Andrzej Sobota

Phosphorylation of FcgammaRIIA is required for the receptor-induced actin rearrangement and capping: the role of membrane rafts.

Activation of Fcγ receptor II (FcγRII) induces rearrangement of the actin-based cytoskeleton that serves as a driving force for FcγRII-mediated phagocytosis and FcγRII capping. To get insight into the signaling events that lead to the actin reorganization we investigated the role of raft-associated Src family tyrosine kinases in capping of FcγRII in U937 cells. After crosslinking, FcγRII was found to be recruited to detergent-resistant membrane domains (DRMs), rafts, where it coexisted with Lyn kinase and underwent tyrosine phosphorylation. Lyn was displaced from DRMs under the influence of DL-α-hydroxymyristic acid and 2-bromopalmitic acid, agents blocking N-terminal myristoylation and palmitoylation of proteins, respectively, and after disruption of DRM integrity by depletion of plasma membrane cholesterol withβ -cyclodextrin. Under these conditions, phosphorylation of the crosslinked FcγRII was diminished and assembly of FcγRII caps was blocked. The similar reduction of FcγRII cap formation correlated with inhibition of receptor phosphorylation was achieved with the use of PP1 and herbimycin A, specific inhibitors of Src family tyrosine kinases. Phosphorylation of FcγRIIA expressed in BHK cells, lacking endogenous FcγRs, was abolished by substitution of tyrosine 298 by phenylalanine in the ITAM of the receptor. The mutant receptor did not undergo translocation towards cap-like structures and failed to promote the receptor-mediated spreading of the cells, as compared to BHK cells transfected with the wild-type FcγRIIA. On the basis of these data, we suggest that tyrosine phosphorylation of activated FcγRIIA by raft-residing tyrosine kinases of the Src family triggers signaling pathways that control the rearrangement of the actin cytoskeleton required for FcγRII-mediated motility.

Tyrosine phosphorylation and Fcγ receptor-mediated phagocytosis

Phagocytosis of IgG‐opsonized particulate material in hematopoietic cells is mediated by Fcγ receptors (FcγRs). Interaction of the receptors with Fc domains of IgG triggers transduction of phagocytic signal in which a key role is played by phosphorylation of tyrosine residues of the receptors. These residues are arranged into a specific motif (immunoreceptor tyrosine‐based activation motif; ITAM) which is located either in the cytoplasmic part of FcγRIIA or in γ chains associated with FcγRI and FcγRIIIA. The conserved tyrosine residues are phosphorylated by, and associate with, tyrosine kinases of Src and Syk families. Coordinated action of these components initiates numerous intracellular events leading finally to local rearrangement of the actin‐based cytoskeleton and internalization of the particles.

The clustered Fcγ receptor II is recruited to Lyn‐containing membrane domains and undergoes phosphorylation in a cholesterol‐dependent manner

Phosphorylation of clustered Fcγ receptor II (FcγRII) by Src family tyrosine kinases is the earliest event in the receptor signaling cascade. However, the molecular mechanisms for the interaction between FcγRII and these kinases are not elucidated. To asses this problem we isolated high molecular weight complexes of cross‐linked FcγRII from non‐ionic detergent lysates of U937 monocytic cells. CD55, a glycosylphosphatidylinositol‐anchored protein, a ganglioside GM1 and Lyn, a Src family tyrosine kinase, were also located in these complexes. Gradient centrifugation demonstrated that the complexes containing cross‐linked FcγRII displayed a low buoyant density. The FcγRII present in the complexes underwent tyrosine phosphorylation. Cross‐linked FcγRII and Lyn occupied common 100–200 nm detergent‐resistant membrane fragments, as demonstrated by immunoprecipitation and microscopy studies. Pretreatment of the cells with β‐cyclodextrin, a cholesterol acceptor, depleted membrane cholesterol and released CD55, GM1 and Lyn from the detergent‐resistant complexes. In parallel, the association of Lyn with cross‐linked FcγRII was disrupted and phosphorylation of the receptor inhibited. Reincorporation of cholesterol evoked the relocation of Lyn into the detergent‐resistant membrane fraction and restored both Lyn association with cross‐linked FcγRII and tyrosine phosphorylation of the receptor. Our data demonstrate that cholesterol‐enriched membrane rafts can facilitate tyrosine phosphorylation of clustered FcγRII by Lyn kinase.

How Mycobacterium tuberculosis subverts host immune responses.

Mycobacterium tuberculosis is the causative agent of pulmonary tuberculosis which has infected one third of the mankind and causes 2–3 million deaths worldwide each year. The persistence of the infection ensues from the ability of M. tuberculosis to subvert host immune responses in favor of survival and growth of mycobacteria in macrophages. The mechanisms by which M. tuberculosis manipulates the host immune system have only recently come to light. These activities are attributed to lipoarabinomannans (LAM) and their precursors lipomannans (LM), two predominant glycolipids of M. tuberculosis cell wall. LM are able to skew anti‐mycobacterial immune responses into un‐protective ones, while LAM evoke immunosupression upon binding to macrophage and dendritic cell receptors specialized in binding to “self” host components. A newly emerging idea implicates plasma membrane rafts in LM and LAM signaling. Depending on acylation patterns, the glycolipids may either directly incorporate into the raft membrane via mannosylphosphatidylinositol anchors or interact with raft‐associated proteins to affect the assembly of receptor signaling complexes. BioEssays 30:943–954, 2008.

Lyn and Syk Kinases Are Sequentially Engaged in Phagocytosis Mediated by FcγR

Recent data indicate that phagocytosis mediated by FcγRs is controlled by the Src and Syk families of protein tyrosine kinases. In this study, we demonstrate a sequential involvement of Lyn and Syk in the phagocytosis of IgG-coated particles. The particles isolated at the stage of their binding to FcγRs (4°C) were accompanied by high amounts of Lyn, in addition to the signaling γ-chain of FcγRs. Simultaneously, the particle binding induced rapid tyrosine phosphorylation of numerous proteins. During synchronized internalization of the particles induced by shifting the cell to 37°C, Syk kinase and Src homology 2-containing tyrosine phosphatase-1 (SHP-1) were associated with the formed phagosomes. At this step, most of the proteins were dephosphorylated, although some underwent further tyrosine phosphorylation. Quantitative immunoelectron microscopy studies confirmed that Lyn accumulated under the plasma membrane beneath the bound particles. High amounts of the γ-chain and tyrosine-phosphorylated proteins were also observed under the bound particles. When the particles were internalized, the γ-chain was still detected in the region of the phagosomes, while amounts of Lyn were markedly reduced. In contrast, the vicinity of the phagosomes was heavily decorated with anti-Syk and anti-SHP-1 Abs. The local level of protein tyrosine phosphorylation was reduced. The data indicate that the accumulation of Lyn during the binding of IgG-coated particles to FcγRs correlated with strong tyrosine phosphorylation of numerous proteins, suggesting an initiating role for Lyn in protein phosphorylation at the onset of the phagocytosis. Syk kinase and SHP-1 phosphatase are mainly engaged at the stage of particle internalization.

Binding of IgG-Opsonized Particles to FcγR Is an Active Stage of Phagocytosis That Involves Receptor Clustering and Phosphorylation

FcγR mediate the phagocytosis of IgG-coated particles and the clearance of IgG immune complexes. By dissecting binding from internalization of the particles, we found that the binding stage, rather than particle internalization, triggered tyrosine phosphorylation of FcγR and accompanying proteins. High amounts of Lyn kinase were found to associate with particles isolated at the binding stage from J774 cells. PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), an Src kinase inhibitor, but not piceatannol, an inhibitor of Syk kinase, reduced the amount of Lyn associated with the bound particles and simultaneously diminished the binding of IgG-coated particles. Studies of baby hamster kidney cells transfected with wild-type and mutant FcγRIIA revealed that the ability of the receptor to bind particles was significantly reduced when phosphorylation of the receptor was abrogated by Y298F substitution in the receptor signaling motif. Under these conditions, binding of immune complexes of aggregated IgG was depressed to a lesser extent. A similar effect was exerted on the binding ability of wild-type FcγRIIA by PP2. Moreover, expression of mutant kinase-inactive Lyn K275R inhibited both FcγRIIA phosphorylation and IgG-opsonized particle binding. To gain insight into the mechanism by which protein tyrosine phosphorylation can control FcγR-mediated binding, we investigated the efficiency of clustering of wild-type and Y298F-substituted FcγRIIA upon binding of immune complexes. We found that a lack of FcγRIIA phosphorylation led to an impairment of receptor clustering. The results indicate that phosphorylation of FcγR and accompanying proteins, dependent on Src kinase activity, facilitates the clustering of activated receptors that is required for efficient particle binding.

Lysenin, a unique sphingomyelin-binding protein

Sphingomyelin plays complex structural and signaling functions in the plasma membrane. Of special interest is that hydrolysis of sphingomyelin to ceramide can modulate dynamics of membrane rafts, which serve as signaling platforms for various receptors. This review is focused on a recently discovered sphingomyelin‐binding protein, lysenin, which can be used as a unique probe to trace distribution and turnover of sphingomyelin in cellular membranes. We analyze the primary and secondary structures of lysenin with respect to its interaction with the plasma membrane. The specificity of lysenin binding to sphingomyelin, revealed by both biochemical and cytochemical approaches, is discussed.

Ceramide and Ceramide 1-Phosphate Are Negative Regulators of TNF-α Production Induced by Lipopolysaccharide

LPS is a constituent of cell walls of Gram-negative bacteria that, acting through the CD14/TLR4 receptor complex, causes strong proinflammatory activation of macrophages. In murine peritoneal macrophages and J774 cells, LPS at 1–2 ng/ml induced maximal TNF-α and MIP-2 release, and higher LPS concentrations were less effective, which suggested a negative control of LPS action. While studying the mechanism of this negative regulation, we found that in J774 cells, LPS activated both acid sphingomyelinase and neutral sphingomyelinase and moderately elevated ceramide, ceramide 1-phosphate, and sphingosine levels. Lowering of the acid sphingomyelinase and neutral sphingomyelinase activities using inhibitors or gene silencing upregulated TNF-α and MIP-2 production in J774 cells and macrophages. Accordingly, treatment of those cells with exogenous C8-ceramide diminished TNF-α and MIP-2 production after LPS stimulation. Exposure of J774 cells to bacterial sphingomyelinase or interference with ceramide hydrolysis using inhibitors of ceramidases also lowered the LPS-induced TNF-α production. The latter result indicates that ceramide rather than sphingosine suppresses TNF-α and MIP-2 production. Of these two cytokines, only TNF-α was negatively regulated by ceramide 1-phosphate as was indicated by upregulated TNF-α production after silencing of ceramide kinase gene expression. None of the above treatments diminished NO or RANTES production induced by LPS. Together the data indicate that ceramide negatively regulates production of TNF-α and MIP-2 in response to LPS with the former being sensitive to ceramide 1-phosphate as well. We hypothesize that the ceramide-mediated anti-inflammatory pathway may play a role in preventing endotoxic shock and in limiting inflammation.

Annexin A1 Tethers Membrane Contact Sites that Mediate ER to Endosome Cholesterol Transport

Summary Membrane contact sites between the ER and multivesicular endosomes/bodies (MVBs) play important roles in endosome positioning and fission and in neurite outgrowth. ER-MVB contacts additionally function in epidermal growth factor receptor (EGFR) tyrosine kinase downregulation by providing sites where the ER-localized phosphatase, PTP1B, interacts with endocytosed EGFR before the receptor is sorted onto intraluminal vesicles (ILVs). Here we show that these contacts are tethered by annexin A1 and its Ca2+-dependent ligand, S100A11, and form a subpopulation of differentially regulated contact sites between the ER and endocytic organelles. Annexin A1-regulated contacts function in the transfer of ER-derived cholesterol to the MVB when low-density lipoprotein-cholesterol in endosomes is low. This sterol traffic depends on interaction between ER-localized VAP and endosomal oxysterol-binding protein ORP1L, and is required for the formation of ILVs within the MVB and thus for the spatial regulation of EGFR signaling.

Lysenin-His, a sphingomyelin-recognizing toxin, requires tryptophan 20 for cation-selective channel assembly but not for membrane binding

Lysenin is 297 amino acid long toxin derived from the earthworm Eisenia foetida which specifically recognizes sphingomyelin and induces cell lysis. We synthesized lysenin gene supplemented with a polyhistidine tag, subcloned it into the pT7RS plasmid and the recombinant protein was produced in Escherichia coli. In order to obtain lysenin devoid of its lytic activity, the protein was mutated by substitution of tryptophan 20 by alanine. The recombinant mutant lysenin-His did not evoke cell lysis, although it retained the ability to specifically interact with sphingomyelin, as demonstrated by immunofluorescence microscopy and by dot blot lipid overlay and liposome binding assays. We found that the lytic activity of wild-type lysenin-His was correlated with the protein oligomerization during interaction with sphingomyelin-containing membranes and the amount of oligomers was increased with an elevation of sphingomyelin/lysenin ratio. Blue native gel electrophoresis indicated that trimers can be functional units of the protein, however, lysenin hexamers and nanomers were stabilized by chemical cross-linking of the protein and by sodium dodecyl sulfate. When incorporated into planar lipid bilayers, wild type lysenin-His formed cation-selective channels in a sphingomyelin-dependent manner. We characterized the channel activity by establishing its various open/closed states. In contrast, the mutant lysenin-His did not form channels and its correct oligomerization was strongly impaired. Based on these results we suggest that lysenin oligomerizes upon interaction with sphingomyelin in the plasma membrane, forming cation-selective channels. Their activity disturbs the ion balance of the cell, leading eventually to cell lysis.