Visualizing lipid raft dynamics and early signaling events during antigen receptor-mediated B-lymphocyte activation

Recent biochemical evidence indicates that an early event in signal transduction by the B-cell antigen receptor (BCR) is its translocation to specialized membrane subdomains known as lipid rafts. We have taken a microscopic approach to image lipid rafts and early events associated with BCR signal transduction. Lipid rafts were visualized on primary splenic B lymphocytes from wild-type or anti-hen egg lysozyme BCR transgenic mice, and on a mature mouse B-cell line Bal 17 by using fluorescent conjugates of cholera toxin B subunit or a Lyn-based chimeric protein, which targets green fluorescent protein to the lipid raft compartment. Time-lapse imaging of B cells stimulated via the BCR with the antigen hen egg lysozyme, or surrogate for antigen anti-IgM, demonstrated that lipid rafts are highly dynamic entities, which move laterally on the surface of these cells and coalesce into large regions. These regions of aggregated lipid rafts colocalized with the BCR and tyrosine-phosphorylated proteins. Microscopic imaging of live B cells also revealed an inducible colocalization of lipid rafts with the tyrosine kinase Syk and the receptor tyrosine phosphatase CD45. These two proteins play indispensable roles in BCR-mediated signaling but are not detectable in biochemically purified lipid raft fractions. Strikingly, BCR stimulation also induced the formation of long, thread-like filopodial projections, similar to previously described structures called cytonemes. These B-cell cytonemes are rich in lipid rafts and actin filaments, suggesting that they might play a role in long-range communication and/or transportation of signaling molecules during an immune response. These results provide a window into the morphological and molecular organization of the B-cell membrane during the early phase of BCR signaling.     

Lipid rafts associate with intracellular B cell receptors and exhibit a B cell stage-specific protein composition

Lipid rafts serve as platforms for BCR signal transduction. To better define the molecular basis of these membrane microdomains, we used two-dimensional gel electrophoresis and mass spectrometry to characterize lipid raft proteins from mature as well as immature B cell lines. Of 51 specific raft proteins, we identified a total of 18 proteins by peptide mass fingerprinting. Among them, we found vacuolar ATPase subunits alpha-1 and beta-2, vimentin, gamma-actin, mitofilin, and prohibitin. None of these has previously been reported in lipid rafts of B cells. The differential raft association of three proteins, including a novel potential signaling molecule designated swiprosin-1, correlated with the stage-specific sensitivity of B cells to BCR-induced apoptosis. In addition, MHC class II molecules were detected in lipid rafts of mature, but not immature B cells. This intriguing finding points to a role for lipid rafts in regulating Ag presentation during B cell maturation. Finally, a fraction of the BCR in the B cell line CH27 was constitutively present in lipid rafts. Surprisingly, this fraction was neither expressed at the cell surface nor fully O-glycosylated. Thus, we conclude that partitioning the BCR into lipid rafts occurs in the endoplasmic reticulum/cis-Golgi compartment and may represent a control mechanism for surface transport.     

A requirement for lipid rafts in B cell receptor induced Ca(2+) flux

Although the major biochemical events triggered by ligation of the B-cell receptor (BCR) have been well defined [1] [2], little is known about the spatio-temporal organization of BCR signaling components within the cell membrane and the mechanisms by which signaling specificity is achieved. Partitioning of signaling complexes into specialized domains in the plasma membrane may provide a mechanism for channeling specific stimuli into distinct signaling pathways. Here, we report that multiple tyrosine-phosphorylated proteins accumulate transiently upon BCR activation in detergent-insoluble membrane microdomains known as lipid rafts. We found an activation-dependent translocation to the rafts of the BCR itself, as well as phospholipase Cgamma2 (PLCgamma2), an enzyme critical for BCR-induced Ca(2+) flux in B cells. An intact raft structure was required for BCR-induced tyrosine phosphorylation of PLCgamma2 and the induction of Ca(2+) flux. Taken together, these data provide a functional role for lipid rafts in BCR signaling.     

Lipid raft proteomics: analysis of in-solution digest of sodium dodecyl sulfate-solubilized lipid raft proteins by liquid chromatography-matrix-assisted laser desorption/ionization tandem mass spectrometry

Lipid rafts are glycolipid- and cholesterol-enriched membrane microdomains implicated in membrane signaling and trafficking. The highly hydrophobic nature of lipid raft proteins pose significant problems of solubilization and recovery that hinder analysis by mass spectrometry (MS) and may under-report the composition of lipid rafts. In a previous investigation of the monocyte lipid raft in which proteins were digested with trypsin following polyacrylamide gel electrophoresis we identified 52 proteins. Here we report the development of a sodium dodecyl sulfate (SDS)-aided approach in which proteins are digested in solution and examined by high-performance liquid chromatography-matrix-assisted laser desorption/ionization-tandem mass spectrometry (HPLC-MALDI-MS/MS) using a novel LC-MALDI interface thereby circumventing the need to separate proteins on gels. Using this approach we identified 71 proteins in the lipid raft, 45 of which were not detected using in-gel digestion. Among the new proteins are alpha- and beta-tubulin, tubulinspecific chaperone A, a folding protein involved in tubulin dimer assembly, and KIF13, a microtubule motor protein indicating that proteins involved in microtubule assembly and trafficking are more readily detected using an in-solution approach. To investigate why tubulin was not identified by in-gel digestion, we compared the distribution of alpha-tubulin and the raft marker flotillin-2 in buoyant density gradients before and after separation on SDS-gels. Both proteins were present in the raft fractions, but tubulin was selectively lost following separation on SDS-gels. Assemblies of cytoskeletal proteins with lipid rafts may therefore be resolved using in-solution digestion that would be missed using gel-based approaches.     

Syk-mediated tyrosine phosphorylation is required for the association of hematopoietic lineage cell-specific protein 1 with lipid rafts and B cell antigen receptor signalosome complex

Hematopoietic lineage cell-specific protein 1 (HS1) is an F-actin- and actin-related proteins 2 and 3 (Arp2/3)-binding protein that undergoes a rapid tyrosine phosphorylation upon B cell antigen receptor (BCR) activation. Density gradient centrifugation of Triton X-100 lysates from B lymphocytes demonstrated that HS1 was translocated in response to BCR cross-linking into lipid raft microdomain along with Arp2/3 complex and Wiskott-Aldrich syndrome protein. HS1-green fluorescent protein was localized in membrane patches enriched with GM1 gangliosides and BCR in the cells treated with anti-IgM antibody. Colocalization of HS1-green fluorescent protein with BCR was also correlated with tyrosine phosphorylation of HS1. Interestingly a murine HS1 mutant at the tyrosine residues Tyr388 and Tyr405 targeted by Syk failed to respond to BCR cross-linking for either translocation into lipid rafts or colocalization with BCR within cells. Furthermore HS1 was unable to translocate into lipid rafts in a chicken B cell line deficient in Syk. Reintroducing a Syk construct into the Syk knock-out cells recovered effectively both tyrosine phosphorylation and translocation of HS1 into lipid rafts. In contrast, translocation of HS1 into rafts was normal in a Lyn knock-out B cell line, and an HS1 mutant at the tyrosine residue Tyr222 targeted by Lyn maintained the ability to partition into rafts upon BCR cross-linking. These data indicate that Syk plays an important role in the translocation of HS1 into lipid rafts and may be responsible for actin assembly recruitment to rafts and subsequent antigen presentations.     

SCP-2/SCP-x gene ablation alters lipid raft domains in primary cultured mouse hepatocytes

Although reverse cholesterol transport from peripheral cell types is mediated through plasma membrane microdomains termed lipid rafts, almost nothing is known regarding the existence, protein/lipid composition, or structure of these putative domains in liver hepatocytes, cells responsible for the net removal of cholesterol from the body. Lipid rafts purified from hepatocyte plasma membranes by a nondetergent affinity chromatography method were: i) present at 33 +/- 3% of total plasma membrane protein; ii) enriched in key proteins of the reverse cholesterol pathway [scavenger receptor class B type I (SR-B1), ABCA1, P-glycoprotein (P-gp), sterol carrier protein-2 (SCP-2)]; iii) devoid of caveolin-1; iv) enriched in cholesterol, sphingomyelin, GM1, and phospholipids low in polyunsaturated fatty acid and double bond index; and v) exhibited an intermediate liquid-ordered lipid phase with significant transbilayer fluidity gradient. Ablation of the gene encoding SCP-2 significantly altered lipid rafts to: i) increase the proportion of lipid rafts present, thereby increasing raft total content of ABCA1, P-gp, and SR-B1; ii) increase total phospholipids while decreasing GM1 in lipid rafts; iii) decrease the fluidity of lipid rafts, consistent with the increased intermediate liquid-ordered phase; and iv) abolish the lipid raft transbilayer fluidity gradient. Thus, despite the absence of caveolin-1 in liver hepatocytes, lipid rafts represented nearly one-third of the mouse hepatocyte plasma membrane proteins and displayed unique protein, lipid, and biophysical properties that were differentially regulated by SCP-2 expression.     

Clustering of the B cell receptor is not required for the apoptotic response

We examined the role of BCR cell membrane redistribution in anti-IgM-induced apoptosis in three human B cell lines, RA#1, 2G6, and MC116, that differ in their relative levels of sIgM expression. The apoptotic response was found to be dependent on the nature of the anti-IgM and the cell line. In the cell lines, RA#1 and MC116, sIgM aggregated into patches that were insensitive to the disruption of cholesterol-rich membrane microdomains by nystatin or beta-MCD. The B cell line 2G6 was able to reorganize sIgM into a tight coalescent cap upon anti-IgM treatment. However, in this case, the lipid raft inhibitors nystatin and beta-MCD disrupted the patching. In 2G6 cells, BCR-mediated apoptosis was not affected by nystatin treatment, whereas it increased in beta-MCD pretreated cells. Thus, no evident correlation was found between apoptosis and BCR cell membrane redistribution or lipid raft formation in either of the three cell lines. The data indicate that the apoptotic signal transduction pathway is independent of BCR translocation into lipid rafts and/or aggregation.     

Transient GPI-anchored protein homodimers are units for raft organization and function

Advanced single-molecule fluorescent imaging was applied to study the dynamic organization of raft-associated glycosylphosphatidylinositol-anchored proteins (GPI-APs) in the plasma membrane and their stimulation-induced changes. In resting cells, virtually all of the GPI-APs are mobile and continually form transient (～200 ms) homodimers (termed homodimer rafts) through ectodomain protein interactions, stabilized by the presence of the GPI-anchoring chain and cholesterol. Heterodimers do not form, suggesting a fundamental role for the specific ectodomain protein interaction. Under higher physiological expression conditions , homodimers coalesce to form hetero- and homo-GPI-AP oligomer rafts through raft-based lipid interactions. When CD59 was ligated, it formed stable oligomer rafts containing up to four CD59 molecules, which triggered intracellular Ca(2+) responses that were dependent on GPI anchorage and cholesterol, suggesting a key part played by transient homodimer rafts. Transient homodimer rafts are most likely one of the basic units for the organization and function of raft domains containing GPI-APs.     

Lipid rafts and B-cell activation

The B-cell antigen receptor acts during B-cell activation both to initiate signalling cascades and to transport antigen into the cell for subsequent processing and presentation. Recent evidence indicates that membrane microdomains, termed lipid rafts, have a role in B-cell activation as platforms for B-cell receptor (BCR) signalling and might also act in antigen trafficking. Lipid rafts might facilitate the regulation of the BCR during B-cell development by B-cell co-receptors, and during viral infection. So, lipid rafts seem to be an important new piece of the B-cell signalling puzzle.     

Lipid raft and influenza virus--viral glycoproteins on a raft

Lipid molecules of the plasma membrane are not distributed homogeneously, but form a lateral organization resulting from preferential packaging of sphingolipid and cholesterol into lipid microdomain rafts, in which specific membrane proteins become incorporated. Evidence has accumulated that a variety of viruses including influenza virus use the raft during some steps of their replication cycles. Influenza virus glycoproteins, hemagglutinin (HA) and neuraminidase, associate intrinsically with the rafts. The HA protein is distributed in clusters at the plasma membrane and concentrated in the small area by interacting with the raft. A mutant influenza virus, whose HA protein lacks the ability to associate with the raft, contains reduced amounts of the HA proteins and exhibits a decreased virus to cell fusion activity, resulting in greatly reduced infectivity. Thus, the raft may play an important role in virus production by acting as a concentrating devise or an efficient carrier to transport the HA protein to the site of virus budding.     

Ternary SNARE complexes are enriched in lipid rafts during mast cell exocytosis

Lipid rafts are membrane microdomains rich in cholesterol and glycosphingolipids that have been implicated in the regulation of intracellular protein trafficking. During exocytosis, a class of proteins termed SNAREs mediate secretory granule-plasma membrane fusion. To investigate the role of lipid rafts in secretory granule exocytosis, we examined the raft association of SNARE proteins and SNARE complexes in rat basophilic leukemia (RBL) mast cells. The SNARE protein SNAP-23 co-localized with a lipid raft marker and was present in detergent-insoluble lipid raft microdomains in RBL cells. By contrast, only small amounts (<20%) of the plasma membrane SNARE syntaxin 4 or the granule-associated SNARE vesicle-associated membrane protein (VAMP)-2 were present in these microdomains. Despite this, essentially all syntaxin 4 and most of VAMP-2 in these rafts were present in SNARE complexes containing SNAP-23, while essentially none of these complexes were present in nonraft membranes. Whereas SNAP-23 is membrane anchored by palmitoylation, the association of the transmembrane protein syntaxin 4 with lipid rafts was because of its binding to SNAP-23. After stimulating mast cells exocytosis, the amount of syntaxin 4 and VAMP-2 present in rafts increased twofold, and these proteins were now present in raft-associated phospho-SNAP-23/syntaxin 4/VAMP-2 complexes, revealing differential association of SNARE fusion complexes during the process of regulated exocytosis.     

Fc gamma RIIB1/SHIP-mediated inhibitory signaling in B cells involves lipid rafts

One type of membrane microdomain, enriched in glycosphingolipids and cholesterol and referred to as lipid rafts, has been implicated in the generation of activating signals triggered by a variety of stimuli. Several laboratories, including ours, have recently demonstrated that the B cell receptor (BCR) inducibly localizes to the rafts upon activation and that functional lipid rafts are important for BCR-mediated "positive" signaling. In the later phases of the immune response, coligation of the BCR and the inhibitory receptor Fc gamma RIIB1 leads to potent inhibition of BCR-induced positive signaling through the recruitment of the inositol phosphatase SHIP to Fc gamma RIIB1. One potential model is that the Fc gamma RIIB1 itself might be excluded from the rafts basally and that destabilization of raft-dependent BCR signaling might be part of the mechanism for the Fc gamma RIIB1-mediated negative regulation. We tested this hypothesis and observed that preventing BCR raft localization is not the mechanism for this inhibition. Surprisingly, a fraction of Fc gamma RIIB1 is constitutively localized in the rafts and increases further after BCR + FcR coligation. SHIP is actively recruited to lipid rafts under negative stimulation conditions, and the majority of Fc gamma RIIB1-SHIP complexes localize to lipid rafts compared with non-raft regions of the plasma membrane. This suggested that this negative feedback loop is also initiated in the lipid rafts. Despite its basal localization to the rafts, Fc gamma RIIB1 did not become phosphorylated after BCR alone cross-linking and did not colocalize with the BCR that moves to rafts upon BCR engagement alone (positive signaling conditions), perhaps suggesting the existence of different subsets of rafts. Taken together, these data suggest that lipid rafts play a role in both the positive signaling via the BCR as well as the inhibitory signaling through Fc gamma RIIB1/SHIP.     

Polyunsaturated eicosapentaenoic acid displaces proteins from membrane rafts by altering raft lipid composition

Polyunsaturated fatty acids (PUFAs) such as eicosapentaenoic acid (20:5 (n-3)) inhibit T lymphocyte activation probably by displacing acylated signaling proteins from membrane lipid rafts. Under physiological conditions, saturated fatty acyl residues of such proteins partition into the cytoplasmic membrane lipid leaflet with high affinity for rafts that are enriched in saturated fatty acyl-containing lipids. However, the biochemical alteration causing displacement of acylated proteins from rafts in PUFA-treated T cells is still under debate but could principally be attributed to altered protein acylation or changes in raft lipid composition. We show that treatment of Jurkat T cells with polyunsaturated eicosapentaenoic acid (20:5 (n-3)) results in marked enrichment of PUFAs (20:5; 22:5) in lipids from isolated rafts. Moreover, PUFAs were significantly incorporated into phosphatidylethanolamine that predominantly resides in the cytoplasmic membrane lipid leaflet. Notably, palmitate-labeled Src family kinase Lck and the linker for activation of T cells (LAT) were both displaced from lipid rafts indicating that acylation by PUFAs is not required for protein displacement from rafts in PUFA-treated T cells. In conclusion, these data provide strong evidence that displacement of acylated proteins from rafts in PUFA-treated T cells is predominantly due to altered raft lipid composition.     

Sterol carrier protein-2 selectively alters lipid composition and cholesterol dynamics of caveolae/lipid raft vs nonraft domains in L-cell fibroblast plasma membranes

Although the functional significance of caveolae/lipid rafts in cellular signaling and cholesterol transfer is increasingly recognized, almost nothing is known regarding the lipids, cholesterol dynamics, and factors regulating these properties in caveolae/lipid rafts as opposed to nonlipid raft domains of the plasma membrane. The present findings demonstrate the utility of con-A affinity chromatography for simultaneous isolation of caveolae/lipid raft and nonlipid raft domains from plasma membranes of L-cell fibroblasts. These domains differed markedly in both protein and lipid constituents. Although caveolae/lipid rafts were enriched in total lipid, cholesterol, and phospholipid as well as other markers for these domains, the cholesterol/phospholipid ratio of caveolae/lipid rafts did not differ from that of nonlipid rafts. Nevertheless, spontaneous sterol transfer was 7-12-fold faster from caveolae/lipid raft than nonlipid raft domains of the plasma membrane. This was largely due to the near absence of exchangeable sterol in the nonlipid rafts. SCP-2 dramatically and selectively enhanced sterol transfer from caveolae/lipid rafts, but not from nonlipid rafts. Finally, overexpression of SCP-2 significantly altered the sterol dynamics of caveolae/lipid rafts to facilitate retention of cholesterol within the cell. These results established for the first time that (i) caveolae/lipid rafts, rather than the nonlipid raft domains, contain significant levels of rapidly transferable sterol, consistent with their role in spontaneous sterol transfer from and through the plasma membrane, and (ii) SCP-2 selectively regulates how caveolae/lipid rafts, but not nonlipid raft domains, mediate cholesterol trafficking through the plasma membrane.     

The tetraspanin CD81 is necessary for partitioning of coligated CD19/CD21-B cell antigen receptor complexes into signaling-active lipid rafts

Tetraspanins have been hypothesized to facilitate the organization of functional multimolecular membrane complexes. In B cells the tetraspanin CD81 is a component of the CD19/CD21 complex. When coligated to the B cell Ag receptor (BCR), the CD19/CD21 complex significantly enhances BCR signaling in part by prolonging the association of the BCR with signaling-active lipid rafts. In this study CD81 is shown to associate with lipid rafts upon coligation of the BCR and the CD19/CD21 complex. Using B cells from CD81-deficient mice we demonstrate that in the absence of CD81, coligated BCR and CD19/CD21 complexes fail to partition into lipid rafts and enhance BCR signaling from rafts. Furthermore, a chimeric CD19 protein that associates only weakly if at all with CD81 fails to promote the association of coligated BCR with lipid rafts. The requirement for CD81 to promote lipid raft association may define a novel mechanism by which tetraspanins function as molecular facilitators of signaling receptors.     

Lipid raft disruption by docosahexaenoic acid induces apoptosis in transformed human mammary luminal epithelial cells harboring HER-2 overexpression

In HER-2-overexpressing breast cells, HER-2 receptors exist on the cell surface as monomers, homodimers and heterodimers. For signal activation and transduction to occur, HER-2 must be localized to lipid rafts. Therefore, we hypothesized that the amount of lipid rafts on the cell membrane would be a factor in HER-2 signaling. To test this, we used HB4a (an untransformed human mammary epithelial cell line) and HB4aC5.2 cells. HB4aC5.2 cells are HB4a derivatives that have been transfected with five copies of pJ5E.c-ErbB-2 and express approximately 900 times more HER-2 than HB4a cells. In these cells, HER-2 overexpression was accompanied by increased lipid rafts in cell membranes, a hyperactivation of downstream Akt and ERK1/2 proteins, and an increased rate of cell growth compared to HB4a. In addition, HER-2 overexpression was associated with an increased activation of FASN, a key enzyme involved in cellular lipogenesis. Its final product, palmitate, is frequently used to synthesize lipid rafts. We further hypothesized that treatment with docosahexaenoic acid (DHA), an omega-3 fatty acid, would disrupt the lipid rafts and lead to a growth arrest. In HB4aC5.2 cells, but not HB4a cells, we found that DHA treatment disrupted lipid raft; inhibited HER-2 signaling by decreasing activation of Akt, ERK1/2 and FASN proteins; and induced apoptosis. Although little is known about lipid rafts, our data support the idea that disturbances in these microdomains induced by DHA may represent a useful tool for controlling the signaling initiated by HER-2 receptors and its therapeutic potential in the treatment of HER-2 positive breast cancer.     

The role of cholesterol and glycosylphosphatidylinositol-anchored proteins of erythrocyte rafts in regulating raft protein content and malarial infection

Human erythrocytes are terminally differentiated, nonendocytic cells that lack all intracellular organelles. Here we show that their plasma membranes contain detergent-resistant membrane rafts that constitute a small fraction (4%) of the total membrane protein, with a complex mixture of proteins that differentially associate with rafts. Depletion of raft-cholesterol abrogates association of all proteins with no significant effect on cholesterol:protein ratios in the rest of the membrane, lipid asymmetry, deformability, or transport properties of the bilayer, indicating that cholesterol is critical for protein assembly into rafts and suggesting that rafts have little influence on several erythrocyte functions. Erythrocytes from patients with paroxysmal nocturnal hemoglobinuria, which lack glycosylphosphatidylinositol-anchored proteins, show significant elevation in raft-cholesterol but no increase in raft protein association, suggesting that raft assembly does not require glycosylphosphatidylinositol-anchored proteins, raft proteins do not bind directly to cholesterol, and only threshold levels of raft-cholesterol are critical for protein recruitment. Loss of glycosylphosphatidylinositol-anchored proteins had no effect on erythrocytic infection by malarial parasite or movement of raft markers into the parasite's vacuole. However, infection is blocked following raft-cholesterol disruption, suggesting that erythrocyte rafts can be functionally exploited and providing the first evidence for the involvement of host rafts in an apicomplexan infection.     

Vav1/Rac-dependent actin cytoskeleton reorganization is required for lipid raft clustering in T cells.

Formation of the immunological synapse (IS) in T cells involves large scale molecular movements that are mediated, at least in part, by reorganization of the actin cytoskeleton. Various signaling proteins accumulate at the IS and are localized in specialized membrane microdomains, known as lipid rafts. We have shown previously that lipid rafts cluster and localize at the IS in antigen-stimulated T cells. Here, we provide evidence that lipid raft polarization to the IS depends on an intracellular pathway that involves Vav1, Rac, and actin cytoskeleton reorganization. Thus, lipid rafts did not translocate to the IS in Vav1-deficient (Vav1-/-) T cells upon antigen stimulation. Similarly, T cell receptor transgenic Jurkat T cells also failed to translocate lipid rafts to the IS when transfected with dominant negative Vav1 mutants. Raft polarization induced by membrane-bound cholera toxin cross-linking was also abolished in Jurkat T cells expressing dominant negative Vav1 or Rac mutants and in cells treated with inhibitors of actin polymerization. However, Vav overexpression that induced F-actin polymerization failed to induce lipid rafts clustering. Therefore, Vav is necessary, but not sufficient, to regulate lipid rafts clustering and polarization at the IS, suggesting that additional signals are required.     

Down-regulation of lipid raft-associated onco-proteins via cholesterol-dependent lipid raft internalization in docosahexaenoic acid-induced apoptosis

Lipid rafts, plasma membrane microdomains, are important for cell survival signaling and cholesterol is a critical lipid component for lipid raft integrity and function. DHA is known to have poor affinity for cholesterol and it influences lipid rafts. Here, we investigated a mechanism underlying the anti-cancer effects of DHA using a human breast cancer cell line, MDA-MB-231. We found that DHA decreased cell surface levels of lipid rafts via their internalization, which was partially reversed by cholesterol addition. With DHA treatment, caveolin-1, a marker for rafts, and EGFR were colocalized with LAMP-1, a lysosomal marker, in a cholesterol-dependent manner, indicating that DHA induces raft fusion with lysosomes. DHA not only displaced several raft-associated onco-proteins, including EGFR, Hsp90, Akt, and Src, from the rafts but also decreased total levels of those proteins via multiple pathways, including the proteasomal and lysosomal pathways, thereby decreasing their activities. Hsp90 overexpression maintained its client proteins, EGFR and Akt, and attenuated DHA-induced cell death. In addition, overexpression of Akt or constitutively active Akt attenuated DHA-induced apoptosis. All these data indicate that the anti-proliferative effect of DHA is mediated by targeting of lipid rafts via decreasing cell surface lipid rafts by their internalization, thereby decreasing raft-associated onco-proteins via proteasomal and lysosomal pathways and decreasing Hsp90 chaperone function.