Copper promotes the trafficking of the amyloid precursor protein

Accumulation of the amyloid β peptide in the cortical and hippocampal regions of the brain is a major pathological feature of Alzheimer disease. Amyloid β peptide is generated from the sequential protease cleavage of the amyloid precursor protein (APP). We reported previously that copper increases the level of APP at the cell surface. Here we report that copper, but not iron or zinc, promotes APP trafficking in cultured polarized epithelial cells and neuronal cells. In SH-SY5Y neuronal cells and primary cortical neurons, copper promoted a redistribution of APP from a perinuclear localization to a wider distribution, including neurites. Importantly, a change in APP localization was not attributed to an up-regulation of APP protein synthesis. Using live cell imaging and endocytosis assays, we found that copper promotes an increase in cell surface APP by increasing its exocytosis and reducing its endocytosis, respectively. This study identifies a novel mechanism by which copper regulates the localization and presumably the function of APP, which is of major significance for understanding the role of APP in copper homeostasis and the role of copper in Alzheimer disease.     

Intramembrane proteolysis promotes trafficking of hepatitis C virus core protein to lipid droplets

Hepatitis C virus (HCV) is the major causative pathogen associated with liver cirrhosis and hepatocellular carcinoma. The virus has a positive-sense RNA genome encoding a single polyprotein with the virion components located in the N-terminal portion. During biosynthesis of the polyprotein, an internal signal sequence between the core protein and the envelope protein E1 targets the nascent polypeptide to the endoplasmic reticulum (ER) membrane for translocation of E1 into the ER. Following membrane insertion, the signal sequence is cleaved from E1 by signal peptidase. Here we provide evidence that after cleavage by signal peptidase, the signal peptide is further processed by the intramembrane-cleaving protease SPP that promotes the release of core protein from the ER membrane. Core protein is then free for subsequent trafficking to lipid droplets. This study represents an example of a potential role for intramembrane proteolysis in the maturation of a viral protein.     

IL-5 promotes eosinophil trafficking to the esophagus

Eosinophil infiltration into the esophagus occurs in a wide range of diseases; however, the underlying pathophysiological mechanisms involved are largely unknown. We now report that the Th2 cytokine, IL-5, is necessary and sufficient for the induction of eosinophil trafficking to the esophagus. We show that transgenic mice overexpressing IL-5 under the control of a T cell (CD2) or a small intestinal enterocyte (fatty acid-binding protein) promoter have markedly increased eosinophil numbers in the esophagus. For example, esophageal eosinophil levels are 1.9 +/- 0.9 and 121 +/- 14 eosinophils/mm(2) in wild-type and CD2-IL-5-transgenic mice, respectively. Consistent with this effect being mediated by a systemic mechanism, pharmacological administration of IL-5 via a miniosmotic pump in the peritoneal cavity resulted in blood and esophageal eosinophilia. To examine the role of IL-5 in oral Ag-induced esophageal eosinophilia, eosinophilic esophagitis was induced by allergen exposure in IL-5-deficient and wild-type mice. Importantly, IL-5-deficient mice were resistant to eosinophilic esophagitis. Finally, we examined the role of eotaxin when IL-5 was overproduced in vivo. Esophageal eosinophil levels in CD2-IL-5-transgenic mice were found to decrease 15-fold in the absence of the eotaxin gene; however, esophageal eosinophil numbers in eotaxin-deficient IL-5-transgenic mice still remained higher than wild-type mice. In conclusion, these studies demonstrate a central role for IL-5 in inducing eosinophil trafficking to the esophagus.     

Differential association of membrane-bound and non-membrane-bound polysomes with the cytoskeleton

We report here a differential release of specific mRNAs from the cytoskeleton by cytochalasin D treatment. Non-membrane-bound polysomal mRNAs, such as histone mRNA and c-fos mRNA, are readily released from the cytoskeleton of HeLa cells during cytochalasin D treatment. Over 90% of H3 and H4 histone mRNA is associated with the cytoskeleton in control cells and only 25% in cells treated with cytochalasin D (40 micrograms/ml). In contrast, the membrane-bound polysomal mRNAs for HLA-B7 and chorionic gonadotropin-alpha are inefficiently released from the cytoskeletal framework by cytochalasin D alone; approximately 98% of the HLA-B7 mRNA in control cells is associated with the cytoskeleton, whereas approximately 65% of the HLA-B7 mRNA is retained on the cytoskeleton in cells treated with cytochalasin D (40 micrograms/ml). Disruption of polysome structure with puromycin during cytochalasin D treatment results in the efficient release of HLA-B7 mRNA from the cytoskeleton. Under these conditions, only 25% of the HLA-B7 mRNA remains associated with the cytoskeletal framework. Thus, membrane-bound polysomes appear to be attached to the cytoskeleton through a cytochalasin D-sensitive site as well as through association with the nascent polypeptide and/or ribosome. These results demonstrate a complex association of polysomes with the cytoskeleton and elements of the endoplasmic reticulum.     

RNA-binding protein HuR promotes growth of small intestinal mucosa by activating the Wnt signaling pathway

Inhibition of growth of the intestinal epithelium, a rapidly self-renewing tissue, is commonly found in various critical disorders. The RNA-binding protein HuR is highly expressed in the gut mucosa and modulates the stability and translation of target mRNAs, but its exact biological function in the intestinal epithelium remains unclear. Here, we investigated the role of HuR in intestinal homeostasis using a genetic model and further defined its target mRNAs. Targeted deletion of HuR in intestinal epithelial cells caused significant mucosal atrophy in the small intestine, as indicated by decreased cell proliferation within the crypts and subsequent shrinkages of crypts and villi. In addition, the HuR-deficient intestinal epithelium also displayed decreased regenerative potential of crypt progenitors after exposure to irradiation. HuR deficiency decreased expression of the Wnt coreceptor LDL receptor–related protein 6 (LRP6) in the mucosal tissues. At the molecular level, HuR was found to bind the Lrp6 mRNA via its 3′-untranslated region and enhanced LRP6 expression by stabilizing Lrp6 mRNA and stimulating its translation. These results indicate that HuR is essential for normal mucosal growth in the small intestine by altering Wnt signals through up-regulation of LRP6 expression and highlight a novel role of HuR deficiency in the pathogenesis of intestinal mucosal atrophy under pathological conditions.     

Dynamic protein trafficking to the cell wall

Recently we have studied the secretion pattern of a pectin methylesterase inhibitor protein (PMEI1) and a polygalacturonase inhibitor protein (PGIP2) in tobacco protoplast using the protein fusions, secGFP-PMEI1 and PGIP2-GFP. Both chimeras reach the cell wall by passing through the endomembrane system but using distinct mechanisms and through a pathway distinguishable from the default sorting of a secreted GFP. After reaching the apoplast, sec-GFP-PMEI1 is stably accumulated in the cell wall, while PGIP2-GFP undergoes endocytic trafficking. Here we describe the final localization of PGIP2-GFP in the vacuole, evidenced by co-localization with the marker Aleu-RFP, and show a graphic elaboration of its sorting pattern. A working model taking into consideration the presence of a regulated apoplast-targeted secretion pathway is proposed.Key words: cell wall trafficking, endocytosis, GPI-anchor, PGIP2, PMEI1, secretion pathway, vacuole fluorescent markerCell wall biogenesis, growth, differentiation and remodeling, as well as wall-related signaling and defense responses depend on the functionality of the secretory pathway. Matrix polysaccharides, synthesized in the Golgi stacks, and cell wall proteins, synthesized in the ER, are packaged into secretory vesicles that fuse with the plasma membrane (PM) releasing their cargo into the cell wall. Also the synthesis and deposition of cellulose itself are driven by the endomembrane system which controls the assembly, within the Golgi, and the export to the plasma membrane of rosette complexes of cellulose synthase.¹ Secretion to the cell wall has always been considered a default pathway² but recent studies have evidenced a complex regulation of wall component trafficking that does not seem to follow the default secretion model. Recent evidence that several cell wall proteins are retained in the Golgi stacks until specific signals at the N-terminal domain are proteolitically removed is a case in point.^3–5 Moreover, it has previously been reported that secretion of exogenous marker proteins (secGFP and secRGUS) and cell wall polysaccharides reach the PM through different pathways.⁶ More recently, we have reported that cell wall protein trafficking also occurs through mechanisms distinguishable from that of a secreted GFP suggesting that more complex events than the mechanisms of bulk flow control cell wall growth and differentiation.⁷ To follow cell wall protein trafficking we used a Phaseolus vulgaris polygalacturonase inhibitor protein (PGIP2) and an Arabidopsis pectin methylesterase inhibitor protein (PMEI1) fused to GFP (PGIP2-GFP and secGFP-PMEI1). Both apoplastic proteins are involved in the remodeling of pectin network with different mechanisms. PGIP2 specifically inhibits exogenous fungal polygalacturonases (PGs) and is involved in the plant defense mechanisms against pathogenic fungi.^8,9 PMEI1 counteracts endogenous PME and takes part in the physiological synthesis and remodeling of the cell wall during growth and differentiation.^10,11 The specific functions of the two apoplastic proteins seem to be strictly related to the distinct mechanisms that control their secretion and stability in the cell wall. In fact, while secGFP-PMEI1 moves through ER and Golgi stacks linked to a glycosyl phosphatidylinositol (GPI)-anchor, PGIP2-GFP moves as a cargo soluble protein. Furthermore, secGFP-PMEI1 is stably accumulated in the cell wall, while PGIP2-GFP, over the time, is internalized into endosomes and targeted to vacuole, likely for degradation. After reaching the cell wall, the different fate of the two proteins seems to be strictly related to the presence/absence of their physiological counteractors. PMEI regulates the demethylesterification of homogalacturonan by inhibiting pectin methyl esterase (PME) activity through the formation of a reversible 1:1 complex which is stable in the acidic cell wall environment.¹² Stable wall localization of PMEI1 is likely related to its interaction with endogenous PME, always present in the wall. Unlike PMEs, fungal polygalacturonases (PGs), the physiological interactors of PGIP2, are present in the cell wall only during a pathogen attack. The absence of PGs may determine PGIP2 internalization. Internalization events have been already reported for PM proteins,^13–16 while cell wall protein internalization is surely a less well-known event. To date, only internalization of an Arabidopsis pollen-specific PME^4,5,17 and PGIP2 ⁷ has been reported.To further confirm the internalization of PGIP2-GFP and its final localization into the vacuole, we constructed a red fluorescent variant (RFP) of the green fluorescent marker protein that accumulates in lytic or acidic vacuole because of the barley aleurain sorting determinants (Aleu-RFP).¹⁸ The localization of PGIP2-GFP was compared to that of Aleu-RFP by confocal microscopy in tobacco protoplasts transiently expressing both fusions. Sixty hours after transformation, PGIP2-GFP labeled the central vacuole as indicated by complete co-localization with the vacuolar marker (Fig. 1A–D). Instead, at the same time point, secGFP-PMEI1 still labeled the cell wall (Fig. 1E–H) and never reached the vacuolar compartment. To summarize PGIP2-GFP secretion pattern, a graphic elaboration of confocal images is reported describing the sorting of PGIP2GFP in tobacco protoplast (Fig. 1I). The protein transits through the endomembrane system (green) and reaches the cell wall which is rapidly regenerating as evidenced by immunostaining with the red monoclonal antibody JIM7 that binds to methylesterified pectins.¹⁹ PGIP2-GFP is then internalized in endosomes, labeled in yellow because of the co-localization with the styryl dye FM4-64, a red marker of the endocytic pathway.Open in a separate windowFigure 1PGIP2-GFP, but not secGFP-PMEI1, is internalized and reaches the vacuole in tobacco leaf protoplasts. (A) Approximately 60 h after transformation, PGIP2-GFP labeled the central vacuole as indicated by co-localization with the vacuole marker Aleu-RFP (B). (C) Merged image of (A and B). (D) Differential interference contrast (DIC) image of (A–C). On the contrary, secGFP-PMEI1 still labeled cell wall (E). (F) No co-localization is present in the vacuole labeled by Aleu-RFP. (G) Merged image of (E and F). (H) DIC image of (E–G). (I) Graphic elaboration of confocal images describing the sorting of PGIP2. The protein is sorted by the endomembrane system (green) to the cell wall (red) that is regenerated by the protoplast. Lacking the specific ligand, it is then internalized in endosome (yellow). Details are reported in the text.In Figure 2 we propose a model of the mechanism of secGFP-PMEI1 and PGIP2-GFP secretion derived from the different lines of evidence previously reported in reference ⁷. SecGFPPMEI1 (Fig. 2-1), but not PGIP2-GFP (Fig. 2-2), carries a GPI-anchor, required for its secretion to the cell wall. When the anchorage of GPI is inhibited by mannosamine (Fig. 2-a) or by the fusion of GFP to the C-terminus of PMEI1 (Fig. 2-b), the two non-anchored proteins accumulate in the Golgi stacks. Evidence of retention in Golgi stacks has already been reported for other two cell wall proteins.^3–5 Unlike secGFP-PMEI1, PGIP2-GFP is not stably accumulated in the cell wall and undergoes endocytic trafficking (Fig. 2-3). PGIP2-GFP internalization, likely due to the absence of PGs, might also be related with its ability to interact with homogalacturonan and oligogalacturonides,²⁰ which have been reported to internalize^21,22 (Fig. 2-4). Since SYP 121, a Qa-SNARE, is involved in the default secretion of secGFP,²³ but not in secretion of PGIP2-GFP and secGFP-PMEI1, trafficking mechanisms underlying secretion into the apoplast are likely different from those underlying the default route (Figs. 2-5). Taken as a whole, evidence suggests the existence of currently undefined signals that control apoplast-targeted secretion.Open in a separate windowFigure 2Schematic illustration for secGFP-PMEI1 and PGIP2-GFP trafficking. See text for details.     

Dephosphorylation of HuR Protein during Alphavirus Infection Is Associated with HuR Relocalization to the Cytoplasm

We have demonstrated previously that the cellular HuR protein binds U-rich elements in the 3′ untranslated region (UTR) of Sindbis virus RNA and relocalizes from the nucleus to the cytoplasm upon Sindbis virus infection in 293T cells. In this study, we show that two alphaviruses, Ross River virus and Chikungunya virus, lack the conserved high-affinity U-rich HuR binding element in their 3′ UTRs but still maintain the ability to interact with HuR with nanomolar affinities through alternative binding elements. The relocalization of HuR protein occurs during Sindbis infection of multiple mammalian cell types as well as during infections with three other alphaviruses. Interestingly, the relocalization of HuR is not a general cellular reaction to viral infection, as HuR protein remained largely nuclear during infections with dengue and measles virus. Relocalization of HuR in a Sindbis infection required viral gene expression, was independent of the presence of a high-affinity U-rich HuR binding site in the 3′ UTR of the virus, and was associated with an alteration in the phosphorylation state of HuR. Sindbis virus-induced HuR relocalization was mechanistically distinct from the movement of HuR observed during a cellular stress response, as there was no accumulation of caspase-mediated HuR cleavage products. Collectively, these data indicate that virus-induced HuR relocalization to the cytoplasm is specific to alphavirus infections and is associated with distinct posttranslational modifications of this RNA-binding protein.     

Cross-linking of F508-CFTR promotes its trafficking to the plasma membrane

CFTR is a cAMP-activated chloride channel responsible for agonist stimulated chloride and fluid transport across epithelial surfaces.¹ Mutations in the CFTR gene lead to cystic fibrosis (CF) which affects the function of secretory organs like the intestine, the pancreas, the airways and the sweat glands. Most of the morbidity and mortality in CF has been linked to a decrease in airway function.² The ΔF508 mutation is the most common CF-related mutation in the Caucasian population and represents 90% of CF alleles. Homozygote carriers of this mutation present with a severe CF phenotype.³ The ΔF508 mutation causes misfolding of the nascent CFTR polypeptide, which leads to inefficient export from the endoplasmic reticulum (ER) and rapid degradation by the proteasome.⁴Key words: cystic fibrosis, endoplasmic reticulum, oligomer, processing mutation, curcuminGiven the frequency of the ΔF508 processing mutation and the severity of its corresponding phenotype, much research has focused on identifying compounds that restore the trafficking and function of this mutant at the plasma membrane. Several synthetic ‘correctors’ of ΔF508 mis-processing and ‘potentiators’ of mutant channel activity have been identified.^5,6 Natural compounds such as curcumin also have generated interest. Curcumin is an organic phenolic compound abundant in turmeric, an Indian spice extracted from the rhizome of Curcuma longa.⁷ Earlier studies performed using ΔF508/ΔF508 mouse models and human airway epithelial cell lines suggested that curcumin may act as a ΔF508-CFTR trafficking corrector.⁸ Also, we and others showed that curcumin stimulates CFTR channel activity in excised membrane patches.^9,10 This stimulation occurs in the absence of ATP binding, which is normally required for channel opening.¹⁰ Binding sites of correctors and potentiators within the CFTR polypeptide as well as the molecular mechanisms underlying the rescue of CFTR trafficking and function remain to be elucidated. In our attempt to understand how curcumin could circumvent the normally critical step of ATP binding to promote CFTR channel activity we investigated the effect of curcumin on CFTR conformation by using biochemical assays. We showed that curcumin caused dimerization of several CFTR channel constructs (including ΔF508-CFTR) in a dose- and time-dependent manner both in microsomes and within intact cells. This effect of curcumin on CFTR oligomerization is attributable to its reactive β-diketone groups, which may undergo an oxidation reaction with CFTR nucleophilic amino acid residues.¹¹ Importantly, CFTR channel activation by curcumin is unrelated to its cross-linking effect. We identified cyclic derivatives of curcumin that lack this cross-linking activity but still promote CFTR channel function.¹¹Here we examined the possibility that the cross-linking of ΔF508-CFTR channels by curcumin promotes the delivery of this ER processing mutant to the cell surface. We were motivated to test this possibility for three reasons: (i) our previous evidence that curcumin-induced dimers of wild-type CFTR polypeptides were detected at the cell surface where they remained over an hour after the removal of curcumin;¹¹ (ii) the very efficient cross-linking of the immature (ER) forms of wild-type CFTR and the ΔF508-CFTR mutant that we observed earlier¹¹ and (iii) prior evidence from our group that the ER export and cell surface delivery of ΔF508-CFTR polypeptides could be promoted by the co-expression of this mutant with certain CFTR fragments (trans-complementation).¹² The latter result might be due to the existence of ER retention ‘signals’ that are exposed on the ΔF508-CFTR polypeptide but become buried by interacting (complementing) fragments.Figure 1 provides evidence that ΔF508-CFTR oligomers that form in response to curcumin treatment do indeed appear at the surfaces of cultured airway epithelial cells (CF bronchial epithelial (CFBE) cells stably transfected with this CFTR mutant). Surface biotinylation assays were performed to detect the appearance of ΔF508-CFTR polypeptides at the cell surface. MESNA, a cell impermeant reducing agent that cleaves the biotin label, was used to verify the surface accessibility of the labeled ΔF508-CFTR polypeptides. ΔF508-CFTR polypeptides were precipititated with streptavidinagarose (surface pool) or with a CFTR monoclonal antibody (total pool). In the absence of curcumin treatment the great majority of the ΔF508-CFTR protein existed as the ER form (monomeric band B), as previously observed by many investigators (Fig. 1, lane 5). No band B was detected in the surface pool before or after curcumin treatment (Fig. 1, lanes 1, 2). As we reported earlier, treatment of the cells with 50 µM curcumin for 15 mins at 37°C cross-linked nearly all of the ΔF508-CFTR polypeptides into higher order complexes (e.g., dimers, termed band D here; lanes 6–8 in Fig. 1). Interestingly, these higher order forms of ΔF508-CFTR were readily apparent in the surface pool (Fig. 1, lane 2).Open in a separate windowFigure 1ΔF508-CFTR oligomers detected at the surfaces of airway epithelial cells after curcumin treatment. ΔF508-CFTR expressing CFBE cells were treated with curcumin (50 µM) for 15 min at 37°C. Cell surface proteins were then biotinylated (Sulfo-NHS-SS-Biotin, 1 mg/ml) for 30 min at 4°C followed by cell lysis with 1% Triton X-100. Surface proteins were isolated by streptavidin pulldown and ΔF508-CFTR was isolated from the total cell protein pool by immunoprecipitation with an anti-CFTR C-terminus antibody (clone 24-1, R&D systems). After SDS-PAGE the ΔF508-CFTR signal was detected by immunoblotting using the 24-1 antibody described above. (SP: streptavidin pulldown; IP: immunoprecipitation). As an additional control curcumin-treated cells were treated with the cell impermeant MESNA after biotinylation to strip the biotin off the cell surface proteins with which it had reacted.CFTR oligomers also can be generated by standard chemical cross-linkers such as DSS, as previously reported by others and confirmed by us.¹³ Figure 2 shows that oligomers of ΔF508-CFTR that are induced by DSS treatment also appear in the surface pool. These experiments were performed using transiently transfected HEK-293T cells with 30 µM curcumin as a positive control. Quantitative densitometry results are shown in Figure 3. By titrating the DSS concentration we observed a dose-dependent disappearance of the monomeric band B form, a corresponding increase in the band D (dimer) pool and the appearance of higher order oligomers (band E) which prevailed at higher DSS concentrations (see total cell pool data in right-hand). A small amount of the band D form was detected in the absence of DSS or curcumin treatment, which might represent some spontaneous cross-linking of ΔF508-CFTR polypeptides under these conditions. The DSS and curcumin-induced ΔF508-CFTR oligomers were readily detected in the surface pool. The densitometry analysis revealed that 20 ± 5% and 33 ± 19% of the total oligomer pool (combined bands D and E) was found in the surface pool after treatment with 0.1 mM DSS (n = 3) or 30 µM curcumin (n = 3), respectively, which corresponded to a 17 ± 7 and 26 ± 20 fold increase compared to the control condition (i.e., no DSS or no curcumin).Open in a separate windowFigure 2ΔF508-CFTR oligomers detected at the surfaces of HEK cells after DSS or curcumin treatment. ΔF508-CFTR expressing HEK cells were treated with the indicated concentrations of DSS or with 30 µM curcumin (*) for 15 min at 37°C. Cell surface proteins were then biotinylated and isolated by streptavidin pulldown as described above. ΔF508-CFTR was immunoprecipitated from the total cell protein pool with the 24-1 antibody and detected by immunoblotting as before (SP: streptavidin pulldown; IP: immunoprecipitation). Band B corresponds to ΔF508 monomer (ER form). Band D corresponds to ΔF508 dimer. Band E corresponds to a higher degree of ΔF508 oligomerization. Each panel corresponds to a different exposure of the same blot.Open in a separate windowFigure 3Dose-dependent expression of ΔF508-CFTR oligomers at the surfaces of HEK cells after DSS treatment. CFTR signals detected by the 24-1 antibody from three different experiments as the one described in Figure 2 were analyzed using the ImageJ software (from the National Institute of Health). (A) band B signal intensity is plotted as a function of the DSS concentrations. Signals analyzed correspond to ΔF508-CFTR band B immunoprecipitated by the 24-1 antibody. (B) band D plus band E signal intensities are plotted as a function of the DSS concentration. Signals analyzed correspond to the sum of ΔF508-CFTR band D and band E immunoprecipitated by the 24-1 antibody. (C) band D plus band E signal intensities at the cell surface are plotted as a function of the DSS concentration. Signals analyzed correspond to the sum of ΔF508-CFTR band D and band E isolated from the surfaces of ΔF508-CFTR expressing HEK cells by biotinylation and streptavidin pulldown. (D) the ratio between the amount of band E and D at the surfaces of ΔF508-CFTR expressing HEK cells is plotted as a function of the DSS concentration. Error bars are SEMs.Altogether these data indicate that the cross-linking of ΔF508-CFTR band B into oligomers by curcumin or DSS allows ΔF508-CFTR to traffic to the cell surface. This effect might be caused by the burial of ER retention motifs within the oligomer, which also could explain our previous trans-complementation results in which we observed that certain CFTR fragments promote the cell surface delivery of this processing mutant.¹² Although non-specific protein cross-linkers like DSS would not be therapeutically beneficial, more specific CFTR cross-linkers (perhaps curcumin?) may be worth considering for treating CF disease linked to ER processing mutations in CFTR. In this regard, we note that cross-linked CFTR polypeptides appear to retain chloride channel activity. Namely, in our prior excised patch clamp studies we observed stable CFTR channel activity when these patches were exposed to curcumin at doses and times that promote robust cross-linking of CFTR polypeptides.^10,11     

PRA1 promotes the intracellular trafficking and NF-kappaB signaling of EBV latent membrane protein 1

Latent membrane protein 1 (LMP1), which is an Epstein-Barr virus (EBV)-encoded oncoprotein, induces nuclear factor-kappa B (NF-kappaB) signaling by mimicking the tumor necrosis factor receptor (TNFR). LMP1 signals primarily from intracellular compartments in a ligand-independent manner. Here, we identify a new LMP1-interacting molecule, prenylated Rab acceptor 1 (PRA1), which interacts with LMP1 for the first time through LMP1's transmembrane domain, and show that PRA1 is involved in intracellular LMP1 trafficking and LMP1-induced NF-kappaB activity. Immunofluorescence and biochemical analyses revealed that LMP1 physically interacted with PRA1 at the Golgi apparatus, and the colocalization of LMP1 and PRA1 to the Golgi was sensitive to nocodazole and brefeldin A. Coexpression of a PRA1 export mutant or knockdown of PRA1 led to redistribution of LMP1 and its associated signaling molecules to the endoplasmic reticulum and subsequent impairment of LMP1-induced NF-kappaB activation, but had no effect on CD40- and TNFR1-mediated signaling or the functional integrity of the Golgi apparatus. These novel findings provide important new insights into LMP1, and identify an unexpected new role for PRA1 in cellular signaling.     

Non-muscle myosin IIA transports a Golgi glycosyltransferase to the endoplasmic reticulum by binding to its cytoplasmic tail

The mechanism of the Golgi-to-ER transport of Golgi glycosyltransferases is not clear. We utilize a cell line expressing the core 2 N-acetylglucosaminyltransferase-M (C2GnT-M) tagged with c-Myc to explore this mechanism. By immunoprecipitation using anti-c-Myc antibodies coupled with proteomics analysis, we have identified several proteins including non-muscle myosin IIA (NMIIA), heat shock protein (HSP)-70 and ubiquitin activating enzyme E1 in the immunoprecipitate. Employing yeast-two-hybrid analysis and pulldown experiments, we show that the C-terminal region of the NMIIA heavy chain binds to the 1-6 amino acids in the cytoplasmic tail of C2GnT-M. We have found that NMIIA co-localizes with C2GnT-M at the periphery of the Golgi. In addition, inhibition or knockdown of NMIIA prevents the brefeldin A-induced collapse of the Golgi as shown by the inhibition of the migration of both Giantin, a Golgi matrix protein, and C2GnT-M, a Golgi non-matrix protein, to the ER. In contrast, knockdown of HSP70 retains Giantin in the Golgi but moves C2GnT-M to the ER, a process also blocked by inhibition or knockdown of NMIIA. Also, the intracellular distribution of C2GnT-M is not affected by knockdown of β-coatomer protein with or without inhibition of HSPs, suggesting that the Golgi-to-ER trafficking of C2GnT-M does not depend on coat protein complex-I. Further, inhibition of proteasome results in accumulation of ubiquitinated C2GnT-M, suggesting its degradation by proteasome. Therefore, NMIIA and not coat protein complex-I is responsible for transporting the Golgi glycosyltransferase to the ER for proteasomal degradation. The data suggest that NMIIA is involved in the Golgi remodeling.     

Interaction of c-Cbl with myosin IIA regulates Bleb associated macropinocytosis of Kaposi's sarcoma-associated herpesvirus

KSHV is etiologically associated with Kaposi's sarcoma (KS), an angioproliferative endothelial cell malignancy. Macropinocytosis is the predominant mode of in vitro entry of KSHV into its natural target cells, human dermal microvascular endothelial (HMVEC-d) cells. Although macropinocytosis is known to be a major route of entry for many viruses, the molecule(s) involved in the recruitment and integration of signaling early during macropinosome formation is less well studied. Here we demonstrate that tyrosine phosphorylation of the adaptor protein c-Cbl is required for KSHV induced membrane blebbing and macropinocytosis. KSHV induced the tyrosine phosphorylation of c-Cbl as early as 1 min post-infection and was recruited to the sites of bleb formation. Infection also led to an increase in the interaction of c-Cbl with PI3-K p85 in a time dependent manner. c-Cbl shRNA decreased the formation of KSHV induced membrane blebs and macropinocytosis as well as virus entry. Immunoprecipitation of c-Cbl followed by mass spectrometry identified the interaction of c-Cbl with a novel molecular partner, non-muscle myosin heavy chain IIA (myosin IIA), in bleb associated macropinocytosis. Phosphorylated c-Cbl colocalized with phospho-myosin light chain II in the interior of blebs of infected cells and this interaction was abolished by c-Cbl shRNA. Studies with the myosin II inhibitor blebbistatin demonstrated that myosin IIA is a biologically significant component of the c-Cbl signaling pathway and c-Cbl plays a new role in the recruitment of myosin IIA to the blebs during KSHV infection. Myosin II associates with actin in KSHV induced blebs and the absence of actin and myosin ubiquitination in c-Cbl ShRNA cells suggested that c-Cbl is also responsible for the ubiquitination of these proteins in the infected cells. This is the first study demonstrating the role of c-Cbl in viral entry as well as macropinocytosis, and provides the evidence that a signaling complex containing c-Cbl and myosin IIA plays a crucial role in blebbing and macropinocytosis during viral infection and suggests that targeting c-Cbl could lead to a block in KSHV infection.