Fyn kinase regulates the association between amyloid precursor protein and Dab1 by promoting their localization to detergent-resistant membranes

The adaptor protein Disabled1 (Dab1) interacts with amyloid precursor protein (APP) and decreases its pathological processing, an effect mediated by Fyn tyrosine kinase. Fyn is highly enriched in lipid rafts, a major site of pathological APP processing. To investigate the role of Fyn in the localization and phosphorylation of APP and Dab1 in lipid rafts, we isolated detergent-resistant membrane (DRM) fractions from wild-type and Fyn knock-out mice. In wild-type mice, all of the Fyn kinase, 17% of total APP, and 33% of total Dab1 were found in DRMs. Nearly all of the tyrosine phosphorylated forms of APP and Dab1 were in DRMs. APP and Dab1 co-precipitated both in and out of DRM fractions, indicating an association that is independent of subcellular localization. Fyn knock-out mice had decreased APP, Dab1, and tyrosine-phosphorylated Dab1 in DRMs but increased co-immunoprecipitation of DRM APP and Dab1. Expression of phosphorylation deficient APP or Dab1 constructs revealed that phosphorylation of APP increases, whereas phosphorylation of Dab1 decreases, the interaction between APP and Dab1. Consistent with these observations, Reelin treatment led to increased Dab1 phosphorylation and decreased association between APP and Dab1. Reelin also caused increased localization of APP and Dab1 to DRMs, an effect that was not seen in Fyn knock-out neurons. These findings suggest that Reelin treatment promotes the localization of APP and Dab1 to DRMs, and affects their phosphorylation by Fyn, thus regulating their interaction.     

FAT/CD36-mediated long-chain fatty acid uptake in adipocytes requires plasma membrane rafts

We previously reported that lipid rafts are involved in long-chain fatty acid (LCFA) uptake in 3T3-L1 adipocytes. The present data show that LCFA uptake does not depend on caveolae endocytosis because expression of a dominant negative mutant of dynamin had no effect on uptake of [3H]oleic acid, whereas it effectively prevented endocytosis of cholera toxin. Isolation of detergent-resistant membranes (DRMs) from 3T3-L1 cell homogenates revealed that FAT/CD36 was expressed in both DRMs and detergent-soluble membranes (DSMs), whereas FATP1 and FATP4 were present only in DSMs but not DRMs. Disruption of lipid rafts by cyclodextrin and specific inhibition of FAT/CD36 by sulfo-N-succinimidyl oleate (SSO) significantly decreased uptake of [3H]oleic acid, but simultaneous treatment had no additional or synergistic effects, suggesting that both treatments target the same mechanism. Indeed, subcellular fractionation demonstrated that plasma membrane fatty acid translocase (FAT/CD36) is exclusively located in lipid rafts, whereas intracellular FAT/CD36 cofractionated with DSMs. Binding assays confirmed that [3H]SSO predominantly binds to FAT/CD36 within plasma membrane DRMs. In conclusion, our data strongly suggest that FAT/CD36 mediates raft-dependent LCFA uptake. Plasma membrane lipid rafts might control LCFA uptake by regulating surface availability of FAT/CD36.     

Active gamma-secretase is localized to detergent-resistant membranes in human brain

Several lines of evidence suggest that polymerization of the amyloid beta-peptide (Abeta) into amyloid plaques is a pathogenic event in Alzheimer's disease (AD). Abeta is produced from the amyloid precursor protein as the result of sequential proteolytic cleavages by beta-secretase and gamma-secretase, and it has been suggested that these enzymes could be targets for treatment of AD. gamma-Secretase is an aspartyl protease complex, containing at least four transmembrane proteins. Studies in cell lines have shown that gamma-secretase is partially localized to lipid rafts, which are detergent-resistant membrane microdomains enriched in cholesterol and sphingolipids. Here, we studied gamma-secretase in detergent-resistant membranes (DRMs) prepared from human brain. DRMs prepared in the mild detergent CHAPSO and isolated by sucrose gradient centrifugation were enriched in gamma-secretase components and activity. The DRM fraction was subjected to size-exclusion chromatography in CHAPSO, and all of the gamma-secretase components and a lipid raft marker were found in the void volume (> 2000 kDa). Co-immunoprecipitation studies further supported the notion that the gamma-secretase components are associated even at high concentrations of CHAPSO. Preparations from rat brain gave similar results and showed a postmortem time-dependent decline in gamma-secretase activity, suggesting that DRMs from fresh rat brain may be useful for gamma-secretase activity studies. Finally, confocal microscopy showed co-localization of gamma-secretase components and a lipid raft marker in thin sections of human brain. We conclude that the active gamma-secretase complex is localized to lipid rafts in human brain.     

Role of phosphorylation in conformational adaptability of bovine myelin basic protein.

Controlled thrombic digestion of a preparation of components 2 + 3 isolated from the 18.5 kDa bovine myelin basic protein (MBP) yielded a polypeptide that was monophosphorylated on threonine 97 (component 3pT97). This is the first posttranslationally phosphorylated MBP isolated in pure form. We studied the effect of this single phosphate on the conformational adaptability of 18.5 kDa bovine MBP by comparing the circular dichroism (CD) spectrum of component 3pT97 with the spectra of highly purified nonphosphorylated components 1 and 2. The CD spectra of nonphosphorylated component 1 and component 2 [monodeamidated form(s) of component 1] were indistinguishable, while component 3pt97 exhibited a different spectrum. The singly phosphorylated MBP component exhibited 13% more ordered conformations than that adopted by nonphosphorylated MBP in dilute aqueous solutions. This was estimated from the CD spectra, and apparently involved about 17 additional amino acid residues in beta-structure(s).     

Sperm capacitation induces an increase in lipid rafts having zona pellucida binding ability and containing sulfogalactosylglycerolipid

Sperm gain full ability to bind to the zona(e) pellucida(e) (ZP) during capacitation. Since lipid rafts are implicated in cell adhesion, we determined whether capacitated sperm lipid rafts had affinity for the ZP. We demonstrated that lipid rafts, isolated as low-density detergent resistant membranes (DRMs), from capacitated pig sperm had ability to bind to homologous ZP. This binding was dependent on pig ZPB glycoprotein, a major participant in sperm binding. Capacitated sperm DRMs were also enriched in the male germ cell specific sulfogalactosylglycerolipid (SGG), which contributed to DRMs-ZP binding. Furthermore, SGG may participate in the formation of sperm DRMs due to its interaction with cholesterol, an integral component of lipid rafts, as shown by infrared spectroscopic studies. Since sperm capacitation is associated with cholesterol efflux from the sperm membrane, we questioned whether the formation of DRMs was compromised in capacitated sperm. Our studies indeed revealed that capacitation induced increased levels of sperm DRMs, with an enhanced ZP affinity. These results corroborated the implication of lipid rafts and SGG in cell adhesion and strongly suggested that the enhanced ZP binding ability of capacitated sperm may be attributed to increased levels and a greater ZP affinity of lipid rafts in the sperm plasma membrane.     

Brij detergents reveal new aspects of membrane microdomain in erythrocytes

Membrane microdomains enriched in cholesterol, sphingolipids (rafts), and specific proteins are involved in important physiological functions. However their structure, size and stability are still controversial. Given that detergent-resistant membranes (DRMs) are in the liquid-ordered state and are rich in raft-like components, they might correspond to rafts at least to some extent. Here we monitor the lateral order of biological membranes by characterizing DRMs from erythrocytes obtained with Brij-98, Brij-58, and TX-100 at 4?°C and 37?°C. All DRMs were enriched in cholesterol and contained the raft markers flotillin-2 and stomatin. However, sphingomyelin (SM) was only found to be enriched in TX-100-DRMs – a detergent that preferentially solubilizes the membrane inner leaflet – while Band 3 was present solely in Brij-DRMs. Electron paramagnetic resonance spectra showed that the acyl chain packing of Brij-DRMs was lower than TX-100-DRMs, providing evidence of their diverse lipid composition. Fatty acid analysis revealed that the SM fraction of the DRMs was enriched in lignoceric acid, which should specifically contribute to the resistance of SM to detergents. These results indicate that lipids from the outer leaflet, particularly SM, are essential for the formation of the liquid-ordered phase of DRMs. At last, the differential solubilization process induced by Brij-98 and TX-100 was monitored using giant unilamellar vesicles. This study suggests that Brij and TX-100-DRMs reflect different degrees of lateral order of the membrane microdomains. Additionally, Brij DRMs are composed by both inner and outer leaflet components, making them more physiologically relevant than TX-100-DRMs to the studies of membrane rafts.     

Adhesion of acidic lipid vesicles by 21.5 kDa (recombinant) and 18.5 kDa isoforms of myelin basic protein

Myelin basic protein (MBP) is thought to be responsible for adhesion of the intracellular surfaces of compact myelin to give the major dense line. The 17 and 21.5 kDa isoforms containing exon II have been reported by others to localize to the cytoplasm and nucleus of murine oligodendrocytes and HeLa cells while the 14 and 18.5 kDa isoforms lacking exon II are confined to the plasma membrane. However, we show that the exon II(-) 18.5 kDa form and a recombinant exon II(+) 21.5 kDa isoform both caused similar aggregation of acidic lipid vesicles, indicating that they should have similar abilities to bind to the intracellular lipid surface of the plasma membrane and to cause adhesion of those surfaces to each other. The circular dichroism spectra of the two isoforms indicated that both had a similar secondary structure. Thus, both isoforms should be able to bind to and cause adhesion of the cytosolic surfaces of compact myelin. The fact that they do not could be due to differences in post-translational modification in vivo, trafficking through the cell and/or subcellular location of synthesis, but it is not due to differences in their lipid binding.     

Myelin management by the 18.5‐kDa and 21.5‐kDa classic myelin basic protein isoforms

The classic myelin basic protein (MBP) splice isoforms range in nominal molecular mass from 14 to 21.5 kDa, and arise from the gene in the oligodendrocyte lineage (Golli) in maturing oligodendrocytes. The 18.5‐kDa isoform that predominates in adult myelin adheres the cytosolic surfaces of oligodendrocyte membranes together, and forms a two‐dimensional molecular sieve restricting protein diffusion into compact myelin. However, this protein has additional roles including cytoskeletal assembly and membrane extension, binding to SH3‐domains, participation in Fyn‐mediated signaling pathways, sequestration of phosphoinositides, and maintenance of calcium homeostasis. Of the diverse post‐translational modifications of this isoform, phosphorylation is the most dynamic, and modulates 18.5‐kDa MBP's protein‐membrane and protein‐protein interactions, indicative of a rich repertoire of functions. In developing and mature myelin, phosphorylation can result in microdomain or even nuclear targeting of the protein, supporting the conclusion that 18.5‐kDa MBP has significant roles beyond membrane adhesion. The full‐length, early‐developmental 21.5‐kDa splice isoform is predominantly karyophilic due to a non‐traditional P‐Y nuclear localization signal, with effects such as promotion of oligodendrocyte proliferation. We discuss in vitro and recent in vivo evidence for multifunctionality of these classic basic proteins of myelin, and argue for a systematic evaluation of the temporal and spatial distributions of these protein isoforms, and their modified variants, during oligodendrocyte differentiation.     

NGF causes TrkA to specifically attract microtubules to lipid rafts

Membrane protein sorting is mediated by interactions between proteins and lipids. One mechanism that contributes to sorting involves patches of lipids, termed lipid rafts, which are different from their surroundings in lipid and protein composition. Although the nerve growth factor (NGF) receptors, TrkA and p75(NTR) collaborate with each other at the plasma membrane to bind NGF, these two receptors are endocytosed separately and activate different cellular responses. We hypothesized that receptor localization in membrane rafts may play a role in endocytic sorting. TrkA and p75(NTR) both reside in detergent-resistant membranes (DRMs), yet they responded differently to a variety of conditions. The ganglioside, GM1, caused increased association of NGF, TrkA, and microtubules with DRMs, but a decrease in p75(NTR). When microtubules were induced to polymerize and attach to DRMs by in vitro reactions, TrkA, but not p75(NTR), was bound to microtubules in DRMs and in a detergent-resistant endosomal fraction. NGF enhanced the interaction between TrkA and microtubules in DRMs, yet tyrosine phosphorylated TrkA was entirely absent in DRMs under conditions where activated TrkA was detected in detergent-sensitive membranes and endosomes. These data indicate that TrkA and p75(NTR) partition into membrane rafts by different mechanisms, and that the fraction of TrkA that associates with DRMs is internalized but does not directly form signaling endosomes. Rather, by attracting microtubules to lipid rafts, TrkA may mediate other processes such as axon guidance.     

Detergent-resistant membranes should not be identified with membrane rafts

Three originally distinct concepts - lipid rafts, detergent-resistant membranes (DRMs) and liquid-ordered (lo) lipid phases - are often confused in current literature; many researchers have assumed that all three names refer to the same chemico-biological entity. In fact, theoretical and experimental findings provide strong evidence against identifying DRMs with rafts and lo domains. Because much of what we think we know about lipid rafts is based on their unjustified identification as DRMs, functional domains in biological membranes might differ markedly from the generally accepted picture.     

Assembly of tubulin by classic myelin basic protein isoforms and regulation by post-translational modification

Myelin basic protein (MBP), a highly cationic protein that maintains the structure of the myelin sheath, associates with tubulin in vivo. The in vitro assembly of tubulin by MBP was examined here using several assays. The unmodified C1 component of 18.5 kDa bovine MBP (bC1) assembled tubulin into microtubules in a dose-dependent manner via filamentous intermediates, and was able simultaneously to promote the formation of microtubule bundles. The critical tubulin concentration in the presence of bC1 was 0.69 +/- 0.05 microM. The effects of post-translational modifications (such as deamidation and phosphorylation) were assayed by comparing the bC1-bC6 components of 18.5 kDa bovine MBP; an increasing level of modification enhanced the ability of MBP to assemble tubulin. The effects of charge reduction via deimination were examined using recombinant murine isoforms emulating the unmodified C1 and deiminated C8 isoforms of 18.5 kDa MBP; both rmC1 and rmC8 exhibited a comparable ability to assemble tubulin. The effects of alternate exon recombination of the classic MBP variants were tested using the recombinant murine 21.5, 17.22, and 14 kDa isoforms. The isoforms containing regions derived from exon II of the classic MBP gene, 21.5 and 17.22 kDa MBP, showed no substantial difference in the extent of tubulin polymerization and bundling when compared to those of 18.5 kDa MBP. The 14 kDa isoform and two terminal deletion mutants of rmC1 were able to induce microtubule polymerization, but not bundling, to the same degree as the longer proteins. Finally, bC1 was shown to disrupt and aggregate planar sheets of crystalline tubulin stabilized by paclitaxel, establishing that these structures are not suitable substrates for the formation of MBP cocrystals.     

Differential solubilization of inner plasma membrane leaflet components by Lubrol WX and Triton X-100

A commonly-used method for analysing raft membrane domains is based on their resistance to extraction by non-ionic detergents at 4 degrees C. However, the selectivity of different detergents in defining raft membrane domains has been questioned. We have compared the lipid composition of detergent-resistant membranes (DRMs) obtained after Triton X-100 or Lubrol WX extraction in MDCK cells in order to understand the differential effect of these detergents on membranes and their selectivity in solubilizing or not proteins. Both Lubrol and Triton DRMs were enriched with cholesterol over the lysate, thus exhibiting characteristics consistent with the properties of membrane rafts. However, the two DRM fractions differed considerably in the ratio between lipids of the inner and outer membrane leaflets. Lubrol DRMs were especially enriched with phosphatidylethanolamine, including polyunsaturated species with long fatty acyl chains. Lubrol and Triton DRMs also differed in the amount of raft transmembrane proteins and raft proteins anchored to the cytoplasmic leaflet. Our results suggest that the inner side of rafts is enriched with phosphatidylethanolamine and cholesterol, and is more solubilized by Triton X-100 than by Lubrol WX.     

Evidence for a physiological role for membrane rafts in human platelets.

We have investigated raft formation in human platelets in response to cell activation. Lipid phase separation and domain formation were detected using the fluorescent dye 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (diI-C(18)) that preferentially partitions into gel-like lipid domains. We showed that when human platelets are activated by cold and physiological agonists, rafts coalesce into visible aggregates. These events were disrupted by depletion of membrane cholesterol. Using Fourier transform infrared spectroscopy (FTIR), we measured a thermal phase transition at around 30 degrees C in intact platelets, which we have assigned as the liquid-ordered to the liquid-disordered phase transition of rafts. Phase separation of the phospholipid and the sphingomyelin-enriched rafts could be observed as two phase transitions at around 15 and 30 degrees C, respectively. The higher transition, assigned to the rafts, was greatly enhanced with removal of membrane cholesterol. Detergent-resistant membranes (DRMs) were enriched in cholesterol (50%) and sphingomyelin (20%). The multi-functional platelet receptor CD36 selectively partitioned into DRMs, whereas the GPI-linked protein CD55 and the major platelet integrin alpha(IIb)beta(3a) did not, which suggests that the clustering of proteins within rafts is a regulated process dependent on specific lipid protein interactions. We suggest that raft aggregation is a dynamic, reversible physiological event triggered by cell activation.     

Cholesterol dependence of Newcastle Disease Virus entry

Lipid rafts are membrane microdomains enriched in cholesterol, sphingolipids, and glycolipids that have been implicated in many biological processes. Since cholesterol is known to play a key role in the entry of some other viruses, we investigated the role of cholesterol and lipid rafts in the host cell plasma membrane in Newcastle Disease Virus (NDV) entry. We used methyl-β-cyclodextrin (MβCD) to deplete cellular cholesterol and disrupt lipid rafts. Our results show that the removal of cellular cholesterol partially reduces viral binding, fusion and infectivity. MβCD had no effect on the expression of sialic acid containing molecule expression, the NDV receptors in the target cell. All the above-described effects were reversed by restoring cholesterol levels in the target cell membrane. The HN viral attachment protein partially localized to detergent-resistant membrane microdomains (DRMs) at 4°C and then shifted to detergent-soluble fractions at 37°C. These results indicate that cellular cholesterol may be required for optimal cell entry in NDV infection cycle.     

Differential solubilization of inner plasma membrane leaflet components by Lubrol WX and Triton X-100

A commonly-used method for analysing raft membrane domains is based on their resistance to extraction by non-ionic detergents at 4 °C. However, the selectivity of different detergents in defining raft membrane domains has been questioned. We have compared the lipid composition of detergent-resistant membranes (DRMs) obtained after Triton X-100 or Lubrol WX extraction in MDCK cells in order to understand the differential effect of these detergents on membranes and their selectivity in solubilizing or not proteins. Both Lubrol and Triton DRMs were enriched with cholesterol over the lysate, thus exhibiting characteristics consistent with the properties of membrane rafts. However, the two DRM fractions differed considerably in the ratio between lipids of the inner and outer membrane leaflets. Lubrol DRMs were especially enriched with phosphatidylethanolamine, including polyunsaturated species with long fatty acyl chains. Lubrol and Triton DRMs also differed in the amount of raft transmembrane proteins and raft proteins anchored to the cytoplasmic leaflet. Our results suggest that the inner side of rafts is enriched with phosphatidylethanolamine and cholesterol, and is more solubilized by Triton X-100 than by Lubrol WX.     

Detergent-resistant membrane fractions contribute to the total 1H NMR-visible lipid signal in cells.

Leukocytes and other cells show an enhanced intensity of mobile lipid in their 1H NMR spectra under a variety of conditions. Such conditions include stimulation, which has recently been shown to involve detergent-resistant, plasma membrane domains (DRMs) often called lipid rafts. As there is much speculation surrounding the origin of cellular NMR-visible lipid, we analysed subcellular fractions, including DRMs, by NMR spectroscopy. We demonstrated that DRMs isolated by density gradient centrifugation from lymphoid (CEM-T4, stimulated Jurkat cells), and monocytoid (THP-1) cells produced NMR-visible, lipid signals. Large scale subfractionation of THP-1 cells determined that while cytoplasmic lipid droplets constituted much of the total NMR-visible lipid, the contribution of DRMs was significant. Qualitative and quantitative lipid analyses revealed that DRMs and lipid droplets differed in their lipid composition. DRMs were enriched in cholesterol and ganglioside GM1, and contained relatively unsaturated fatty acids compared with the lipid droplets. Both lipid droplets and DRMs contained neutral lipids (triacylgycerols, cholesterol ester, fatty acids in THP-1 cells) that could, in addition to phospholipids, contribute to the NMR-visible lipid. The lipid droplets also exhibited different protein profiles and contained 500-fold less protein than DRMs, confirming that DRMs and droplets were fractionated as separate entities. The NMR-visible lipid in DRMs is therefore unlikely to be a contaminant from lipid droplets. We propose a micropartitioning of the NMR-visible mobile lipid of whole cells between intracellular lipid droplets, where most of this lipid resides, and detergent-resistant plasma membrane domains.     

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.     

Quantitative proteome analysis of detergent‐resistant membranes identifies the differential regulation of protein kinase C isoforms in apoptotic T cells

Several lines of evidence suggest that detergent‐resistant membranes (DRMs) (also known as lipid rafts and glycosphingolipid‐enriched microdomains) may have a role in signaling pathways of apoptosis. Here, we developed a method that combines DRMs isolation and methanol/chloroform extraction with stable isotope labeling with amino acids in cell culture‐based quantitative proteome analysis of DRMs from control and cisplatin‐induced apoptotic Jurkat T cells. This approach enabled us to enrich proteins with a pivotal role in cell signaling of which several were found with increased or decreased amounts in DRMs upon induction of apoptosis. Specifically, we show that three isoforms of protein kinase C (PKC) are regulated differently upon apoptosis. Although PKCα which belongs to the group of conventional PKCs is highly up‐regulated in DRMs, the levels of two novel PKCs, PKCη and PKCθ, are significantly reduced. These alterations/differences in PKC regulation are verified by immunoblotting and confocal microscopy. In addition, a specific enrichment of PKCα in apoptotic blebs and buds is shown. Furthermore, we observe an increased expression of ecto‐PKCα as a result of exposure to cisplatin using flow cytometry. Our results demonstrate that in‐depth proteomic analysis of DRMs provides a tool to study differential localization and regulation of signaling molecules important in health and disease.