylated at its N-terminus conferring membrane targeting and concentration in plasma membrane lipid rafts, which are lipid-based organisational platforms. Confocal fluorescence microscopy shows that Lck colocalises in rafts with GPI-linked proteins, the adaptor protein LAT and Ras, but not with non-raft membrane proteins including the protein tyrosine phosphatase CD45. The TCR also associates with lipid rafts and its cross-linking causes coaggregation of raft-associated proteins including Lck, but not of CD45. Cross-linking of either the TCR or rafts strongly induces specific tyrosine phosphorylation of the TCR in the rafts. Remarkably, raft patching alone induces signalling events analogous to TCR stimulation, with the same dependence on expression of key TCR signalling molecules. Our results indicate a mechanism whereby TCR engagement promotes aggregation of lipid rafts, which facilitates colocalisation of signaling proteins including Lck, LAT, and the TCR, while excluding CD45, thereby potentiating protein tyrosine phosphorylation and downstream signaling. We are currently testing this hypothesis as well as using imaging techniques such as fluorescence resonance energy transfer (FRET) microscopy to study the dynamics of proteins and lipids in lipid rafts in living cells undergoing signaling events. Recent data show that the key phosphoinositide PI(4,5)P2 is concentrated in T cell lipid rafts and that on stimulation of the cells it is rapidly converted to PI(3,4,5)P3 and diacylglycerol within rafts. Thus rafts are hotspots for both protein and lipid signalling pathways.

Key terms: lipid rafts; Lck; tyrosine kinase; T cell; signalling; phosphatase

(*)Abbreviations: CT, cholera toxin; DAG, diacylglycerol; DIGs, detergent-insoluble glycolipid-enriched domains; DRMs, detergent-resistant membranes; FRET, fluorescence resonance energy transfer; GPI, glycosylphosphatidyl inositol; ITAMs, immunoreceptor tyrosine-based activation motifs; LAT, linker for activation of T cells; PTK, protein tyrosine kinase; PTPase, protein tyrosine phosphatase; TCR, T cell receptor; TIFF, Triton-insoluble floating fraction.

INTRODUCTION

The Src family tyrosine kinase (PTK) Lck is essential for T cell development and T cell receptor (TCR) signalling. Lck is post-translationally modified by fatty acylation with amide-linked myristate at its N-terminus and with longer chain fatty acids (mainly palmitate) thioester-linked at nearby cysteine residues, conferring membrane targeting (Kabouridis et al., 1997). Biochemical studies, involving cold non-ionic detergent extraction followed by sucrose density gradient flotation, show that Lck and many other signaling proteins are concentrated in a low density detergent-insoluble membrane fraction that is enriched in glycolipids and cholesterol.

These structures have been called by many names, including detergent-resistant membranes (DRMs), detergent-insoluble glycolipid-enriched domains (DIGs), Triton-insoluble floating fraction (TIFF), etc. They are believed to be derived from lipid rafts in the plasma membrane: cholesterol- and sphingolipid-enriched domains with lower fluidity than the bulk membrane forming a so-called liquid-ordered (l_o) phase (Simons and Ikonen, 1997; Brown and London, 1998). These structures form spontaneously because the long saturated hydrophobic fatty tails of sphingolipids tend to associate together and pack with cholesterol. Phospholipids with saturated acyl chains also tend to partition into rafts, but in most cells the majority of plasma membrane phospholipids have unsaturated acyl groups and form a more fluid bulk phase. In some cells, e.g. neurons, where the content of glycolipids is particularly high, lipid rafts may actually comprise more than 50% of the plasma membrane area but usually it is much less than this, perhaps in the region of a few up to 10%. These structures are highly dynamic, because lipids are their main structural components and they have an intrinsically fast rate of lateral movement in the bilayer; nevertheless, lipid rafts have a biologically significant lifetime. Their dynamic behaviour makes them ideally suited to responding rapidly to changes in cell physiology. Many transmembrane proteins are excluded from lipid rafts although some partition into them; the reasons for these differences are largely unknown. However, the proteins most commonly found in lipid rafts carry one or more lipid modifications, such as acylation or glycosylphosphatidylinositol (GPI) anchors; the saturated acyl groups favour packing into the l_o phase. Many of these proteins are involved in signaling and hence lipid rafts are frequently used by cells as supramolecular platforms for coordinating and enhancing signaling events.

Lipid raft aggregation contributes to T cell signaling

Lipid rafts are too small (<100 nm diameter) for resolution by standard microscopy techniques which are limited by the wavelength of illuminating light to at least 200-300nm across (Varma and Mayor, 1998). Therefore, we have investigated lipid rafts in Jurkat T cells after cross-linking of the raft component GM1 ganglioside with fluorescent cholera toxin (CT) B subunit (for which it is the receptor) and anti-CTB antibody, to produce aggregated rafts that can be seen in the confocal microscope (Janes et al., 1999). The resulting visible membrane patches have the expected properties of lipid rafts, colocalising with GPI-linked proteins and the adaptor protein Linker of Activated T cells (LAT), which are enriched in rafts, but not with non-raft membrane proteins including the protein tyrosine phosphatase (PTPase) CD45 and transferrin receptor. An Lck-EGFP fusion protein also colocalised to CTB-patched lipid rafts, as did the TCR, although this was sensitive to non-ionic detergent in contrast to Lck and GPI-linked proteins. Specific TCR cross-linking with activating anti-TCR antibody caused coaggregation of raft-associated proteins including Lck, but not of CD45. Cross-linking of either the TCR or GM1 strongly induced tyrosine phosphorylation of TCR immunoreceptor tyrosine-based activation motifs (ITAMs) in the rafts. Remarkably, CTB-induced raft patching induced signaling events analogous to TCR stimulation, with the same dependence on expression of key TCR signaling molecules as shown by the use of a panel of mutant cell lines defective in various steps of TCR signal transduction pathways. Targeting of Lck to rafts was necessary for these events, as a raft-excluded transmembrane Lck chimera could not reconstitute CTB-induced signaling (Kabouridis et al., 1997).

Extraction of the key raft component cholesterol with methyl beta-cyclodextrin perturbs TCR signaling with complex kinetics (Kabouridis et al., 2000). In the first few minutes of cholesterol extraction several signaling pathways are transiently activated but as cholesterol depletion proceeds further rafts are destabilised and signaling is turned off. We interpret these data to mean that initial cholesterol removal causes partial disruption of rafts and mixing of raft-associated and non-raft components, that are normally segregated to maintain homeostasis, resulting in inappropriate signaling. Longer term cholesterol depletion causes complete mixing of components and attainment of a new steady state in which signaling is down-regulated.

Our results indicate a mechanism whereby TCR engagement promotes aggregation of lipid rafts, which facilitates colocalisation of signaling proteins including Lck, LAT and the TCR, while excluding CD45, thereby potentiating protein tyrosine phosphorylation (Janes et al., 1999; Janes et al., 2000). Further studies to test this hypothesis are in progress. Our studies are complimentary to analysis of immune synapses between T cells and antigen-presenting cells where lipid rafts are found in the contact region (see Faroudi et al., 2002).

Targeting function of lipid modification motifs

Lck is able to reconstitute TCR signaling in the Lck-deficient Jurkat-derived cell line JCam1. However, these cells already contain the related Src family kinase Fyn which is unable to compensate for the loss of Lck. We are studying the reasons for the functional differences between these two kinases, especially the role of the N-terminal dual acylation (SH4) motifs of these proteins, which differ in charge and the positions of S-acylations. Preliminary data suggest that the SH4 motifs of Fyn and Lck confer different functional properties on the rest of the molecule. We are currently using fluorescence resonance energy transfer (FRET) microscopy between the cyan and yellow variants of green fluorescent protein with alternative membrane targeting signals to study the domain organisation of these kinases and other components of TCR signaling cascades on the nanometer scale.

Lipid metabolism in the inner raft leaflet

Little is currently known about the lipid composition of the cytoplasmic leaflet of rafts. Interestingly, in most cell types examined cholesterol has been reported to be about three-fold more abundant in the cytoplasmic leaflet of the plasma membrane compared with the exoplasmic leaflet (Schroeder et al., 1996). Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a negatively charged lipid that has a well established role in the generation of second messengers and is also involved in signaling to the actin cytoskeleton and in vesicle trafficking (Caroni, 2001; Holz et al., 2000). PI(4,5)P2 and its metabolites, phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3, the product of PI 3-kinase action) and diacylglycerol (DAG, the product of phospholipase Cg hydrolysis) localised to CTB-aggregated lipid rafts as investigated using EGFP fusions of appropriate lipid-binding domains (Parmryd et al., submitted for publication; see also Faroudi et al., 2002). Imaging of live cells confirmed that PI(3,4,5)P3 is produced in rafts upon their aggregation. This demonstrates spatial coupling between an outer leaflet raft lipid (GM1) and inner leaflet lipids (PI(4,5)P2, PI(3,4,5)P3 and DAG). This may be mediated by interdigitation of long ceramide and acyl chains found in raft lipids (Simons and Ikonen, 1997). The association is surprisingly stable, despite the generally high lateral mobility of lipids and this is likely to be due to a long residence time of these lipids in rafts.

Association of Ras proteins with lipid rafts

Ras proteins are small G-proteins with oncogenic potential. There are four members of the mammalian Ras family, H-, N- and K-Ras, of which the latter exists in two splice variants, the major 4B and the minor 4A. Throughout the majority of their sequences, the isoforms are almost identical, the main differences residing in a short stretch at the C-terminus. In order to become associated with the plasma membrane, Ras proteins require either S-acylation (H-, N- and K-Ras(4A)) or a polybasic domain (K-Ras(4B)) in addition to prenylation at their C-termini (Hancock et al., 1990). There is controversy over the presence of Ras proteins in lipid rafts and the related structures caveolae, that may be formed from lipid rafts by the insertion of the membrane protein caveolin (Furuchi and Anderson, 1998; Roy et al., 1999). It has been proposed that prenylated proteins are reluctant to partition into rafts due to the bulkiness of the prenyl group, based on DRM reconstitution studies (Melkonian et al., 1999; Moffett et al., 2000). However, it has been demonstrated that there are differences in the strength of the association of various proteins with rafts and, consequently, loosely-associated raft proteins may not be retained in isolated DRMs (Janes et al., 1999). We have used the imaging approach to address this question in Jurkat T cells that have lipid rafts but do not have caveolin or caveolae. The localisation of Ras proteins was investigated using confocal microscopy imaging of intact Jurkat T cells that were labelled for the raft marker lipid GM1 which was then cross-linked to allow raft visualisation (Janes et al., 1999). Neither endogenous Ras proteins (mainly N- and K-Ras(4B)) nor transiently expressed EGFP fusions of Ras isoforms were excluded from aggregated lipid rafts. EGFP fusions of H- and K(4B)-Ras proteins were localised to the plasma membrane of transfected Jurkat cells and were not excluded from rafts, although they colocalised with them imperfectly. For H-Ras the activation status of the protein affected its level of association with patched rafts, activated H-Ras being if anything more highly enriched in rafts, but for K-Ras(4B) it did not. A fusion protein consisting of EGFP with the membrane-targeting C-terminal 20 amino acids of H-Ras showed an indistinguishable distribution to that of the full-length Ras proteins, suggesting that the targeting information was contained entirely in the lipid modification motif. The mechanisms of interaction of Ras proteins with the inner leaflet of raft domains remain to be elucidated, but as mentioned above, we have shown that the highly negatively-charged lipids PI(4,5)P2 and PI(3,4,5)P3 are concentrated in rafts suggesting that ionic interactions with a polybasic region could play a role in the case of the K-Ras(4B) protein.

Our data differ substantially from data obtained in the caveolin/caveolae-positive BHK cell line where H-Ras but not K-Ras(4B) was found to associate with cholesterol-dependent membrane domains and GTP loading of H-Ras caused it to move out of these domains into the bulk membrane (Roy et al., 1999; Prior et al., 2001). The differences between the data may reflect real differences in the organisation of membrane domains in cells with or without caveolin/caveolae.

SUMMARY

Strong evidence is accumulating for lipid rafts playing major roles in many cellular processes. The unique properties of rafts enable them to coordinate the activities of both protein and lipid components of cellular membranes. Our own data support an involvement of rafts in T cell signaling, by maintaining enhanced concentration and organisation of interacting components, and segregation of negative factors such as the phosphatase CD45. Further detailed studies will concentrate on the functional aspects of raft dynamics in regulating signaling processes.

ACKNOWLEDGEMENTS

The work reported in this manuscript was supported by the UK Medical Research Council (AIM, SNP, NP), EC FP5 programme grant VASCAN-2000 (QLG1-1999-00084) (JA, SNP), the Swedish Medical Research Council in collaboration with the Wellcome Trust (058922) and later the Swedish Cancer Fund (4350-B01-02SAA) (IP).

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Received: June 17, 2002. In revised form: June 26, 2002. Accepted: July 11, 2002