Selective accumulation of raft-associated membrane protein LAT in T cell receptor signaling assemblies

Activation of T cell antigen receptor (TCR) induces tyrosine phosphorylations that mediate the assembly of signaling protein complexes. Moreover, cholesterol-sphingolipid raft membrane domains have been implicated to play a role in TCR signal transduction. Here, we studied the assembly of TCR with signal transduction proteins and raft markers in plasma membrane subdomains of Jurkat T leukemic cells. We employed a novel method to immunoisolate plasma membrane subfragments that were highly concentrated in activated TCR-CD3 complexes and associated signaling proteins. We found that the raft transmembrane protein linker for activation of T cells (LAT), but not a palmitoylation-deficient non-raft LAT mutant, strongly accumulated in TCR-enriched immunoisolates in a tyrosine phosphorylation-dependent manner. In contrast, other raft-associated molecules, including protein tyrosine kinases Lck and Fyn, GM1, and cholesterol, were not highly concentrated in TCR-enriched plasma membrane immunoisolates. Many downstream signaling proteins coisolated with the TCR/LAT-enriched plasma membrane fragments, suggesting that LAT/TCR assemblies form a structural scaffold for TCR signal transduction proteins. Our results indicate that TCR signaling assemblies in plasma membrane subdomains, rather than generally concentrating raft-associated membrane proteins and lipids, form by a selective protein-mediated anchoring of the raft membrane protein LAT in vicinity of TCR.     

Combined spatial and enzymatic regulation of Csk by cAMP and protein kinase a inhibits T cell receptor signaling

Raft-associated Csk controls signaling through the T cell receptor (TCR) and was mainly anchored to Cbp/PAG (phosphoprotein associated with glycosphingolipid-enriched membrane domains). Treatment of cells with the cAMP-elevating agent prostaglandin E(2) (PGE(2)) augmented the level of Cbp/PAG phosphorylation with a concomitant increase in amounts of Csk bound to Cbp/PAG. While TCR-triggering resulted in transient dissociation of Csk from Cbp/PAG/rafts allowing TCR-induced tyrosine phosphorylation to occur, pretreatment with PGE(2) reduced Csk dissociation upon TCR triggering. This correlated with lowered TCR-induced phosphorylation of CD3 zeta-chain and linker for activation of T cells. Moreover, competition of endogenous Csk from lipid rafts abolished PGE(2)-mediated inhibition of TCR-induced zeta-chain phosphorylation and activation of the nuclear factor of activated T cells (NFAT) activator protein 1 (AP-1). Finally, raft-associated Csk already activated via Cbp/PAG binding, gained additional increase in phosphotransferase activity upon protein kinase A-mediated phosphorylation of Csk. We propose that cAMP regulates Csk via both spatial and enzymatic mechanisms, thereby inhibiting signaling through the TCR.     

Lipid rafts: cell surface platforms for T cell signaling

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 colocalizes 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 colocalization 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.     

Cutting edge: Fyn is essential for tyrosine phosphorylation of Csk-binding protein/phosphoprotein associated with glycolipid-enriched microdomains in lipid rafts in resting T cells

In resting T cells, Csk is constitutively localized in lipid rafts by virtue of interaction with a phosphorylated adaptor protein, Csk-binding protein (Cbp)/phosphoprotein associated with glycolipid-enriched microdomains, and sets an activation threshold in TCR signaling. In this study, we examined a kinase responsible for Cbp phosphorylation in T cell membrane rafts. By analyzing T cells from Fyn-/- mice, we clearly demonstrated that Fyn, but not Lck, has its kinase activity in membrane rafts, and plays a critical role in Cbp phosphorylation, Cbp-Csk interaction, and Csk kinase activity. Naive CD44(low)CD62 ligand(high) T cells were substantially reduced in Fyn-/- mice, presumably due to the inhibition of Cbp phosphorylation. Thus, Fyn mediates Cbp-Csk interaction and recruits Csk to rafts by phosphorylating Cbp. Csk recruited to rafts would then be activated and inhibit the kinase activity of Lck to keep resting T cells in a quiescent state. Our results elucidate a negative regulatory role for Fyn in proximal TCR signaling in lipid rafts.     

Membrane lipid rafts: new targets for immunoregulation

Engagement of immune receptors by antigen may lead to activation, cell proliferation, differentiation and effector functions. It has recently been proposed that the initiation and propagation of the signaling events taking place in immune cells occur in specialized membrane regions called lipid rafts. These detergent-insoluble glycolipid domains are specialized membrane compartments enriched in cholesterol and glycolipids. They also contain many lipid-modified signaling proteins such as tyrosine kinases of the Src family, GPI (glycosylphosphatidylinositol)-linked proteins as well as adaptor proteins. The confinement of signaling molecules in membrane subdomains suggests that lipid rafts function as platforms for the formation of multicomponent transduction complexes. Indeed, upon receptor binding, immune receptors become raft-associated and additional components of the signaling pathways are recruited to rafts in order to form signaling complexes. It has been speculated that the entry of immune receptors into rafts can regulate cell activation. Accordingly, numerous experiments have provided substantial evidence that raft integrity is crucial for the initiation and maintenance of intracellular signals. Recent studies have also shown that the access and translocation of immune receptors to lipid rafts are developmentally regulated (immature versus mature cells, Th1 versus Th2 lymphocytes) and sensitive to pharmacological agents. The aim of the present review is to summarize the current knowledge of immune receptor signal transduction with particular emphasis on the role of membrane compartments in immune activation. Finally, experimental evidences indicating that these membrane structures may represent clinically relevant potential targets for immune regulation, will be discussed.     

n-3 Fatty acids uniquely affect anti-microbial resistance and immune cell plasma membrane organization

It is now well established that dietary lipids are incorporated into macrophage and T-cell membrane microdomains, altering their structure and function. Within cell membranes, there are specific detergent-resistant domains in which key signal transduction proteins are localized. These regions are classified as “lipid rafts”. Rafts are composed mostly of cholesterol and sphingolipids and therefore do not integrate well into the fluid phospholipid bilayers causing them to form microdomains. Upon cell activation, rafts compartmentalize signal-transducing molecules, thus providing an environment conducive to signal transduction. In this review, we discuss recent novel data describing the effects of n−3 PUFA on alterations in the activation and functions of macrophages and T-cells. We believe that the modifications in these two disparate immune cell types are linked by fundamentally similar changes in membrane lipid composition and transmembrane signaling functions. We conclude that the outcomes of n−3 PUFA-mediated immune cell alterations may be beneficial (e.g., anti-inflammatory) or detrimental (e.g., loss of microbial immunity) depending upon the cell type interrogated.     

Dynamic association of CD45 with detergent-insoluble microdomains in T lymphocytes

The receptor-like protein tyrosine phosphatase CD45 is essential for TCR signal transduction. Substrates of CD45 include the protein tyrosine kinases p56(lck) and p59(fyn), both of which have been shown to be enriched in detergent-insoluble microdomains. Here we find that there is a cholesterol-dependent association between CD45 and the raft-associated protein linker for activation of T cells, suggesting that CD45 and linker for activation of T cells may colocalize in lipid rafts. Consistent with this observation, we find that approximately 5% of total CD45 can be detected in Triton X-100-insoluble buoyant fractions of sucrose gradients, demonstrating that CD45 is not excluded from lipid rafts. Upon stimulation of T cells with anti-CD3, there is a reduction in the amount of CD45 found associating with lipid rafts. Our data suggest that CD45 is present in lipid rafts in T cells before activation, perhaps to activate raft-associated p56(lck), allowing membrane-proximal signaling events to proceed. Furthermore, the reduction in CD45 content of lipid rafts after CD3 stimulation may serve to limit the amounts of activated p56(lck) in rafts and thus possibly the duration of T cell responses.     

Removal of C-terminal SRC kinase from the immune synapse by a new binding protein

The Csk tyrosine kinase negatively regulates the Src family kinases Lck and Fyn in T cells. Engagement of the T-cell antigen receptor results in a removal of Csk from the lipid raft-associated transmembrane protein PAG/Cbp. Instead, Csk becomes associated with an approximately 72-kDa tyrosine-phosphorylated protein, which we identify here as G3BP, a phosphoprotein reported to bind the SH3 domain of Ras GTPase-activating protein. G3BP reduced the ability of Csk to phosphorylate Lck at Y505 by decreasing the amount of Csk in lipid rafts. As a consequence, G3BP augmented T-cell activation as measured by interleukin-2 gene activation. Conversely, elimination of endogenous G3BP by RNA interference increased Lck Y505 phosphorylation and reduced TCR signaling. In antigen-specific T cells, endogenous G3BP moved into a intracellular location adjacent to the immune synapse, but deeper inside the cell, upon antigen recognition. Csk colocalization with G3BP occurred in this "parasynaptic" location. We conclude that G3BP is a new player in T-cell-antigen receptor signaling and acts to reduce the amount of Csk in the immune synapse.     

Spatiotemporal control of cyclic AMP immunomodulation through the PKA-Csk inhibitory pathway is achieved by anchoring to an Ezrin-EBP50-PAG scaffold in effector T cells

A variety of immunoregulatory signals to effector T cells from monocytes, macrophages and regulatory T cells act through cyclic adenosine monophosphate. In the effector T cell, the protein kinase A (PKA) type I isoenzyme localizes to lipid rafts during T cell activation and modulates directly the proximal events that take place after engagement of the T cell receptor. The most proximal target for PKA phosphorylation is C-terminal Src kinase (Csk), which initiates a negative signal pathway that fine-tunes the T cell activation process. The A kinase anchoring protein Ezrin colocalizes PKA and Csk by forming a supramolecular signaling complex consisting of PKA, Ezrin, Ezrin/radixin/moesin (ERM) binding protein of 50 kDa (EBP50), phosphoprotein associated with glycosphingolipid-enriched membrane microdomains (GEMs) (PAG) and Csk.     

The Csk-binding protein PAG regulates PDGF-induced Src mitogenic signaling via GM1

Spatial regulation is an important feature of signal specificity elicited by cytoplasmic tyrosine kinases of the Src family (SRC family protein tyrosine kinases [SFK]). Cholesterol-enriched membrane domains, such as caveolae, regulate association of SFK with the platelet-derived growth factor receptor (PDGFR), which is needed for kinase activation and mitogenic signaling. PAG, a ubiquitously expressed member of the transmembrane adaptor protein family, is known to negatively regulate SFK signaling though binding to Csk. We report that PAG modulates PDGFR levels in caveolae and SFK mitogenic signaling through a Csk-independent mechanism. Regulation of SFK mitogenic activity by PAG requires the first N-terminal 97 aa (PAG-N), which include the extracellular and transmembrane domains, palmitoylation sites, and a short cytoplasmic sequence. We also show that PAG-N increases ganglioside GM1 levels at the cell surface and, thus, displaces PDGFR from caveolae, a process that requires the ganglioside-specific sialidase Neu-3. In conclusion, PAG regulates PDGFR membrane partitioning and SFK mitogenic signaling by modulating GM1 levels within caveolae independently from Csk.     

Glucocorticoids alter the lipid and protein composition of membrane rafts of a murine T cell hybridoma

Glucocorticoids (GC) are widely used anti-inflammatory agents known to suppress T cell activation by interfering with the TCR activation cascade. The attenuation of early TCR signaling events by these compounds has been recently attributed to a selective displacement of key signaling proteins from membrane lipid rafts. In this study, we demonstrate that GC displace the acyl-bound adaptor proteins linker for activation of T cells and phosphoprotein associated with glycosphingolipid-enriched microdomains from lipid rafts of murine T cell hybridomas, possibly by inhibiting their palmitoylation status. Analysis of the lipid content of the membrane rafts revealed that GC treatment led to a significant decrease in palmitic acid content. Moreover, we found an overall decrease in the proportion of raft-associated saturated fatty acids. These changes were consistent with a decrease in fluorescence anisotropy of isolated lipid rafts, indicating an increase in their fluidity. These findings identify the mechanisms underlying the complex inhibitory effects of glucocorticoids on early TCR signaling and suggest that some of the inhibitory properties of GC on T cell responses may be related to their ability to affect the membrane lipid composition and the palmitoylation status of important signaling molecules.     

Mechanism of Csk-mediated down-regulation of Src family tyrosine kinases in epidermal growth factor signaling

The Src family tyrosine kinases (SFKs) play pivotal roles as molecular switches that link a variety of extracellular cues to intracellular signaling pathway. The function of SFK is regulated by phosphorylation at the C-terminal regulatory site mediated by Csk. Recently a novel SFK target Cbp (or PAG) was identified as a membrane-anchored scaffold protein for Csk. To establish the mechanism of Csk/Cbp-mediated regulation of SFK in vivo, we observed dynamic changes in the interaction of Csk with Cbp by utilizing fusion proteins with modified green fluorescent proteins: cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP). Upon SFK activation induced by epidermal growth factor stimulation, fluorescent resonance energy transfer (FRET) response was detected transiently at membrane ruffles in COS1 cells co-expressing CFP-Csk and Cbp-YFP and in cells expressing a single-molecule FRET indicator consisting of CskSH2 and Cbp. Suppression of SFK by PP2 or use of a mutant Cbp that lacks the Csk binding site abolished the FRET response, although a dominant-negative form of Csk enhanced and sustained the FRET response, demonstrating that the FRET response is dependent upon the SFK activity. These observations show that Csk/Cbp-mediated down-regulation of SFK takes place at membrane ruffles in an early stage of epidermal growth factor signaling and suggest that the Csk/Cbp-based FRET indicators are useful for monitoring the status of SFK in living cells.     

CD40 signaling in human dendritic cells is initiated within membrane rafts

Despite CD40's role in stimulating dendritic cells (DCs) for efficient specific T-cell stimulation, its signal transduction components in DCs are still poorly documented. We show that CD40 receptors on human monocyte-derived DCs associate with sphingolipid- and cholesterol-rich plasma membrane microdomains, termed membrane rafts. Following engagement, CD40 utilizes membrane raft-associated Lyn Src family kinase, and possibly other raft-associated Src family kinases, to initiate tyrosine phosphorylation of intracellular substrates. CD40 engagement also leads to a membrane raft-restricted recruitment of tumor necrosis factor (TNF) receptor-associated factor (TRAF) 3 and, to a lesser extent, TRAF2, to CD40's cytoplasmic tail. Thus, the membrane raft structure plays an integral role in proximal events of CD40 signaling in DCs. We demonstrate that stimulation of Src family kinase within membrane rafts initiates a pathway implicating ERK activation, which leads to interleukin (IL)-1alpha/beta and IL-1Ra mRNA production and contributes to p38-dependent IL-12 mRNA production. These results provide the first evidence that membrane rafts play a critical role in initiation of CD40 signaling in DCs, and delineate the outcome of CD40-mediated pathways on cytokine production.     

Dynamic changes in the mobility of LAT in aggregated lipid rafts upon T cell activation

Lipid rafts are known to aggregate in response to various stimuli. By way of raft aggregation after stimulation, signaling molecules in rafts accumulate and interact so that the signal received at a given membrane receptor is amplified efficiently from the site of aggregation. To elucidate the process of lipid raft aggregation during T cell activation, we analyzed the dynamic changes of a raft-associated protein, linker for activation of T cells (LAT), on T cell receptor stimulation using LAT fused to GFP (LAT-GFP). When transfectants expressing LAT-GFP were stimulated with anti-CD3-coated beads, LAT-GFP aggregated and formed patches at the area of bead contact. Photobleaching experiments using live cells revealed that LAT-GFP in patches was markedly less mobile than that in nonpatched regions. The decreased mobility in patches was dependent on raft organization supported by membrane cholesterol and signaling molecule binding sites, especially the phospholipase C gamma 1 binding site in the cytoplasmic domain of LAT. Thus, although LAT normally moves rapidly at the plasma membrane, it loses its mobility and becomes stably associated with aggregated rafts to ensure organized and sustained signal transduction required for T cell activation.     

Activation of c-Src and Fyn kinases by protein-tyrosine phosphatase RPTPalpha is substrate-specific and compatible with lipid raft localization

Src family tyrosine kinases (SFKs) function in multiple signaling pathways, raising the question of how appropriate regulation and substrate choice are achieved. SFK activity is modulated by several protein-tyrosine phosphatases, among which RPTPalpha and SHP2 are the best established. We studied how RPTPalpha affects substrate specificity and regulation of c-Src and Fyn in response to epidermal growth factor and platelet-derived growth factor. We find that RPTPalpha, in a growth factor-specific manner, directs the specificity of these kinases toward a specific subset of SFK substrates, particularly the focal adhesion protein Paxillin and the lipid raft scaffolding protein Cbp/PAG. A significant fraction of RPTPalpha is present in lipid rafts, where its targets Fyn and Cbp/PAG reside, and growth factor-mediated SFK activation within this compartment is strictly dependent on RPTPalpha. Forced concentration of RPTPalpha into lipid rafts is compatible with activation of Fyn. Finally, RPTPalpha-induced phosphorylation of Paxillin and Cbp/PAG induces recruitment of the SFK inhibitory kinase Csk, indicative of negative feedback loops limiting SFK activation by RPTPalpha. Our findings indicate that individual SFK-controlling PTPs play important and specific roles in dictating SFK substrate specificity and regulatory mechanism.     

Lipid raft heterogeneity in human peripheral blood T lymphoblasts: a mechanism for regulating the initiation of TCR signal transduction

Lateral mobility and spatial organization of proteins within the plasma membrane are likely to mediate the initial events coordinating T cell activation. Lipid rafts, distinct cholesterol/sphingolipid-rich membrane microdomains, provide a mechanism for this regulation by concentrating or excluding signaling proteins. We demonstrate in peripheral blood T cell lymphoblasts that immediate early phosphotyrosine signal transduction through the TCR complex is functionally dependent on a distinct population of lipid rafts. Specifically, cholesterol extraction destabilizes the membrane microdomains containing Lck, while the rafts containing the adapter protein linker for activation of T cells remain intact. Heterogeneity in the partitioning of these proteins in resting cells was confirmed by immunoelectron microscopy. After T cell activation, both Lck and the linker for activation of T cells colocalize to 50-100 nm microdomains in the plasma membrane, indicating that sequestration of these proteins into distinct lipid rafts may function to regulate the initiation of T cell signal transduction.     

The ubiquitously expressed Csk adaptor protein Cbp is dispensable for embryogenesis and T-cell development and function

Regulation of Src family kinase (SFK) activity is indispensable for a functional immune system and embryogenesis. The activity of SFKs is inhibited by the presence of the carboxy-terminal Src kinase (Csk) at the cell membrane. Thus, recruitment of cytosolic Csk to the membrane-associated SFKs is crucial for its regulatory function. Previous studies utilizing in vitro and transgenic models suggested that the Csk-binding protein (Cbp), also known as phosphoprotein associated with glycosphingolipid microdomains (PAG), is the membrane adaptor for Csk. However, loss-of-function genetic evidence to support this notion was lacking. Herein, we demonstrate that the targeted disruption of the cbp gene in mice has no effect on embryogenesis, thymic development, or T-cell functions in vivo. Moreover, recruitment of Csk to the specialized membrane compartment of "lipid rafts" is not impaired by Cbp deficiency. Our results indicate that Cbp is dispensable for the recruitment of Csk to the membrane and that another Csk adaptor, yet to be discovered, compensates for the loss of Cbp.     

T-Cell Receptor Microclusters Critical for T-Cell Activation Are Formed Independently of Lipid Raft Clustering

We studied the function of lipid rafts in generation and signaling of T-cell receptor microclusters (TCR-MCs) and central supramolecular activation clusters (cSMACs) at immunological synapse (IS). It has been suggested that lipid raft accumulation creates a platform for recruitment of signaling molecules upon T-cell activation. However, several lipid raft probes did not accumulate at TCR-MCs or cSMACs even with costimulation and the fluorescence resonance energy transfer (FRET) between TCR or LAT and lipid raft probes was not induced at TCR-MCs under the condition of positive induction of FRET between CD3ζ and ZAP-70. The analysis of LAT mutants revealed that raft association is essential for the membrane localization but dispensable for TCR-MC formation. Careful analysis of the accumulation of raft probes in the cell interface revealed that their accumulation occurred after cSMAC formation, probably due to membrane ruffling and/or endocytosis. These results suggest that lipid rafts control protein translocation to the membrane but are not involved in the clustering of raft-associated molecules and therefore that the lipid rafts do not serve as a platform for T-cell activation.Lipid rafts are specialized liquid-ordered membrane microdomains that are enriched in cholesterol and sphingolipids. Many studies using various methodologies have shown that lipid rafts exist as leaflets less than 200 nm in size and float on the plasma membrane (6, 10, 24, 28, 32). They have been implied to play a role in protein sorting and cell activation as a platform by recruiting various signaling molecules such as Src family kinases, G proteins, and adaptor molecules. Because of size limitation, all of the raft-associated molecules could not be accommodated on the same lipid raft, and heterogeneity of lipid rafts both in size and in the repertoire of resident molecules has been suggested (22). The functional importance of lipid rafts in signal transduction has been particularly appreciated in T-cell activation through the T-cell receptor (TCR). Some of the initial observations in this area included the findings that cross-linking of the raft-associated ganglioside GM1 induces T-cell activation (12) and that a mutant of LAT, a membrane adaptor protein, that was unable to localize to rafts failed to induce activation signals (33). Since then, increasing data have demonstrated that lipid raft accumulation creates a platform to stabilize the signaling complex for T-cell activation (13, 29).T cells are activated upon recognition of peptide-major histocompatibility complex (MHC) complexes expressed on antigen-presenting cells (APC). An immunological synapse (IS) is formed at the interface between the T cell and the APC where a specialized segregated structure of T-cell surface receptors is generated. This supramolecular activation cluster (SMAC) contains the TCR in the central region (cSMAC) and lymphocyte function-associated antigen 1 (LFA-1) in the peripheral region (pSMAC). The accumulation of lipid rafts at this interface, particularly in the cSMAC, has been suggested to create a transient structure to mediate signal transduction (13, 17). In addition, CD28-mediated costimulation has been suggested to enhance lipid raft accumulation and TCR activation (29). However, the idea that lipid rafts accumulated in the cSMAC serve as the platform for T-cell activation has been controversial; the accumulation of the lipid raft was only partial in the contact area (3), or the concentration of lipid raft was constant even in the area of T-cell activation (5, 8, 28, 32). These variations could be partly attributed to differences in experimental approaches such as the cell systems being analyzed, stimulation conditions, and detection methods, including imaging and biochemical fractionation. The idea that the cSMAC is the site responsible for inducing signals for T-cell activation has been recently revised based on analysis of the dynamic assembly of signaling complexes upon TCR stimulation. Analysis of T-cell activation using a planar membrane system containing glycosylphosphatidylinositol (GPI)-anchored MHC-peptide complexes and the LFA-1 ligand intercellular adhesion molecule 1 (ICAM-1) revealed that small clusters containing approximately a hundred TCRs, kinases, and adaptors, which we termed TCR microclusters (MCs), were generated at the initial contact sites. This was followed by translocation of the MCs to the center of the interface to generate a cSMAC (31). Since protein phosphorylation, including that of ZAP-70, was induced in the TCR-MCs and Ca²⁺ mobilization was induced in parallel with the formation of TCR-MCs, these MCs appear to be the very first and minimum unit for generating TCR activation signals (31). Furthermore, a major costimulatory receptor, CD28, forms clusters which are also colocalized in TCR-MCs to regulate costimulatory signals (30).Among these TCR proximal signaling molecules, LAT is a well-studied raft-associated membrane adaptor protein that is indispensable for TCR activation. LAT is phosphorylated by ZAP-70 and then behaves as a signal scaffold, recruiting various signaling adaptors and effector molecules such as phospholipase Cγ (PLCγ), SLP-76, and Grb2/Gads. Because mutation of LAT palmitoylation sites (C26,29A) resulted in its dislocation from lipid rafts and defective signaling, it was concluded that the association with lipid rafts is essential for the function of LAT (33). However, a recent study showed that this mutant LAT has impaired trafficking to the plasma membrane in the Jurkat T-cell line (27), raising the question of whether the impaired signaling resulting from this LAT mutation was due to dislocation from the raft or defective trafficking to the membrane.Here, we analyzed the role of lipid rafts in T-cell activation, particularly their relationship with immunological synapse formation (9). Provided that lipid raft functions as a platform for T-cell activation, the new idea that TCR-MCs serve as the signal unit for activation would predict that lipid raft could be accumulated in or interact with TCR-MCs (29).Utilizing several lipid raft probes, which retain the capability of raft localization but lack signaling capacity, we found that the full-length LAT generated MCs, but none of the raft probes formed visible clusters at TCR-MCs or cSMAC, even in conjunction with CD28-mediated costimulation. Furthermore, no significant interaction between lipid rafts and TCR-MCs was revealed by fluorescence resonance energy transfer (FRET) analysis. Conversely, the non-raft-localizing LAT mutant showed MC formation upon TCR stimulation. These results suggest that lipid rafts do not serve as a platform for TCR signaling but rather regulate the traffic/recruitment of proteins to the plasma membrane. Furthermore, our data indicate that the previous observation of lipid raft accumulation at the cSMAC may reflect membrane ruffling and endocytosis rather than active formation of signal platform.     

Cyclodextrins but not compactin inhibit the lateral diffusion of membrane proteins independent of cholesterol

Cholesterol and glycosphingolipid-enriched membrane domains, termed lipid rafts, were proposed to play important roles in trafficking and signaling events. These functions are inhibited following putative disruption of rafts by cholesterol depletion, commonly induced by treatment with methyl-beta-cyclodextrin (MbetaCD). However, several studies showed that the lateral diffusion of membrane proteins is inhibited by MbetaCD, suggesting that it may have additional effects on membrane organization unrelated to cholesterol removal. Here, we investigated this possibility by comparison of the effects of cholesterol depletion by MbetaCD and by metabolic inhibition (compactin), and of treatment with alpha-CD, which does not bind cholesterol. The studies employed two series of proteins (Ras and influenza hemagglutinin), each containing as internal controls related mutants that differ in raft association. Mild MbetaCD treatment retarded the lateral diffusion of both raft and non-raft mutants, whereas similar cholesterol reduction (30-33%) by metabolic inhibition enhanced selectively the diffusion of the raft-associated mutants. Moreover, alpha-CD also inhibited the diffusion of raft and non-raft mutants, despite its lack of effect on cholesterol content. These findings suggest that the widely used treatment with CD to reduce cholesterol has additional, cholesterol-independent effects on membrane protein mobility, which do not necessarily distinguish between raft and non-raft proteins.