Localization of Src homology 2 domain-containing phosphatase 1 (SHP-1) to lipid rafts in T lymphocytes: functional implications and a role for the SHP-1 carboxyl terminus

The protein tyrosine phosphatase Src homology 2 domain-containing phosphatase 1 (SHP-1) has previously been shown to be a negative regulator of signaling mediated via the TCR. A growing body of evidence indicates that the regulated localization of proteins within certain membrane subdomains, referred to as lipid rafts, is important for the successful transduction of signaling events downstream of the TCR. However, considerably less is known about the localization of negative regulators during these lipid raft-dependent signaling events. In this study we have investigated the subcellular localization of SHP-1 and its role in regulation of TCR-mediated signaling. Our studies demonstrate that in a murine T cell hybridoma as well as in primary murine thymocytes, a fraction of SHP-1 localizes to the lipid rafts, both basally and after TCR stimulation. Interestingly, although SHP-1 localized in the nonraft fractions is tyrosine phosphorylated, the SHP-1 isolated from the lipid rafts lacks the TCR-induced tyrosine phosphorylation, suggesting physical and/or functional differences between these two subpopulations. We identify a requirement for the C-terminal residues of SHP-1 in optimal localization to the lipid rafts. Although expression of SHP-1 that localizes to lipid rafts potently inhibits TCR-mediated early signaling events and IL-2 production, the expression of lipid raft-excluded SHP-1 mutants fails to elicit any of the inhibitory effects. Taken together these studies reveal a key role for lipid raft localization of SHP-1 in mediating the inhibitory effects on T cell signaling events.     

Targeting Src homology 2 domain-containing tyrosine phosphatase (SHP-1) into lipid rafts inhibits CD3-induced T cell activation

To study the mechanism by which protein tyrosine phosphatases (PTPs) regulate CD3-induced tyrosine phosphorylation, we investigated the distribution of PTPs in subdomains of plasma membrane. We report here that the bulk PTP activity associated with T cell membrane is present outside the lipid rafts, as determined by sucrose density gradient sedimentation. In Jurkat T cells, approximately 5--10% of Src homology 2 domain-containing tyrosine phosphatase (SHP-1) is constitutively associated with plasma membrane, and nearly 50% of SHP-2 is translocated to plasma membrane after vanadate treatment. Similar to transmembrane PTP, CD45, the membrane-associated populations of SHP-1 and SHP-2 are essentially excluded from lipid rafts, where other signaling molecules such as Lck, linker for activation of T cells, and CD3 zeta are enriched. We further demonstrated that CD3-induced tyrosine phosphorylation of these substrates is largely restricted to lipid rafts, unless PTPs are inhibited. It suggests that a restricted partition of PTPs among membrane subdomains may regulate protein tyrosine phosphorylation in T cell membrane. To test this hypothesis, we targeted SHP-1 into lipid rafts by using the N-terminal region of Lck (residues 1--14). The results indicate that the expression of Lck/SHP-1 chimera inside lipid rafts profoundly inhibits CD3-induced tyrosine phosphorylation of CD3 zeta/epsilon, IL-2 generation, and nuclear mobilization of NF-AT. Collectively, these results suggest that the exclusion of PTPs from lipid rafts may be a mechanism that potentiates TCR/CD3 activation.     

A molecular framework for two-step T cell signaling: Lck Src homology 3 mutations discriminate distinctly regulated lipid raft reorganization events

Costimulation by CD28 or lipid-raft-associated CD48 potentiate TCR-induced signals, cytoskeletal reorganization, and IL-2 production. We and others have proposed that costimulators function to construct a raft-based platform(s) especially suited for TCR engagement and sustained and processive signal transduction. Here, we characterize TCR/CD48 and TCR/CD28 costimulation in T cells expressing Lck Src homology 3 (SH3) mutants. We demonstrate that Lck SH3 functions after initiation of TCR-induced tyrosine phosphorylation and concentration of transducers within rafts, to regulate the costimulation-dependent migration of rafts to the TCR contact site. Expression of kinase-active/SH3-impaired Lck mutants disrupts costimulation-dependent raft recruitment, sustained TCR protein tyrosine phosphorylation, and IL-2 production. However, TCR-induced apoptosis, shown only to require "partial" TCR signals, is unaffected by expression of kinase-active/SH3-impaired Lck mutants. Therefore, two distinctly regulated raft reorganization events are required for processive and sustained "complete" TCR signal transduction and T cell activation. Together with recent characterization of CD28 and CD48 costimulatory activities, these findings provide a molecular framework for two signal models of T cell activation.     

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.     

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.     

Caspase-8 regulation by direct interaction with TRAF6 in T cell receptor-induced NF-kappaB activation

Triggering of lymphocyte antigen receptors is the critical first step in the adaptive immune response against pathogens. T cell receptor (TCR) ligation assembles a large membrane signalosome, culminating in NF-kappaB activation [1,2]. Recently, caspase-8 was found to play a surprisingly prominent role in lymphocyte activation in addition to its well-known role in apoptosis [3]. Caspase-8 is activated after TCR stimulation and nucleates a complex with B cell lymphoma 10 (BCL10), paracaspase MALT1, and the inhibitors of kappaB kinase (IKK) complex [4]. We now report that the ubiquitin ligase TRAF6 binds to active caspase-8 upon TCR stimulation and facilitates its movement into lipid rafts. We identified in silico two putative TRAF6 binding motifs in the caspase-8 sequence and found that mutation of critical residues within these sites abolished TRAF6 binding and diminished TCR-induced NF-kappaB activation. Moreover, RNAi-mediated silencing of TRAF6 abrogated caspase-8 recruitment to the lipid rafts. Protein kinase Ctheta (PKCtheta), CARMA1, and BCL10 are also required for TCR-induced caspase-8 relocation, but only PKCtheta and BCL10 control caspase-8 activation. Our results suggest that PKCtheta independently controls CARMA1 phosphorylation and BCL10-dependent caspase-8 activation and unveil an essential role for TRAF6 as a critical adaptor linking these two convergent signaling events.     

A lipid raft environment enhances Lyn kinase activity by protecting the active site tyrosine from dephosphorylation

The plasma membrane contains ordered lipid domains, commonly called lipid rafts, enriched in cholesterol, sphingolipids, and certain signaling proteins. Lipid rafts play a structural role in signal initiation by the high affinity receptor for IgE. Cross-linking of IgE-receptor complexes by antigen causes their coalescence with lipid rafts, where they are phosphorylated by the Src family tyrosine kinase, Lyn. To understand how lipid rafts participate in functional coupling between Lyn and FcepsilonRI, we investigated whether the lipid raft environment influences the specific activity of Lyn. We used differential detergent solubility and sucrose gradient fractionation to isolate Lyn from raft and nonraft regions of the plasma membrane in the presence or absence of tyrosine phosphatase inhibitors. We show that Lyn recovered from lipid rafts has a substantially higher specific activity than Lyn from nonraft environments. Furthermore, this higher specific activity correlates with increased tyrosine phosphorylation at the active site loop of the kinase domain. Based on these results, we propose that lipid rafts exclude a phosphatase that negatively regulates Lyn kinase activity by constitutive dephosphorylation of the kinase domain tyrosine residue of Lyn. In this model, cross-linking of FcepsilonRI promotes its proximity to active Lyn in a lipid raft environment.     

Lipid rafts in T cell receptor signalling

The molecular events and the protein components that are involved in signalling by the T cell receptor (TCR) for antigen have been extensively studied. Activation of signalling cascades following TCR stimulation depends on the phosphorylation of the receptor by the tyrosine kinase Lck, which localizes to the cytoplasmic face of the plasma membrane by virtue of its post-translational modification. However, the precise order of events during TCR phosphorylation at the plasma membrane, remains to be defined. A current theory that describes early signalling events incorporates the function of lipid rafts, microdomains at the plasma membrane with distinct lipid and protein composition. Lipid rafts have been implicated in diverse biological functions in mammalian cells. In T cells, molecules with a key role in TCR signalling, including Lck, localize to these domains. Importantly, mutant versions of these proteins which fail to localise to raft domains were unable to support signalling by the TCR. Biochemical studies using purified detergent-resistant membranes (DRM) and confocal microscopy have suggested that upon stimulation, the TCR and Lck-containing lipid rafts may come into proximity allowing phosphorylation of the receptor. Further, there are data suggesting that phosphorylation of the TCR could depend on a transient increase in Lck activity that takes place within lipid rafts to initiate signalling. Current results and a model of how lipid rafts may regulate TCR signalling are discussed.     

CD5 negatively regulates the T-cell antigen receptor signal transduction pathway: involvement of SH2-containing phosphotyrosine phosphatase SHP-1

The negative regulation of T- or B-cell antigen receptor signaling by CD5 was proposed based on studies of thymocytes and peritoneal B-1a cells from CD5-deficient mice. Here, we show that CD5 is constitutively associated with phosphotyrosine phosphatase activity in Jurkat T cells. CD5 was found associated with the Src homology 2 (SH2) domain containing hematopoietic phosphotyrosine phosphatase SHP-1 in both Jurkat cells and normal phytohemagglutinin-expanded T lymphoblasts. This interaction was increased upon T-cell receptor (TCR)-CD3 cell stimulation. CD5 co-cross-linking with the TCR-CD3 complex down-regulated the TCR-CD3-increased Ca2+ mobilization in Jurkat cells. In addition, stimulation of Jurkat cells or normal phytohemagglutinin-expanded T lymphoblasts through TCR-CD3 induced rapid tyrosine phosphorylation of several protein substrates, which was substantially diminished after CD5 cross-linking. The CD5-regulated substrates included CD3zeta, ZAP-70, Syk, and phospholipase Cgammal but not the Src family tyrosine kinase p56(lck). By mutation of all four CD5 intracellular tyrosine residues to phenylalanine, we found the membrane-proximal tyrosine at position 378, which is located in an immunoreceptor tyrosine-based inhibitory (ITIM)-like motif, crucial for SHP-1 association. The F378 point mutation ablated both SHP-1 binding and the down-regulating activity of CD5 during TCR-CD3 stimulation. These results suggest a critical role of the CD5 ITIM-like motif, which by binding to SHP-1 mediates the down-regulatory activity of this receptor.     

Selective down-regulation of angiotensin II receptor type 1A signaling by protein tyrosine phosphatase SHP-2 in vascular smooth muscle cells

The heptahelical AT(1) G-protein-coupled receptor lacks inherent tyrosine kinase activity. Angiotensin II binding to AT(1) nevertheless activates several tyrosine kinases and stimulates both tyrosine phosphorylation and phosphatase activity of the SHP-2 tyrosine phosphatase in vascular smooth muscle cells. Since a balance between tyrosine kinase and tyrosine phosphatase activities is essential in angiotensin II signaling, we investigated the role of SHP-2 in modulating tyrosine kinase signaling pathways by stably transfecting vascular smooth muscle cells with expression vectors encoding wild-type SHP-2 protein or a catalytically inactive SHP-2 mutant. Our data indicate that SHP-2 is an efficient negative regulator of angiotensin II signaling. SHP-2 inhibited c-Src catalytic activity by dephosphorylating a positive regulatory tyrosine 418 within the Src kinase domain. Importantly, SHP-2 expression also abrogated angiotensin II-induced activation of ERK, whereas expression of catalytically inactive SHP-2 caused sustained ERK activation. Thus, SHP-2 likely regulates angiotensin II-induced MAP kinase signaling by inactivating c-Src. These SHP-2 effects were specific for a subset of angiotensin II signaling pathways, since SHP-2 overexpression failed to influence Jak2 tyrosine phosphorylation or Fyn catalytic activity. These data show SHP-2 represents a critical negative regulator of angiotensin II signaling, and further demonstrate a new function for this phosphatase in vascular smooth muscle cells.     

Dynamic recruitment of human CD2 into lipid rafts. Linkage to T cell signal transduction

CD2 mediates T cell adhesion via its ectodomain and signal transduction utilizing its 117-amino acid cytoplasmic tail. Here we show that a significant fraction of human CD2 molecules is inducibly recruited into lipid rafts upon CD2 cross-linking by a specific pair of mitogenic anti-CD2 monoclonal antibodies (anti-T11(2) + anti-T11(3)) or during cellular conjugate formation by CD58, the physiologic ligand expressed on antigen-presenting cells. Translocation to lipid microdomains is independent of the T cell receptor (TCR) and, unlike inducible TCR-raft association, requires no tyrosine phosphorylation. Structural integrity of rafts is necessary for CD2-stimulated elevation of intracellular free calcium and tyrosine phosphorylation of cellular substrates. Whereas murine CD2 contains two membrane-proximal intracellular cysteines, partitioning CD2 into cholesterol-rich lipid rafts constitutively, human CD2 has no cytoplasmic cysteines. Mapping studies using CD2 point mutation, deletion, and chimeric molecules suggest that conformational change in the CD2 ectodomain participates in inducible raft association and excludes the membrane-proximal N-linked glycans, the transmembrane segment, and the CD2 cytoplasmic region (residues 8-117) as necessary for translocation. Translocation of CD2 into lipid rafts may reorganize the membrane into an activation-ready state prior to TCR engagement by a peptide associated with a major histocompatibility complex molecule, accounting for synergistic T cell stimulation by CD2 and the TCR.     

Transient translocation of the B cell receptor and Src homology 2 domain-containing inositol phosphatase to lipid rafts: evidence toward a role in calcium regulation

Membrane microdomains (lipid rafts) are enriched in selected signaling molecules and may compartmentalize receptor-mediated signals. Here, we report that in primary human B lymphocytes and in Ramos B cells B cell receptor (BCR) stimulation induces rapid and transient redistribution of a subset of engaged BCRs to lipid rafts and phosphorylation of raft-associated tyrosine kinase substrates. Cholesterol sequestration disrupted the lipid rafts, preventing BCR redistribution, but did not inhibit tyrosine kinase activation or phosphorylation of mitogen-activated protein kinase/extracellular regulated kinase. However, raft disruption enhanced the release of calcium from intracellular stores, suggesting that rafts may sequester early signaling events that down-regulate calcium flux. Consistent with this, BCR stimulation induced rapid and transient translocation of the Src homology 2 domain-containing inositol phosphatase, SHIP, into lipid rafts.     

The tyrosine phosphatase SHP-2 controls urokinase-dependent signaling and functions in human vascular smooth muscle cells

The urokinase (uPA)/urokinase receptor (uPAR) multifunctional system is an important mediator of functional behaviour of human vascular smooth muscle cells (VSMC). uPAR associates with platelet-derived growth factor receptor β (PDGFR-β), which serves as a transmembrane adaptor for uPAR in VSMC, to transduce intracellular signaling and initiate functional changes. The precise and rapid propagation of these signaling cascades demands both strict and flexible regulatory mechanisms that remain unexplored. We provide evidence that the tyrosine phosphatase SHP-2 mediates these processes. uPA regulated SHP-2 phosphorylation, catalytic activity, and its co-localization and association with the PDGFR-β. Active PDGFR-β was required for the uPA-induced SHP-2 phosphorylation. uPAR-directed STAT1 pathway was disturbed in cells expressing SHP-2 inactive mutant. Both, cell proliferation and migration were impaired in VSMC with downregulated SHP-2. Elucidating the underlying mechanisms, we found that uPA induced SHP-2 recruitment to lipid rafts. Disruption of rafts abolished uPA-related control of SHP-2 phosphorylation, its association with PDGFR-β and finally the VSMC functional responses. Our results demonstrate that SHP-2 plays an important role in uPA-directed signaling and functional control of human VSMC and suggest that this phosphatase might contribute to the pathogenesis of the uPA-related vascular remodeling.     

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.     

Reactive oxygen species promote raft formation in T lymphocytes

Lipid rafts are involved in many cell biology events, yet the molecular mechanisms on how rafts are formed are poorly understood. In this study we probed the possible requirement of reactive oxygen species (ROS) for T-cell receptor (TCR)-induced lipid raft formation. Microscopy and biochemical analyses illustrated that blockage of ROS production, by superoxide dismutase-mimic MnTBAP, significantly reduced partitioning of LAT, phospho-LAT, and PLC-gamma in lipid rafts. Another antioxidant N-acetylcysteine (NAC) displayed a similar suppressive effect on the entry of phospho-LAT into raft microdomains. The involvement of ROS in TCR-mediated raft assembly was observed in T-cell hybridomas, T leukemia cells, and normal T cells. Removal of ROS was accompanied by an attenuated activation of LAT and PKCtheta, with reduced production of IL-2. Consistently, treating T cells with the ROS-producer tert-butyl hydrogen peroxide (TBHP) greatly enhanced membrane raft formation, distribution of phospho-LAT into lipid rafts, and increased IL-2 production. Our results indicate for the first time that ROS contribute to TCR-induced membrane raft formation.     

Aggregation of lipid rafts accompanies signaling via the T cell antigen receptor.

The role of lipid rafts in T cell antigen receptor (TCR) signaling was investigated using fluorescence microscopy. Lipid rafts labeled with cholera toxin B subunit (CT-B) and cross-linked into patches displayed characteristics of rafts isolated biochemically, including detergent resistance and colocalization with raft-associated proteins. LCK, LAT, and the TCR all colocalized with lipid patches, although TCR association was sensitive to nonionic detergent. Aggregation of the TCR by anti-CD3 mAb cross-linking also caused coaggregation of raft-associated proteins. However, the protein tyrosine phosphatase CD45 did not colocalize to either CT-B or CD3 patches. Cross-linking of either CD3 or CT-B strongly induced tyrosine phosphorylation and recruitment of a ZAP-70(SH2)(2)-green fluorescent protein (GFP) fusion protein to the lipid patches. Also, CT-B patching induced signaling events analagous to TCR stimulation, with the same dependence on expression of key TCR signaling molecules. Targeting of LCK to rafts was necessary for these events, as a nonraft- associated transmembrane LCK chimera, which did not colocalize with TCR patches, could not reconstitute CT-B-induced signaling. Thus, our results indicate a mechanism whereby TCR engagement promotes aggregation of lipid rafts, which facilitates colocalization of LCK, LAT, and the TCR whilst excluding CD45, thereby triggering protein tyrosine phosphorylation.     

Role of Amphipathic Helix of a Herpesviral Protein in Membrane Deformation and T Cell Receptor Downregulation

Lipid rafts are membrane microdomains that function as platforms for signal transduction and membrane trafficking. Tyrosine kinase interacting protein (Tip) of T lymphotropic Herpesvirus saimiri (HVS) is targeted to lipid rafts in T cells and downregulates TCR and CD4 surface expression. Here, we report that the membrane-proximal amphipathic helix preceding Tip''s transmembrane (TM) domain mediates lipid raft localization and membrane deformation. In turn, this motif directs Tip''s lysosomal trafficking and selective TCR downregulation. The amphipathic helix binds to the negatively charged lipids and induces liposome tubulation, the TM domain mediates oligomerization, and cooperation of the membrane-proximal helix with the TM domain is sufficient for localization to lipid rafts and lysosomal compartments, especially the mutivesicular bodies. These findings suggest that the membrane-proximal amphipathic helix and TM domain provide HVS Tip with the unique ability to deform the cellular membranes in lipid rafts and to downregulate TCRs potentially through MVB formation.     

Ligand binding to inhibitory killer cell Ig-like receptors induce colocalization with Src homology domain 2-containing protein tyrosine phosphatase 1 and interruption of ongoing activation signals

Interaction of NK cells with target cells leads to formation of an immunological synapse (IS) at the contact site. NK cells form two distinctly different IS, the inhibitory NK cell IS (NKIS) and the cytolytic NKIS. Cognate ligand binding is sufficient to induce clustering of inhibitory killer cell Ig-like receptors (KIR) and phosphorylation of both the receptor and the phosphatase Src homology domain 2-containing protein tyrosine phosphatase 1 (SHP-1). Recruitment and activation of SHP-1 by a signaling competent inhibitory receptor are essential early events for NK cell inhibition. We have in the present study used three-dimensional immunofluorescence microscopy to analyze distribution of inhibitory KIR, SHP-1, LFA-1, and lipid rafts within the NKIS during cytolytic and noncytolytic interactions. NK clones retrovirally transduced with the inhibitory KIR2DL3 gene fused to GFP demonstrate colocalization of KIR2DL3 with SHP-1 in the center of early inhibitory NKIS. Ligand binding translocates the receptor to the center of the IS where activation signals are accumulating and provides a docking site for SHP-1. SHP-1 and rafts cluster in the center of early inhibitory NKIS and late cytolytic NKIS, and whereas rafts continue to increase in size in cytolytic conjugates, they are rapidly dissolved in inhibitory conjugates. Furthermore, rafts are essential only for cytolytic, not for inhibitory, outcome. These results indicate that the outcome of NK cell-target cell interactions is dictated by early quantitative differences in cumulative activating and inhibitory signals.     

Lipid raft distribution of CD4 depends on its palmitoylation and association with Lck,and evidence for CD4-induced lipid raft aggregation as an additional mechanism to enhance CD3 signaling

By mutagenesis, we demonstrated that the palmitoylation of the membrane-proximal Cys(396) and Cys(399)of CD4, and the association of CD4 with Lck contribute to the enrichment of CD4 in lipid rafts. Ab cross-linking of CD4 induces an extensive membrane patching on the T cell surface, which is related to lipid raft aggregation. The lipid raft localization of CD4 is critical for CD4 to induce the aggregation of lipid rafts. The localization of CD4 in lipid rafts also correlates to the ability of CD4 to enhance receptor tyrosine phosphorylation. Thus, our data suggest that CD4-induced aggregation of lipid rafts may play an additional role in CD4 signaling besides its adhesion to MHC molecules and association with Lck.