Spatial raft coalescence represents an initial step in Fc gamma R signaling

Characterization of lipid rafts as separated membrane microdomains consist of heterogeneous proteins suggesting that lateral assembly of rafts after Ag receptor cross-linking represents the earliest signal generating process. In line with the concept, cross-linked Ag receptors have been shown to associate with detergent-insoluble raft fraction without the aid of Src family kinases. However, it has not been established whether spatial raft coalescence could also precede Src family kinase activation. In this study, we showed that spatial raft coalescence after low-affinity FcgammaR cross-linking in RAW264.7 macrophages is independent of Src family kinase activity. The lateral raft assembly was found to be ascribed to the action of ligand-binding subunits, rather than to immunoreceptor tyrosine-based activation motif-bearing signal subunits, because monomeric murine FcgammaRIIb expressed in rat basophilic leukemia cells successfully induced spatial raft reorganization after cross-linking. We also showed that extracellular and transmembrane region of FcgammaRIIb is sufficient for raft stabilization. Moreover, this receptor fragment triggers rapid calcium mobilization and linker for activation of T cells phosphorylation, in a manner sensitive to Src family kinase inhibition and to cholesterol depletion. Presence of immunoreceptor tyrosine-based inhibitory motif and addition of immunoreceptor tyrosine-based activation motif to the receptor fragment abolished and enhanced the responses, respectively, but did not affect raft stabilization. These findings support the concept that ligand-binding subunit is responsible for raft coalescence, and that this event triggers initial biochemical signaling.     

The HIV-1 pathogenicity factor Nef interferes with maturation of stimulatory T-lymphocyte contacts by modulation of N-Wasp activity

The Nef protein is a key determinant of human immunodeficiency virus (HIV) pathogenicity that, among other activities, sensitizes T-lymphocytes for optimal virus production. The initial events by which Nef modulates the T-cell receptor (TCR) cascade are poorly understood. TCR engagement triggers actin rearrangements that control receptor clustering for signal initiation and dynamic organization of signaling protein complexes to form an immunological synapse. Here we report that Nef potently interferes with cell spreading and formation of actin-rich circumferential rings in T-lymphocytes upon surface-supported TCR stimulation. These effects were conserved among Nef proteins from different lentiviruses and occurred in HIV-1-infected primary human T-lymphocytes. This novel Nef activity critically depended on its Src homology 3 domain binding motif and required efficient association with Pak2 activity. Notably, whereas overall signaling microcluster formation immediately following TCR engagement occurred normally in Nef-expressing cells, the viral protein inhibited the concomitant activation of the actin organizer N-Wasp. During the subsequent maturation phase of the stimulatory contact, Nef interfered with the translocation of N-Wasp to the cell periphery, the overall induction of tyrosine phosphorylation, and the selective recruitment of phosphorylated LAT to stimulatory contacts. Consistent with such a critical role of N-Wasp in this process, Nef also blocked morphological changes induced by the known N-Wasp regulators Rac1 and Cdc42. Together, our results demonstrate that Nef alters both the amount and composition of signaling microclusters. We propose modulation of actin dynamics as an important mechanism for Nef-induced alterations of TCR signaling.     

TCR signal initiation machinery is pre-assembled and activated in a subset of membrane rafts

Recent studies suggest that rafts are involved in numerous cell functions, including membrane traffic and signaling. Here we demonstrate, using a polyoxyethylene ether Brij 98, that detergent-insoluble microdomains possessing the expected biochemical characteristics of rafts are present in the cell membrane at 37 degrees C. After extraction, these microdomains are visualized as membrane vesicles with a mean diameter of approximately 70 nm. These findings provide further evidence for the existence of rafts under physiological conditions and are the basis of a new isolation method allowing more accurate analyses of raft structure. We found that main components of T cell receptor (TCR) signal initiation machinery, i.e. TCR-CD3 complex, Lck and ZAP-70 kinases, and CD4 co-receptor are constitutively partitioned into a subset of rafts. Functional studies in both intact cells and isolated rafts showed that upon ligation, TCR initiates the signaling in this specialized raft subset. Our data thus strongly indicate an important role of rafts in organizing TCR early signaling pathways within small membrane microdomains, both prior to and following receptor engagement, for efficient TCR signal initiation upon stimulation.     

Distinct roles of cellular Lck and p80 proteins in herpesvirus saimiri Tip function on lipid rafts

Lipid rafts are proposed to function as platforms for both receptor signaling and trafficking. Following interaction with antigenic peptides, the T-cell receptor (TCR) rapidly translocates to lipid rafts, where it transmits signals and subsequently undergoes endocytosis. The Tip protein of herpesvirus saimiri (HVS), which is a T-lymphotropic tumor virus, interacts with cellular Lck tyrosine kinase and p80, a WD domain-containing endosomal protein. Interaction of Tip with p80 induces enlarged vesicles and recruits Lck and TCR complex into these vesicles for trafficking. We report here that Tip is constitutively present in lipid rafts and that Tip interaction with p80 but not with Lck is necessary for its efficient localization in lipid rafts. The Tip-Lck interaction was required for recruitment of the TCR complex to lipid rafts, and the Tip-p80 interaction was critical for the aggregation and internalization of lipid rafts. These results suggest the potential mechanism for Tip-mediated TCR downregulation: Tip interacts with Lck to recruit TCR complex to lipid rafts, and it subsequently interacts with p80 to initiate the aggregation and internalization of the lipid raft domain and thereby downregulate the TCR complex. Thus, the signaling and targeting functions of HVS Tip rely on two functionally and genetically separable mechanisms that independently target cellular Lck tyrosine kinase and p80 endosomal protein.     

Cell membrane extensions, generated by mechanical constraint, are associated with a sustained lipid raft patching and an increased cell signaling

Platelet activation triggers an imbalance in plasma membrane phospholipids by a specific aminophospholipid outflux, resulting in filopodia formation. Similarly, the addition of a phospholipid excess in the outer leaflet of the plasma membrane induces cellular extensions and actin polymerization. The implication of membrane microdomains in sustaining these mechanical constraints remains, however, unknown and was investigated in human platelets and mouse fibroblasts. The disruption of lipid rafts by cholesterol depletion prevents actin polymerization and formation of cellular extensions. Phospholipid excess triggers raft patching underneath the cell extensions, recruitment of protein raft markers and increase of tyrosine phosphorylation of raft proteins. Using a mass spectrometric analysis of isolated platelet rafts, we identified tyrosine kinases and proteins implicated in the formation of cell membrane extensions, cell adhesion and motility. They are recruited to rafts in response to a mechanical constraint. Taken together, our results demonstrate that exogenous phospholipid addition causes a modulation of the lateral plasma membrane organization and an activation of the cell signaling triggering actin remodeling and the formation of cellular protrusions. Raft disruption abolishes these processes, demonstrating that their integrity is crucial for cell shape changes in response to a mechanical constraint on plasma membrane.     

HIV-1 Nef employs two distinct mechanisms to modulate Lck subcellular localization and TCR induced actin remodeling

The Nef protein acts as critical factor during HIV pathogenesis by increasing HIV replication in vivo via the modulation of host cell vesicle transport and signal transduction processes. Recent studies suggested that Nef alters formation and function of immunological synapses (IS), thereby modulating exogenous T-cell receptor (TCR) stimulation to balance between partial T cell activation required for HIV-1 spread and prevention of activation induced cell death. Alterations of IS function by Nef include interference with cell spreading and actin polymerization upon TCR engagement, a pronounced intracellular accumulation of the Src kinase Lck and its reduced IS recruitment. Here we use a combination of Nef mutagenesis and pharmacological inhibition to analyze the relative contribution of these effects to Nef mediated alterations of IS organization and function on TCR stimulatory surfaces. Inhibition of actin polymerization and IS recruitment of Lck were governed by identical Nef determinants and correlated well with Nef's association with Pak2 kinase activity. In contrast, Nef mediated Lck endosomal accumulation was separable from these effects, occurred independently of Pak2, required integrity of the microtubule rather than the actin filament system and thus represents a distinct Nef activity. Finally, reduction of TCR signal transmission by Nef was linked to altered actin remodeling and Lck IS recruitment but did not require endosomal Lck rerouting. Thus, Nef affects IS function via multiple independent mechanisms to optimize virus replication in the infected host.     

Human immunodeficiency virus type 1 Nef recruits the guanine exchange factor Vav1 via an unexpected interface into plasma membrane microdomains for association with p21-activated kinase 2 activity

Alterations of T-cell receptor signaling by human immunodeficiency virus type 1 (HIV-1) Nef involve its association with a highly active subpopulation of p21-activated kinase 2 (PAK2) within a dynamic signalosome assembled in detergent-insoluble membrane microdomains. Nef-PAK2 complexes contain the GTPases Rac and Cdc42 as well as a factor providing guanine nucleotide exchange factor (GEF) activity for Rac/Cdc42. However, the identity of this GEF has remained controversial. Previous studies suggested the association of Nef with at least three independent GEFs, Vav, DOCK2/ELMO1, and βPix. Here we used a broad panel of approaches to address which of these GEFs is involved in the functional interaction of Nef with PAK2 activity. Biochemical fractionation and confocal microscopy revealed that Nef recruits Vav1, but not DOCK2/ELMO1 or βPix, to membrane microdomains. Transient RNAi knockdown, analysis of cell lines defective for expression of Vav1 or DOCK2 as well as use of a βPix binding-deficient PAK2 variant confirmed a role for Vav1 but not DOCK2 or βPix in Nef's association with PAK2 activity. Nef-mediated microdomain recruitment of Vav1 occurred independently of the Src homology 3 domain binding PxxP motif, which is known to connect Nef to many cellular signaling processes. Instead, a recently described protein interaction surface surrounding Nef residue F195 was identified as critical for Nef-mediated raft recruitment of Vav1. These results identify Vav1 as a relevant component of the Nef-PAK2 signalosome and provide a molecular basis for the role of F195 in formation of a catalytically active Nef-PAK2 complex.     

Epstein-Barr Virus LMP1 Modulates Lipid Raft Microdomains and the Vimentin Cytoskeleton for Signal Transduction and Transformation

The Epstein-Barr virus (EBV) is an important human pathogen that is associated with multiple cancers. The major oncoprotein of the virus, latent membrane protein 1 (LMP1), is essential for EBV B-cell immortalization and is sufficient to transform rodent fibroblasts. This viral transmembrane protein activates multiple cellular signaling pathways by engaging critical effector molecules and thus acts as a ligand-independent growth factor receptor. LMP1 is thought to signal from internal lipid raft containing membranes; however, the mechanisms through which these events occur remain largely unknown. Lipid rafts are microdomains within membranes that are rich in cholesterol and sphingolipids. Lipid rafts act as organization centers for biological processes, including signal transduction, protein trafficking, and pathogen entry and egress. In this study, the recruitment of key signaling components to lipid raft microdomains by LMP1 was analyzed. LMP1 increased the localization of phosphatidylinositol 3-kinase (PI3K) and its activated downstream target, Akt, to lipid rafts. In addition, mass spectrometry analyses identified elevated vimentin in rafts isolated from LMP1 expressing NPC cells. Disruption of lipid rafts through cholesterol depletion inhibited PI3K localization to membranes and decreased both Akt and ERK activation. Reduction of vimentin levels or disruption of its organization also decreased LMP1-mediated Akt and ERK activation and inhibited transformation of rodent fibroblasts. These findings indicate that LMP1 reorganizes membrane and cytoskeleton microdomains to modulate signal transduction.     

MHC class II molecules traffic into lipid rafts during intracellular transport

There have been many studies demonstrating that a portion of MHC class II molecules reside in detergent-insoluble membrane domains (commonly referred to as lipid rafts). We have proposed that the function of raft association is to concentrate specific MHC class II-peptide complexes in plasma membrane microdomains that can facilitate efficient T cell activation. We now show that MHC class II becomes lipid raft associated before binding antigenic peptides. Using pulse-chase radiolabeling techniques, we find that newly synthesized MHC class II and MHC class II-invariant chain complexes initially reside in a detergent-soluble membrane fraction and acquire detergent insolubility as they traffic to lysosomal Ag processing compartments. Monensin, an inhibitor of protein transport through the Golgi apparatus, blocks association of newly synthesized MHC class II with lipid rafts. Treatment of cells with leupeptin, which inhibits invariant chain degradation, leads to the accumulation of MHC class II in lipid rafts within the lysosome-like Ag-processing compartments. Raft fractionation of lysosomal membranes confirmed the presence of MHC class II in detergent-insoluble microdomains in Ag-processing compartments. These findings indicate that newly synthesized MHC class II complexes are directed to detergent-insoluble lipid raft microdomains before peptide loading, a process that may facilitate the loading of similar peptides on MHC class II complexes in these microdomains.     

Lipid raft-dependent and -independent signaling through HLA-DR molecules

Lipid rafts are plasma membrane microdomains that are highly enriched in signaling molecules and that act as signal transduction platforms for many immune receptors. The involvement of these microdomains in HLA-DR-induced signaling is less well defined. We examined the constitutive presence of HLA-DR molecules in lipid rafts, their possible recruitment into these microdomains, and the role of these microdomains in HLA-DR-induced responses. We detected significant amounts of HLA-DR molecules in the lipid rafts of EBV(+) and EBV(-) B cell lines, monocytic cell lines, transfected HeLa cells, tonsillar B cells, and human monocytes. Localization of HLA-DR in these microdomains was unaffected by the deletion of the cytoplasmic domain of both the alpha and beta chains. Ligation of HLA-DR with a bivalent, but not a monovalent, ligand resulted in rapid tyrosine phosphorylation of many substrates, especially Lyn, and activation of ERK1/2 MAP kinase. However, the treatment failed to induce further recruitment of HLA-DR molecules into lipid rafts. The HLA-DR-induced signaling events were accompanied by the induction of cell-cell adhesion that could be inhibited by PTK and Lyn but not ERK1/2 inhibitors. Disruption of lipid rafts by methyl-beta-cyclodextrin (MbetaCD) resulted in the loss of membrane raft association with HLA-DR molecules, inhibition of HLA-DR-mediated protein tyrosine phosphorylation and cell-cell adhesion. MbetaCD did not affect the activation of ERK1/2, which was absent from lipid rafts. These results indicate that although all the HLA-DR-induced events studied are dependent on HLA-DR dimerization, some require the presence of HLA-DR molecules in lipid rafts, whereas others do not.     

Quantitative proteomics analysis of macrophage rafts reveals compartmentalized activation of the proteasome and of proteasome-mediated ERK activation in response to lipopolysaccharide

Lipopolysaccharide (LPS), a glycolipid component of the outer membrane of Gram-negative bacteria, is a potent initiator of the innate immune response of the macrophage. LPS triggers downstream signaling by selectively recruiting and activating proteins in cholesterol-rich membrane microdomains called lipid rafts. We applied proteomics analysis to macrophage detergent-resistant membranes (DRMs) during an LPS exposure time course in an effort to identify and validate novel events occurring in macrophage rafts. Following metabolic incorporation in cell culture of heavy isotopes of amino acids arginine and lysine ([(13)C(6)]Arg and [(13)C(6)]Lys) or their light counterparts, a SILAC (stable isotope labeling with amino acids in cell culture)-based quantitative, liquid chromatography-tandem mass spectrometry proteomics approach was used to profile LPS-induced changes in the lipid raft proteome of RAW 264.7 macrophages. Unsupervised network analysis of the proteomics data set revealed a marked representation of the ubiquitin-proteasome system as well as changes in proteasome subunit composition following LPS challenge. Functional analysis of DRMs confirmed that LPS causes selective activation of the proteasome in macrophage rafts and proteasome inactivation outside of rafts. Given previous reports of an essential role for proteasomal degradation of IkappaB kinase-phosphorylated p105 in LPS activation of ERK mitogen-activated protein kinase, we tested for a role of rafts in compartmentalization of these events. Immunoblotting of DRMs revealed proteasome-dependent activation of MEK and ERK specifically occurring in lipid rafts as well as proteasomal activity upon raft-localized p105 that was enhanced by LPS. Cholesterol extraction from the intact macrophage with methyl-beta-cyclodextrin was sufficient to activate ERK, recapitulating the LPS-IkappaB kinase-p105-MEK-ERK cascade, whereas both it and the alternate raft-disrupting agent nystatin blocked subsequent LPS activation of the ERK cascade. Taken together, our findings indicate a critical, selective role for raft compartmentalization and regulation of proteasome activity in activation of the MEK-ERK pathway.     

Immature CD4+CD8+ thymocytes do not polarize lipid rafts in response to TCR-mediated signals

TCR-mediated stimulation induces activation and proliferation of mature T cells. When accompanied by signals through the costimulatory receptor CD28, TCR signals also result in the recruitment of cholesterol- and glycosphingolipid-rich membrane microdomains (lipid rafts), which are known to contain several molecules important for T cell signaling. Interestingly, immature CD4(+)CD8(+) thymocytes respond to TCR/CD28 costimulation not by proliferating, but by dying. In this study, we report that, although CD4(+)CD8(+) thymocytes polarize their actin cytoskeleton, they fail to recruit lipid rafts to the site of TCR/CD28 costimulation. We show that coupling of lipid raft mobilization to cytoskeletal reorganization can be mediated by phosphoinositide 3-kinase, and discuss the relevance of these findings to the interpretation of TCR signals by immature vs mature T cells.     

Spatiotemporal analysis of differential Akt regulation in plasma membrane microdomains

As a central kinase in the phosphatidylinositol 3-kinase pathway, Akt has been the subject of extensive research; yet, spatiotemporal regulation of Akt in different membrane microdomains remains largely unknown. To examine dynamic Akt activity in membrane microdomains in living cells, we developed a specific and sensitive fluorescence resonance energy transfer-based Akt activity reporter, AktAR, through systematic testing of different substrates and fluorescent proteins. Targeted AktAR reported higher Akt activity with faster activation kinetics within lipid rafts compared with nonraft regions of plasma membrane. Disruption of rafts attenuated platelet-derived growth factor (PDGF)-stimulated Akt activity in rafts without affecting that in nonraft regions. However, in insulin-like growth factor-1 (IGF)-1 stimulation, Akt signaling in nonraft regions is dependent on that in raft regions. As a result, cholesterol depletion diminishes Akt activity in both regions. Thus, Akt activities are differentially regulated in different membrane microdomains, and the overall activity of this oncogenic pathway is dependent on raft function. Given the increased abundance of lipid rafts in some cancer cells, the distinct Akt-activating characteristics of PDGF and IGF-1, in terms of both effectiveness and raft dependence, demonstrate the capabilities of different growth factor signaling pathways to transduce differential oncogenic signals across plasma membrane.     

Measles virus interacts with and alters signal transduction in T-cell lipid rafts

By a contact-dependent surface interaction, the measles virus (MV) glycoprotein complex induces a pronounced inhibition of T-cell proliferation. We now show that MV directly interacts with glycosphingolipid-enriched membrane microdomains on human primary T cells and alters recruitment and segregation of membrane proximal signaling components. Contact-dependent interference with T-cell receptor-stimulated tyrosine phosphorylation and Ca mobilization is a late event seen 24 h after MV treatment. In contrast, stimulated recruitment of pleckstrin homology domain-containing proteins such as Akt and Vav is inhibited early after MV contact, as is segregation of the activated Akt kinase from rafts. Tyrosine phosphorylation of the regulatory subunit of the phosphatidylinositol 3-kinase (PI3K), p85, is apparently normal then, yet this protein fails to partition to the lipid raft fraction, and this is associated with stable expression of its negative regulator Cbl-b. Thus, by interaction with lipid rafts, MV contact initially targets recruitment of PI3K by preventing stimulated Cbl-b degradation and activation of PI3K-dependent signaling components.     

Signaling proteins in raft-like microdomains are essential for Ca2+ wave propagation in glial cells

The hypothesis that calcium signaling proteins segregate into lipid raft-like microdomains was tested in isolated membranes of rat oligodendrocyte progenitor (OP) cells and astrocytes using Triton X-100 solubilization and density gradient centrifugation. Western blot analysis of gradient fractions showed co-localization of caveolin-1 with proteins involved in the Ca2+ signaling cascade. These included agonist receptors, P2Y1, and M1, TRPC1, IP3R2, ryanodine receptor, as well as the G protein Galphaq and Homer. Membranes isolated from agonist-stimulated astrocytes showed an enhanced recruitment of phospholipase C (PLCbeta1), IP3R2 and protein kinase C (PKC-alpha) into lipid raft fractions. IP3R2, TRPC1 and Homer co-immunoprecipitated, suggesting protein-protein interactions. Disruption of rafts by cholesterol depletion using methyl-beta-cyclodextrin (beta-MCD) altered the distribution of caveolin-1 and GM1 to non-raft fractions with higher densities. beta-MCD-induced disruption of rafts inhibited agonist-evoked Ca2+ wave propagation in astrocytes and attenuated wave speeds. These results indicate that in glial cells, Ca2+ signaling proteins might exist in organized membrane microdomains, and these complexes may include proteins from different cellular membrane systems. Such an organization is essential for Ca2+ wave propagation.     

Association of sterol- and glycosylphosphatidylinositol-linked proteins with Drosophila raft lipid microdomains

In vertebrates, the formation of raft lipid microdomains plays an important part in both polarized protein sorting and signal transduction. To establish a system in which raft-dependent processes could be studied genetically, we have analyzed the protein and lipid composition of these microdomains in Drosophila melanogaster. Using mass spectrometry, we identified the phospholipids, sphingolipids, and sterols present in Drosophila membranes. Despite chemical differences between Drosophila and mammalian lipids, their structure suggests that the biophysical properties that allow raft formation have been preserved. Consistent with this, we have identified a detergent-insoluble fraction of Drosophila membranes that, like mammalian rafts, is rich in sterol, sphingolipids, and glycosylphosphatidylinositol-linked proteins. We show that the sterol-linked Hedgehog N-terminal fragment associates specifically with this detergent-insoluble membrane fraction. Our findings demonstrate that raft formation is preserved across widely separated phyla in organisms with different lipid structures. They further suggest sterol modification as a novel mechanism for targeting proteins to raft membranes and raise the possibility that signaling and polarized intracellular transport of Hedgehog are based on raft association.     

Sensitization of epidermal growth factor-induced signaling by bradykinin is mediated by c-Src. Implications for a role of lipid microdomains

Communication between receptor tyrosine kinase (RTK)- and G protein-coupled receptor (GPCR)-mediated signaling systems has received increasing attention in recent years. Here, we report that activation of G protein-coupled bradykinin B2 receptor induces an up-regulation of cellular responses mediated by epidermal growth factor receptor (EGFR) and provide essential mechanistic characteristics of this sensitization process. EGF, which failed to evoke detectable amount of calcium increase and neurotransmitter release when administrated alone in primary cultures of rat adrenal chromaffin cells and PC12 cells, became capable of inducing these responses specifically after bradykinin pretreatment. Both EGFR and non-receptor tyrosine kinase p60Src, whose kinase activities were required in the sensitization, were found to be enriched in cholesterol-rich lipid rafts. Bradykinin caused activation of p60Src and Src-dependent phosphorylation of the EGFR on Tyr-845 in lipid rafts, as well as recruitment of phospholipase C (PLC) gamma1 to the rafts. Depletion of cholesterol by methyl-beta-cyclodextrin disrupted the raft localization of EGFR and Src, as well as bradykinin-induced translocation of PLCgamma1. Furthermore, sensitization, which was impaired by cholesterol depletion, was restored by repletion of cholesterol. Therefore, we suggest that lipid rafts are essential participants in the regulation of receptor-mediated signal transduction and cross-talk via organizing signaling complexes in membrane microdomains.