Lipid Raft Targeting Research Articles

Messages from Mutant Desmosomes

Single gene disorders are ideally suited to establish robust genotype‒phenotype correlations and provide excellent opportunities to understand molecular pathomechanisms with relevance to complex disorders. The observation that patients diagnosed with the same causative mutation can present with phenotypic disease variability illustrates the significant role of disease modifiers and warns against oversimplification. In a new article in the Journal of Investigative Dermatology, Zimmer etal. (2021) analyze two mutations located in the desmoglein (DSG) 1 transmembrane domain (TMD) and find that both mutants fail to assemble into desmosomes owing to reduced membrane trafficking and lipid raft targeting. One mutation maintained normal protein expression levels and turnover relative to those of wild-type (WT) DSG1, and behaved as a dominant negative. The second mutant showed reduced stability and increased turnover compared with WT DSG1 as well as reduced desmosome size and abundance. A full understanding of the TMD of DSG1 requires cell biological approaches, underscoring the value of cell biology in biomedical research in general.

Subsynaptic Distribution, Lipid Raft Targeting and G Protein-Dependent Signalling of the Type 1 Cannabinoid Receptor in Synaptosomes from the Mouse Hippocampus and Frontal Cortex

Numerous studies have investigated the roles of the type 1 cannabinoid receptor (CB1) in glutamatergic and GABAergic neurons. Here, we used the cell-type-specific CB1 rescue model in mice to gain insight into the organizational principles of plasma membrane targeting and Gαi/o protein signalling of the CB1 receptor at excitatory and inhibitory terminals of the frontal cortex and hippocampus. By applying biochemical fractionation techniques and Western blot analyses to synaptosomal membranes, we explored the subsynaptic distribution (pre-, post-, and extra-synaptic) and CB1 receptor compartmentalization into lipid and non-lipid raft plasma membrane microdomains and the signalling properties. These data infer that the plasma membrane partitioning of the CB1 receptor and its functional coupling to Gαi/o proteins are not biased towards the cell type of CB1 receptor rescue. The extent of the canonical Gαi/o protein-dependent CB1 receptor signalling correlated with the abundance of CB1 receptor in the respective cell type (glutamatergic versus GABAergic neurons) both in frontal cortical and hippocampal synaptosomes. In summary, our results provide an updated view of the functional coupling of the CB1 receptor to Gαi/o proteins at excitatory and inhibitory terminals and substantiate the utility of the CB1 rescue model in studying endocannabinoid physiology at the subcellular level.

Differential Pathomechanisms of Desmoglein 1 Transmembrane Domain Mutations in Skin Disease

Dominant and recessive mutations in the desmosomal cadherin, desmoglein (DSG) 1, cause the skin diseases palmoplantar keratoderma (PPK) and severe dermatitis, multiple allergies, and metabolic wasting (SAM) syndrome, respectively. In this study, we compare two dominant missense mutations in the DSG1 transmembrane domain (TMD), G557R and G562R, causing PPK (DSG1PPK-TMD) and SAM syndrome (DSG1SAM-TMD), respectively, to determine the differing pathomechanisms of these mutants. Expressing the DSG1TMD mutants in a DSG-null background, we use cellular and biochemical assays to reveal the differences in the mechanistic behavior of each mutant. Super-resolution microscopy and functional assays showed a failure by both mutants to assemble desmosomes due to reduced membrane trafficking and lipid raft targeting. DSG1SAM-TMD maintained normal expression levels and turnover relative to wildtype DSG1, but DSG1PPK-TMD lacked stability, leading to increased turnover through lysosomal and proteasomal pathways and reduced expression levels. These results differentiate the underlying pathomechanisms of these disorders, suggesting that DSG1SAM-TMD acts dominant negatively, whereas DSG1PPK-TMD is a loss-of-function mutation causing the milder PPK disease phenotype. These mutants portray the importance of the DSG TMD in desmosome function and suggest that a greater understanding of the desmosomal cadherin TMDs will further our understanding of the role that desmosomes play in epidermal pathophysiology.

Lipid binding by the N-terminal motif mediates plasma membrane localization of Bordetella effector protein BteA

The respiratory pathogens Bordetella pertussis and Bordetella bronchiseptica employ a type III secretion system (T3SS) to inject a 69-kDa BteA effector protein into host cells. This effector is known to contain two functional domains, including an N-terminal lipid raft targeting (LRT) domain and a cytotoxic C-terminal domain that induces nonapoptotic and caspase-1–independent host cell death. However, the exact molecular mechanisms underlying the interaction of BteA with plasma membrane (PM) as well as its cytotoxic activity in the course of Bordetella infections remain poorly understood. Using a protein–lipid overlay assay and surface plasmon resonance, we show here that the recombinant LRT domain binds negatively charged membrane phospholipids. Specifically, we determined that the dissociation constants of the LRT domain–binding liposomes containing phosphatidylinositol 4,5-bisphosphate, phosphatidic acid, and phosphatidylserine were ∼450 nM, ∼490 nM, and ∼1.2 μM, respectively. Both phosphatidylserine and phosphatidylinositol 4,5-bisphosphate were required to target the LRT domain and/or full-length BteA to the PM of yeast cells. The membrane association further involved electrostatic and hydrophobic interactions of LRT and depended on a leucine residue in the L1 loop between the first two helices of the four-helix bundle. Importantly, charge-reversal substitutions within the L1 region disrupted PM localization of the BteA effector without hampering its cytotoxic activity during B. bronchiseptica infection of HeLa cells. The LRT-mediated targeting of BteA to the cytosolic leaflet of the PM of host cells is, therefore, dispensable for effector cytotoxicity.

The Search Continues: Determination of a Lipid Raft Targeting Motif in Cytochromes P450

Cytochromes P450 play an essential role in metabolism of both xenobiotics and endogenous compounds. The liver P450s responsible for drug metabolism are predominantly expressed in hepatocytes, where they closely associate with the endoplasmic reticulum (ER). This membranous organelle is mostly composed of phospholipids, which can assemble into disordered regions (ld) or ordered regions (lo, “lipid rafts”).Our group previously showed that different P450 species show strong preference for either ordered or disordered regions. When expressed in human embryonic kidney (HEK) cells, we demonstrated that CYP1A2 is found in ordered (raft) regions, and that CYP1A1 exhibits a strong preference for the disordered region. This pattern was striking, as the two P450 species have over 70% sequence homology. We then showed that a chimeric CYP1A2 construct containing the first 107 amino acids of CYP1A1 no longer localized in the ordered regions, but mobilized to the disordered region. These findings indicate that a P450 raft targeting sequence exists within the first 107 amino acids of CYP1A2.The length and composition of this motif remain unclear. We hypothesize that a 5–9 amino acid sequence within the first 107 amino acids is responsible for the localization of CYP1A2 into the ordered domains, and that substitution of the corresponding region of CYP1A1 can mobilize full‐length CYP1A2 to the disordered region. To test this, we generated 5‐ to 30‐amino acid chimeras using overhang PCR cloning and expressed these constructs in HEK cells. The disordered membrane regions from these cells were solubilized using Brij98, with the ordered regions remaining in the membrane. The ordered membranes were isolated as a pellet after centrifugation at 100,000 x g for 1 h. We found that insertion of the first 28 amino acids of CYP1A1 into CYP1A2 led to its partial relocation into the disordered regions, and that the first 30 amino acids led to more complete relocation of the chimeric protein. We then generated smaller chimeras in an effort to identify the minimum amino acid segment responsible for membrane relocation. Substitution of amino acids 28–30 led to relocation of CYP1A2 from the ordered to disordered regions. These results demonstrate that a raft‐targeting sequence resides within the first 30 N‐terminal amino acids of CYP1A2 and, when eliminated, permits mobilization of the P450.Support or Funding InformationSupported by NIH GM123253 and ES013648 and by a predoctoral fellowship from the PhRMA Foundation.

The Desmoglein‐1 Transmembrane Domain Drives Lipid Raft Association and Desmosome Assembly

The epidermis is a barrier to the outside world, but mutations in genes encoding desmosomal proteins can inhibit this function, causing human diseases of skin fragility. Severe dermatitis, multiple allergies, and metabolic wasting (SAM) syndrome is a severe homozygous recessive disorder of skin fragility caused by mutations in the gene encoding desmoglein 1 (DSG1), a component of desmosomes. Desmosomes are junctional complexes that function in mechanical stress resistance across tissues. In these junctions, desmosomal cadherins such as DSG1 interact across extracellular spaces and with intracellular adaptor proteins to link cytoskeletal elements of adjacent cells. Both assembly and disassembly of desmosomes have been shown to rely on lipid raft membrane microdomains, highly ordered cholesterol and sphingolipid‐enriched regions of the plasma membrane important in signaling and trafficking. Our current model predicts that raft association promotes clustering of desmosomal proteins to drive assembly and mediate adhesion. However, the mechanisms of raft association and desmosome assembly are not fully understood. We created a DSG‐null A431 cell line to examine the relationship between DSG raft association and desmosomal adhesion. A431s are an immortal human epidermal carcinoma cell line commonly used in desmosomal studies that express a single DSG species, DSG2. The absence of DSG2 in these cells was confirmed by western blot and immunofluorescence. Further characterization of these DSG‐null A431 cells revealed mislocalized desmoplakin, a marker of mature desmosomes, and a lack of strong cell‐cell adhesion. Exogenous stable expression of GFP‐tagged wildtype DSG1 (DSG1WT‐GFP) rescued both desmosome formation and function, suggesting that these DSG‐null cells would be suitable for probing the relationship between DSG raft association and desmosome assembly and function. Recently, we discovered a novel, heterozygous mutation in the transmembrane domain (TMD)‐encoding region of DSG1 that causes a glycine to arginine substitution and SAM syndrome. DSG1SAM is a lipid raft targeting mutant suggesting an important role for the TMD in raft association. Stable expression of DSG1SAM‐GFP could not rescue desmosome formation or function in the DSG‐null cells, suggesting that a loss of raft association also impacts desmosome assembly and adhesive function. Intriguingly, a chimeric DSG1 protein containing the TMD from E‐cadherin (DSG1Ecad‐GFP), a non‐raft associating adhesive protein, only partly rescued desmosome formation and function. Furthermore, these results correlated with the predicted lipid raft affinity of each TMD, suggesting that desmosome assembly and function is dependent on raft association. We are currently analyzing a panel of GFP‐tagged DSG1TMD variants with differential raft targeting affinities and assessing their ability to support desmosome assembly and function. In conclusion, the TMD targets desmogleins to lipid rafts and modulates desmoglein adhesive function.Support or Funding InformationNIH R01AR050501, NIH R01AR048266

SPalmitoylC-PseAAC: A sequence-based model developed via Chou's 5-steps rule and general PseAAC for identifying S-palmitoylation sites in proteins

S-Palmitoylation is a uniquely reversible and biologically important post-translational modification as it plays an essential role in a variety of cellular processes including signal transduction, protein-membrane interactions, neuronal development, lipid raft targeting, subcellular localization and apoptosis. Due to its association with the neuronal development, it plays a pivotal role in a variety of neurodegenerative diseases, mainly Alzheimer's, Schizophrenia and Huntington's disease. It is also essential for developmental life cycles and pathogenesis of Toxoplasma gondii and Plasmodium falciparum, known to cause toxoplasmosis and malaria, respectively. This depicts the strong biological significance of S-Palmitoylation, thus, the timely and accurate identification of S-palmitoylation sites is crucial. Herein, we propose a predictor for S-Palmitoylation sites in proteins namely SPalmitoylC-PseAAC by integrating the Chou's Pseudo Amino Acid Composition (PseAAC) and relative/absolute position-based features. Self-consistency testing and 10-fold cross-validation are performed to evaluate the performance of SPalmitoylC-PseAAC, using accuracy metrics. For self-consistency testing, 99.79% Acc, 99.77% Sp, 99.80% Sn and 1.00 MCC was observed, whereas, for 10-fold cross validation 97.22% Acc, 98.85% Sp, 95.80% Sn and 0.94 MCC was observed. Thus the proposed predictor can help in predicting the palmitoylation sites in an efficient and accurate way. The SPalmitoylC-PseAAC is available at (biopred.org/palm).

544 A mutation in the desmoglein 1 transmembrane domain abrogates lipid raft targeting and causes severe dermatitis, multiple allergies, and metabolic wasting (SAM) syndrome

544 A mutation in the desmoglein 1 transmembrane domain abrogates lipid raft targeting and causes severe dermatitis, multiple allergies, and metabolic wasting (SAM) syndrome

Caveolin interaction governs Kv1.3 lipid raft targeting.

The spatial localization of ion channels at the cell surface is crucial for their functional role. Many channels localize in lipid raft microdomains, which are enriched in cholesterol and sphingolipids. Caveolae, specific lipid rafts which concentrate caveolins, harbor signaling molecules and their targets becoming signaling platforms crucial in cell physiology. However, the molecular mechanisms involved in such spatial localization are under debate. Kv1.3 localizes in lipid rafts and participates in the immunological response. We sought to elucidate the mechanisms of Kv1.3 surface targeting, which govern leukocyte physiology. Kv1 channels share a putative caveolin-binding domain located at the intracellular N-terminal of the channel. This motif, lying close to the S1 transmembrane segment, is situated near the T1 tetramerization domain and the determinants involved in the Kvβ subunit association. The highly hydrophobic domain (FQRQVWLLF) interacts with caveolin 1 targeting Kv1.3 to caveolar rafts. However, subtle variations of this cluster, putative ancillary associations and different structural conformations can impair the caveolin recognition, thereby altering channel’s spatial localization. Our results identify a caveolin-binding domain in Kv1 channels and highlight the mechanisms that govern the regulation of channel surface localization during cellular processes.

Abstract A229: Mechanism of action of perifosine on the mantle cell lymphoma line, Granta-519.

Abstract Lipid rafts are highly ordered membrane domains that are enriched in cholesterol and sphingolipids and provides a scaffold for signal transduction. Altered raft assembly has been implicated in cancer progression. Alkylphospholipids have been used with promising specific cytotoxic effects in different types of cancer cells. These alkylphospholipids act by interaction with cell membranes and protein lipid rafts. Cell viability was determined by trypan blue assay. Cell cycle was evaluated by flow cytometry. Lipid rafts were isolated using sucrose density-gradient centrifugation after 12 hours of treatment with perifosine. Antibody microarray and western blotting was used to determine levels of key cellular proteins in special on cell signaling proteins. We showed that perifosine, an alkylphospholipid, targets raft-like domains in model membranes and induces apoptosis in mantle cell lymphomas (ED-50 20 μM - Granta-519) by activation of both extrinsic and intrinsic pathways. We observed an effect on the organization of the cell cycle, with increase of the population at G2/M phase (8,6% ± 0,6% to 27,4% ± 2,4%). Cyclin D1 decrease was detected at 24 hours of treatment. We also showed that perifosine downregulates NTAL/LAB (Non-T-cell activation linker/ Linker for activation of B-cells), an adaptor protein that is targeted to rafts by palmitoylation and specifically expressed in hematopoietic tissues. Moreover, perifosine induced a loss of NTAL/LAB in the lipid rafts of Granta-519 cells after 12 hours of treatment. Since NTAL/LAB may function as an adaptor protein in AKT signalization, we evaluate the effect of perifosine on the ATK pathway. We showed that perifosine lead to dephosphorylation of AKT and downstream components of AKT signaling. Moreover, in functional experiments, perifosine inhibited AKT activation by CD40L or IL-4 in Granta-519 cells, after few minutes of incubation, in a way similar to the specific PI3K inhibitor wortmannin. Treatment with methyl-β-cyclodextrin, a compound for cholesterol depletion, potentiated this effect. This suggests that NTAL/LAB translation is highly regulated and dependent on a functional AKT pathway. Our results indicate that perifosine acts on NTAL/LAB presumable by interference with its protein-lipid interactions and consequently AKT signaling. Our results demonstrate that a lipid raft targeting drug may present several effects on signal transduction, causing a severe toxicity to mantle cell lymphoma. Moreover, adaptor proteins like NTAL/LAB emerge as possible new therapeutic targets in lymphoma. This research was supported by FAPESP, CNPq and CAPES. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A229. Citation Format: Germano A. Ferreira, Carolina H. Thome, Guilherme A. dos Santos, Priscila S. Scheucher, Andreia M. Leopoldino, Ana M. Simão, Clarice Izume, Rodrigo A. Panepucci, Pietro Ciancaglini, Eduardo M. Rego, Vitor M. Faça, Lewis J. Greene. Mechanism of action of perifosine on the mantle cell lymphoma line, Granta-519. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A229.

The Intracellular Domain of Sortilin Interacts with Amyloid Precursor Protein and Regulates Its Lysosomal and Lipid Raft Trafficking

The processing of Amyloid precursor protein (APP) is multifaceted, comprising of protein transport, internalization and sequential proteolysis. However, the exact mechanism of APP intracellular trafficking and distribution remains unclear. To determine the interaction between sortilin and APP and the effect of sortilin on APP trafficking and processing, we studied the binding site and its function by mapping experiments, colocalization, coimmunoprecipitation and sucrose gradient fractionation. We identified for the first time that sortilin interacts with APP at both N- and C-terminal regions. The sortilin-FLVHRY (residues 787–792) and APP-NPTYKFFE (residues 759–766) motifs are crucial for the C-terminal interaction. We also found that lack of the FLVHRY motif reduces APP lysosomal targeting and increases APP distribution in lipid rafts in co-transfected HEK293 cells. These results are consistent with our in vivo data where sortilin knockout mice showed a decrease of APP lysosomal distribution and an increase of APP in lipid rafts. We further confirmed that overexpression of sortilin-FLVHRY mutants failed to rescue the lysosomal degradation of APP. Thus, our data suggests that sortilin is implicated in APP lysosomal and lipid raft targeting via its carboxyl-terminal F/YXXXXF/Y motif. Our study provides new molecular insights into APP trafficking and processing.

Rnd1 and Rnd3 targeting to lipid raft is required for p190 RhoGAP activation

The Rnd proteins Rnd1, Rnd2, and Rnd3/RhoE are well known as key regulators of the actin cytoskeleton in various cell types, but they comprise a distinct subgroup of the Rho family in that they are GTP bound and constitutively active. Functional differences of the Rnd proteins in RhoA inhibition signaling have been reported in various cell types. Rnd1 and Rnd3 antagonize RhoA signaling by activating p190 RhoGAP, whereas Rnd2 does not. However, all the members of the Rnd family have been reported to bind directly to p190 RhoGAP and equally induce activation of p190 RhoGAP in vitro, and there is no evidence that accounts for the functional difference of the Rnd proteins in RhoA inhibition signaling. Here we report the role of the N-terminal region in signaling. Rnd1 and Rnd3, but not Rnd2, have a KERRA (Lys-Glu-Arg-Arg-Ala) sequence of amino acids in their N-terminus, which functions as the lipid raft-targeting determinant. The sequence mediates the lipid raft targeting of p190 RhoGAP correlated with its activation. Overall, our results demonstrate a novel regulatory mechanism by which differential membrane targeting governs activities of Rnd proteins to function as RhoA antagonists.

Linker of Activation of T Cells – 2 (LAT2), a Lipid Raft-Associated Adaptor Protein, Is a Novel Target for Therapy in Acute Leukemia

Abstract 737Lipid rafts are highly ordered membrane domains that are enriched in cholesterol and sphingolipids and function in providing a scaffold to signal transduction. Altered raft assembly has been implicated in breast and colorectal cancer progression; rafts are essential to sustained p38K activation by GM-CSF and VEGFa in acute myeloid leukemia (Saulle et al. BJH, 2009). In a previous study, we showed that 10-(Octyloxy) decyl-2-(trimethylammonium) ethyl phosphate (ODPC), an alkylphospholipid, targets raft like domains in model membranes and induces apoptosis in leukemia cells, (ED-50 25mM - NB4), but spares normal hematopoietic and epithelial cells (dos Santos et al. BBACTA 2010). Here, we performed an unbiased quantitative proteomic screen of ODPC targets in lipid rafts. We used stable isotope labeling in cell culture (SILAC) to reveal proteins that are modulated by ODPC before the installation of apoptosis, to gain access to the primary mechanisms of drug action (Fig 1A, B and C). We used successive detergent extraction (Fig 1D) to isolate detergent resistant membranes (DRM) enriched in lipid rafts; blots with specific markers prove the quality of fractionation (Fig 1E). Proteins were considered modulated if: fold change of 2/0.5 or greater plus two or more quantification events and a P value <0.1. A small fraction of proteins was modulated by ODPC (Fig 1F). Of the 1426 proteins identified in the total extract, only one protein (0.07%) was upregulated. Of the 1314 proteins identified in the DRM, 6 proteins (0.45%) were donwregulated. Of these, 4 have predicted or validated palmitoylation sites (Fig 1F). We validated that ODPC downregulates LAT-2 (Fig 2A), an adaptor protein that is target to rafts by palmitoylation and is expressed in B cells and myeloid cells. Interesting, LAT-2 was not expressed in K562 (Fig 2A), a cell resistant to ODPC-induced apoptosis (Fig 1A and B). Moreover, ODPC induced a loss of LAT-2 in the lipid rafts of NB4 cells after 3h of treatment (Fig 2B). Since LAT-2 may function as an adaptor protein in AKT signalization (Roget et al. J.Immunol. 2008) we evaluate the effect of ODPC on the ATK pathway. Fig 2C shows that ODPC induces a downregulation of all components from AKT signaling, compatible with a membrane target drug. However, in functional experiments, ODPC inhibited AKT activation in NB4 cells by myeloid growth factors (MGF), after few minutes of incubation, in a similar way of the specific PI3K inhibitor Wortmannin (Fig 2D). Moreover, in these experiments, MGF induced a rapid and translation-dependent up regulation in LAT-2 expression in control treated cells (Fig 2E). This effect was partially suppressed by ODPC treatment and was suppressed by Wortmannin (Fig 2D). This suggests that LAT-2 translation is highly regulated and dependent on a functional AKT pathway. Indeed, phosphorylation of 4EBP-1 and ribossomal S6 kinase (S6K) with consequent upregulation of translation in the ribosome is downstream of PI3K/AKT/mTOR (Mamane et al. Oncogene, 2006). Moreover, we confirmed a hypophosphorilation state of S6K in the presence of ODPC or Wortmannin (Fig 2D). Interesting, we have identified two potential-palmitoylated ribosomal proteins (RP) to be downregulated in the lipid raft fraction of ODPC treated cells (Fig 1F). This finding confirms previous reports that a raft disruption treatment (methyl-β-cyclodextrin) causes downregulation of several RP (Foster, De Hoog and Mann. PNAS, 2003) and that palmitoylation target ribosome proteins to lipid rafts (Wei et al. MCP, 2010). This indicates that ODPC may present also a direct or indirect inhibitory effect on translational apparatus. Our results strongly indicate that ODPC acts on LAT-2 by interference with its protein-lipid interactions, since experimental-induced disturbances in lipid raft physiology recapitulate or increase ODPC effects on LAT-2 expression (Fig 3A and B). To further clarify the mechanism of LAT-2 downregulation we evaluate if it is degraded by the proteasome after ODPC or 2BrPA treatments. Fig 3C and D reveals that this is the case. This confirms the findings of Orr and McVicar (J.Leuk.Biol. 2011) of ubiquitin-proteasomal regulation of LAT-2 stability. Taken together, our results demonstrate that a lipid raft targeting drug may present several effects on signal transduction, causing a severe toxicity to leukemia cells. Moreover, adaptor proteins like LAT-2 emerge as possible new therapeutic targets in leukemia. [Display omitted] [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Cyclin-dependent Kinase-5 Is a Key Molecule in Tumor Necrosis Factor-α-induced Insulin Resistance

The mechanism of TNF-α-induced insulin resistance has remained unresolved with evidence for down-regulation of insulin effector targets effects or blockade of proximal as well as distal insulin signaling events depending upon the dose, time, and cell type examined. To address this issue we examined the acute actions of TNF-α in differentiated 3T3L1 adipocytes. Acute (5-15 min) treatment with 20 ng/ml (~0.8 nm) TNF-α had no significant effect on IRS1-associated phosphatidylinositol 3-kinase. In contrast, TNF-α increased insulin-stimulated cyclin-dependent kinase-5 (CDK5) phosphorylation on tyrosine residue 15 through an Erk-dependent pathway and up-regulated the expression of the CDK5 regulator protein p35. In parallel, TNF-α stimulation also resulted in the phosphorylation and GTP loading of the Rho family GTP-binding protein, TC10α. TNF-α enhanced the depolymerization of cortical F-actin and inhibited insulin-stimulated glucose transporter-4 (GLUT4) translocation. Treatment with the MEK inhibitor, PD98059, blocked the TNF-α-induced increase in CDK5 phosphorylation and the depolymerization of cortical F-actin. Conversely, siRNA-mediated knockdown of CDK5 or treatment with the MEK inhibitor restored the impaired insulin-stimulated GLUT4 translocation induced by TNF-α. Furthermore, siRNA-mediated knockdown of p44/42 Erk also rescued the TNF-α inhibition of insulin-stimulated GLUT4 translocation. Together, these data demonstrate that TNF-α-mediated insulin resistance of glucose uptake can occur through a MEK/Erk-dependent activation of CDK5.

Correction: Inhibition of T Cell Activation by Cyclic Adenosine 5′-Monophosphate Requires Lipid Raft Targeting of Protein Kinase A Type I by the A-Kinase Anchoring Protein Ezrin

Correction: Inhibition of T Cell Activation by Cyclic Adenosine 5′-Monophosphate Requires Lipid Raft Targeting of Protein Kinase A Type I by the A-Kinase Anchoring Protein Ezrin

Capacitation suppression by mouse seminal vesicle autoantigen involves a decrease in plasma membrane Ca2+‐ATPase (PMCA)‐mediated intracellular calcium

Successful fertilization is tightly regulated by capacitation and decapacitation processes. Without appropriate decapacitation regulation, sperm would undergo a spontaneous acrosome reaction which leads to loss of fertilization ability. Seminal plasma is known to negatively regulate sperm capacitation. However, the suppressive mechanisms still remain unclear. In this study, we demonstrate the decapacitation mechanism of mouse seminal vesicle autoantigen (SVA) might target membrane sphingomyelin (SPM) and regulate plasma membrane Ca(2+)-ATPase (PMCA) activity. The SVA was shown to suppress sperm capacitation induced by a broad panel of capacitation factors (bovine serum albumin (BSA), PAF, and cyclodextrin (CD)). Furthermore, SVA significantly decreased [Ca(2+)](i) and NaHCO(3)-induced [cAMP](i). Cyclic AMP agonists bypassed the SVA's suppressive ability. Importantly, the SVA may regulate PMCA activity which was evidenced by the fact that the SVA decreased the [Ca(2+)](i) and intracellular pH (pH(i)) of sperm; meanwhile, a PMCA inhibitor (carboxyeosin) could reverse SVA's suppression of [Ca(2+)](i). The potential target of the SVA on membrane SPM/lipid rafts was highlighted by the high binding affinity of SPM-SVA (with a K(d) of ~3 µM) which was close to the IC(50) of SVA's suppressive activity. Additionally, treatment of mink lung epithelial cells with the SVA enhanced plasminogen activator inhibitor (PAI)-1 expression stimulated by tumor growth factor (TGF)-β and CD. These observations supported the membrane lipid-raft targeting of SVA. In summary, in this paper, we demonstrate that the decapacitation mechanism of the SVA might target membrane sphingolipid SPM and regulate PMCA activity to lower [Ca(2+)](i), thereby decreasing the [cAMP](i) level and preventing sperm pre-capacitation.

Palmitoylation of platelet‐activating factor receptor is required for its lipid raft targeting and signaling

Platelet‐activating factor receptor (PAFR) signaling has been implicated in a broad range of pathologies. Site‐directed mutagenesis, recombinant protein analysis, sucrose density fractionation and radiolabeling studies indicate that the PAFR is targeted to lipid rafts and can be palmitoylated on cysteine 317. Polyunsaturated fatty acids (PUFA) block PAF‐induced apoptosis, ion transport and proinflammatory gene expression. Palmitate incorporation into the PAFR is inhibited in the presence of excess arachidonic acid (AA), docosahexaenoic acid (DHA), and a palmitoylation inhibitor 2‐bromopalmitate (2‐BP). PAFR solubilized from membranes of untreated HT29‐CL19A colonocytes sediments to the 30% sucrose density fraction similar to occludin, a bone fide lipid raft marker in these cells. Both the PAFR and occludin are displaced from this fraction and they are detected in the lighter 15% to 30% interface (both PAFR and occludin) and in the 15% sucrose fraction (occludin) when solubilized from mebranes derived from HT29‐CL19A cells treated with 2‐BP AA or DHA. These data are consistent with the notion that, in addition to their well known effects on prostaglandin biosytnthesis, PUFA can act via a novel mechanism by which they alter posttranslational modification and they displace PAFR from lipid rafts. Supported by NIH DK062960.

Growth hormone receptor targeting to lipid rafts requires extracellular subdomain 2

GH receptor (GHR) is a single membrane-spanning glycoprotein dimer that binds GH in its extracellular domain (ECD). GH activates the GHR intracellular domain (ICD)-associated tyrosine kinase, JAK2, which causes intracellular signaling. We previously found that plasma membrane (PM)-associated GHR was dramatically enriched in the lipid raft (LR) component of the membrane and that localization of GHR within PM regions may regulate GH signaling by influencing the profile of pathway activation. In this study, we examined determinants of LR localization of the GHR using a reconstitution system which lacks endogenous JAK2 and GHR. By non-detergent extraction and multistep fractionation, we found that GHR was highly enriched in the LR fraction independent of JAK2 expression. Various GHR mutants were examined in transfectants harboring JAK2. LR concentration was observed for a GHR in which the native transmembrane domain (TMD) is replaced by that of the unrelated LDL receptor and for a GHR that lacks its ICD. Thus, LR association requires neither the TMD nor the ICD. Similarly, a GHR that lacks the ECD, except for the membrane-proximal ECD stem region, was only minimally LR-concentrated. Mutants with internal stem deletions in the context of the full-length receptor were LR-concentrated similar to the wild-type. A GHR lacking ECD subdomain 1 reached the PM and was LR-concentrated, while one lacking ECD subdomain 2, also reached the PM, but was not LR-concentrated. These data suggest LR targeting resides in ECD subdomain 2, a region relatively uninvolved in GH binding.

TheBordetellatype III secretion system effector BteA contains a conserved N-terminal motif that guides bacterial virulence factors to lipid rafts

The Bordetella type III secretion system (T3SS) effector protein BteA is necessary and sufficient for rapid cytotoxicity in a wide range of mammalian cells. We show that BteA is highly conserved and functionally interchangeable between Bordetella bronchiseptica, Bordetella pertussis and Bordetella parapertussis. The identification of BteA sequences required for cytotoxicity allowed the construction of non-cytotoxic mutants for localization studies. BteA derivatives were targeted to lipid rafts and showed clear colocalization with cortical actin, ezrin and the lipid raft marker GM1. We hypothesized that BteA associates with the cytoplasmic face of lipid rafts to locally modulate host cell responses to Bordetella attachment. B. bronchiseptica adhered to host cells almost exclusively to GM1-enriched lipid raft microdomains and BteA colocalized to these same sites following T3SS-mediated translocation. Disruption of lipid rafts with methyl-β-cyclodextrin protected cells from T3SS-induced cytotoxicity. Localization to lipid rafts was mediated by a 130-amino-acid lipid raft targeting domain at the N-terminus of BteA, and homologous domains were identified in virulence factors from other bacterial species. Lipid raft targeting sequences from a T3SS effector (Plu4750) and an RTX-type toxin (Plu3217) from Photorhabdus luminescens directed fusion proteins to lipid rafts in a manner identical to the N-terminus of BteA.

The C1 and C2 domains target human type 6 adenylyl cyclase to lipid rafts and caveolae

Previous data has shown that adenylyl cyclase type 6 (AC6) is expressed principally in lipid rafts or caveolae of cardiac myocytes and other cell types while certain other isoforms of AC are excluded from these microdomains. The mechanism by which AC6 is localized to lipid rafts or caveolae is unknown. In this study, we show AC6 is localized in lipid rafts of COS-7 cells (expressing caveolin-1) and in HEK-293 cells or cardiac fibroblasts isolated from caveolin-1 knock-out mice (both of which lack prototypical caveolins). To determine the region of AC6 that confers raft localization, we independently expressed each of the major intracellular domains, the N-terminus, C1 and C2 domains, and examined their localization with various approaches. The N-terminus did not associate with lipid rafts or caveolae of either COS-7 or HEK-293 cells nor did it immunoprecipitate with caveolin-1 when expressed in COS-7 cells. By contrast, the C1 and C2 domains each associated with lipid rafts to varying degrees and were present in caveolin-1 immunoprecipitates. There were no differences in the pattern of localization of either the C1 or C2 domains between COS-7 and HEK-293 cells. Further dissection of the C1 domain into four individual proteins indicated that the N-terminal half of this domain is responsible for its raft localization. To probe for a role of a putative palmitoylation motif in the C-terminal portion of the C2 domain, we expressed various truncated forms of AC6 lacking most or all of the C-terminal 41 amino acids. These truncated AC6 proteins were not altered in terms of their localization in lipid rafts or their catalytic activity, implying that this C-terminal region is not required for lipid raft targeting of AC6. We conclude that while the C1 domain may be most important, both the C1 and C2 domains of AC6 play a role in targeting AC6 to lipid rafts.