Ivan R. Nabi

A Viral Phospholipase A2 Is Required for Parvovirus Infectivity

Sequence analysis revealed phospholipase A2 (PLA2) motifs in capsid proteins of parvoviruses. Although PLA2 activity is not known to exist in viruses, putative PLA2s from divergent parvoviruses, human B19, porcine parvovirus, and insect GmDNV (densovirus from Galleria mellonella), can emulate catalytic properties of secreted PLA2. Mutations of critical amino acids strongly reduce both PLA2 activity and, proportionally, viral infectivity, but cell surface attachment, entry, and endocytosis by PLA2-deficient virions are not affected. PLA2 activity is critical for efficient transfer of the viral genome from late endosomes/lysosomes to the nucleus to initiate replication. These findings offer the prospect of developing PLA2 inhibitors as a new class of antiviral drugs against parvovirus infections and associated diseases.

Mammalian staufen is a double-stranded-RNA- and tubulin-binding protein which localizes to the rough endoplasmic reticulum

ABSTRACT Staufen (Stau) is a double-stranded RNA (dsRNA)-binding protein involved in mRNA transport and localization in Drosophila.To understand the molecular mechanisms of mRNA transport in mammals, we cloned human (hStau) and mouse (mStau)staufen cDNAs. In humans, four transcripts arise by differential splicing of the Stau gene and code for two proteins with different N-terminal extremities. In vitro, hStau and mStau bind dsRNA via each of two full-length dsRNA-binding domains and tubulin via a region similar to the microtubule-binding domain of MAP-1B, suggesting that Stau cross-links cytoskeletal and RNA components. Immunofluorescent double labeling of transfected mammalian cells revealed that Stau is localized to the rough endoplasmic reticulum (RER), implicating this RNA-binding protein in mRNA targeting to the RER, perhaps via a multistep process involving microtubules. These results are the first demonstration of the association of an RNA-binding protein in addition to ribosomal proteins, with the RER, implicating this class of proteins in the transport of RNA to its site of translation.

Ganglioside GM1 levels are a determinant of the extent of caveolae/raft-dependent endocytosis of cholera toxin to the Golgi apparatus

Cholera toxin is associated with caveolae and raft domains in various cell types and previous studies have shown that cholera toxin can be internalized by caveolae/raft-dependent endocytosis as well as by other pathways. We undertook the study of cholera toxin endocytosis in CaCo-2 and HeLa cells. CaCo-2 cells do not express detectable levels of caveolin and, relative to HeLa cells, also present significantly reduced expression of ganglioside GM1, the cholera toxin receptor, that remains Triton X-100 insoluble. Amongst the HeLa cell population, caveolin expression is constant, however, GM1 expression is highly variable. Cholera toxin is internalized to the Golgi apparatus via a caveolae/raft-dependent pathway sensitive to methyl-β-cyclodextrin and genistein in high-GM1-expressing HeLa cells but not in low-GM1 HeLa cells or in CaCo-2 cells. Limited cholera toxin endocytosis to endosomes sensitive to neither methyl-β-cyclodextrin nor genistein is also observed in all cells and corresponds to a non-caveolae/raft endocytic pathway. Increasing cell-associated GM1 by adding GM1 to the cell media of both HeLa and CaCo-2 cells selectively enhances the methyl-β-cyclodextrin-, genistein-sensitive delivery of cholera toxin to the Golgi apparatus but not to endosomes. GM1 expression levels are therefore a selective determinant of caveolae/raft-dependent endocytosis of cholera toxin to the Golgi apparatus and variable expression of GM1 between cells can impact on the endocytosis and choice of pathway followed by cholera toxin.

Synaptojanin 2 Functions at an Early Step of Clathrin-Mediated Endocytosis

Synaptojanin 2 is a ubiquitously expressed polyphosphoinositide phosphatase that displays a high degree of homology in its catalytic domains with synaptojanin 1 [1,2]. Neurons of synaptojanin 1-deficient mice display an increase in clathrin-coated vesicles and delayed reentry of recycling vesicles into the fusion-competent vesicle pool, but no defects in early steps of endocytosis [3,4]. Here we show that inhibition of synaptojanin 2 expression via small interfering (si) RNA causes a strong defect in clathrin-mediated receptor internalization in a lung carcinoma cell line. This inhibitory phenotype is rescued by overexpression of wild-type synaptojanin 2, but not of wild-type synaptojanin 1 or mutant synaptojanin 2 that is deficient in 5-phosphatase activity. In addition, electron-microscopic analysis shows that synaptojanin 2 depletion causes a decrease in clathrin-coated pits and vesicles. These results suggest a role for synaptojanin 2 in clathrin-coated pit formation and imply that lipid hydrolysis is required at an early stage of clathrin-mediated endocytosis. Taken together, our results also indicate that synaptojanin 2 is functionally distinct from synaptojanin 1.

A Novel Murine Staufen Isoform Modulates the RNA Content of Staufen Complexes

ABSTRACT Mouse Staufen (mStau) is a double-stranded RNA-binding protein associated with polysomes and the rough endoplasmic reticulum (RER). We describe a novel endogenous isoform of mStau (termed mStaui) which has an insertion of six amino acids within dsRBD3, the major double-stranded RNA (dsRNA)-binding domain. With a structural change of the RNA-binding domain, this conserved and widely distributed isoform showed strongly impaired dsRNA-binding ability. In transfected cells, mStaui exhibited the same tubulovesicular distribution (RER) as mStau when weakly expressed; however, when overexpressed, mStaui was found in large cytoplasmic granules. Markers of the RER colocalized with mStaui-containing granules, showing that overexpressed mStaui could still be associated with the RER. Cotransfection of mStaui with mStau relocalized overexpressed mStaui to the reticular RER, suggesting that they can form a complex on the RER and that a balance between these isoforms is important to achieve proper localization. Coimmunoprecipitation demonstrated that the two mStau isoforms are components of the same complex in vivo. Analysis of the immunoprecipitates showed that mStau is a component of an RNA-protein complex and that the association with mStaui drastically reduces the RNA content of the complex. We propose that this new isoform, by forming a multiple-isoform complex, regulates the amount of RNA in mStau complexes in mammalian cells.

Autocrine Activation of the Hepatocyte Growth Factor Receptor/Met Tyrosine Kinase Induces Tumor Cell Motility by Regulating Pseudopodial Protrusion

The multiple β-actin rich pseudopodial protrusions of the invasive variant of Moloney sarcoma virus (MSV)-transformed epithelial MDCK cells (MSV-MDCK-INV) are strongly labeled for phosphotyrosine. Increased tyrosine phosphorylation among a number of proteins was detected in MSV-MDCK-INV cells relative to untransformed and MSV-transformed MDCK cells, especially for the hepatocyte growth factor receptor (HGF-R), otherwise known as c-met proto-oncogene. Cell surface expression of HGF-R was similar in the three cell lines, indicating that HGF-R is constitutively phosphorylated in MSV-MDCK-INV cells. Both the tyrosine kinase inhibitor herbimycin A and the HGFα antibody abolished HGF-R phosphorylation, induced retraction of pseudopodial protrusions, and promoted the establishment of cell-cell contacts as well as the apparition of numerous stabilizing stress fibers in MSV-MDCK-INV cells. Furthermore, anti-HGFα antibody abolished cell motility among MSV-MDCK-INV cells. Conditioned medium from MSV-MDCK-INV cells induced MDCK cell scattering, indicating that HGF is secreted by MSV-MDCK-INV cells. HGF titration followed by a subsequent washout of the antibodies led to renewed pseudopodial protrusion and cellular movement. We therefore show that activation of the tyrosine kinase activity of HGF-R/Met via an autocrine HGF loop is directly responsible for pseudopodial protrusion, thereby explaining the motile and invasive potential of this model epithelium-derived tumor cell line.

The enzymatic activity of phosphoglucose isomerase is not required for its cytokine function

PGI is a housekeeping gene encoding phosphoglucose isomerase (PGI) a glycolytic enzyme that also functions as a cytokine (autocrine motility factor (AMF)/neuroleukin/maturation factor) upon secretion from the cell and binding to its 78 kDa seven‐transmembrane domain receptor (gp78/AMF‐R). PGI contains a CXXC motif, characteristic of redox proteins and possibly evolutionarily related to the CC and CXC motif of the chemokine gene family. Using site‐directed mutagenesis, single‐ and double‐deletion (CXC, CC) mutants were created by deleting amino acids 331 and 332 of human PGI, respectively. The mutant proteins lost their enzymatic activity; however, neither of the deletions augmented the proteins’ binding affinity to the receptor and all maintained cytokine function. The results demonstrate that the enzymatic activity of PGI is not essential for either receptor binding or cytokine function of human PGI.

Species specificity of the cytokine function of phosphoglucose isomerase

Phosphoglucose isomerase (PGI) is a cytosolic glycolytic enzyme that also functions as an extracellular cytokine (neuroleukin/autocrine motility factor (AMF)/maturation factor). Contrary to mammalian PGI, bacterial PGI was not internalized by the PGI/AMF receptor (gp78/AMF‐R) and neither bacterial nor yeast PGI competed with mammalian PGI for receptor binding and internalization. Furthermore, while the bacterial, yeast and mammalian preparations all exhibited isomerase activity, only mammalian PGI stimulated the motility of NIH‐3T3 fibroblasts. The conserved active site of PGI is therefore not sufficient for receptor binding and cytokine activity of PGI. However, synthetic peptides corresponding to distinct peripheral mammalian PGI sequences did not inhibit internalization of mammalian PGI. Our data therefore argue that the cytokine activity of PGI is specific to mammalian PGI but cannot exclude the possibility that the receptor binding motif of PGI is complex and includes elements within and without the active site.

Expression of the AMF/neuroleukin receptor in developing and adult brain cerebellum

The peptide sequence of autocrine motility factor (AMF), a tumor secreted cytokine that induces cell motility, corresponds to that of the previously identified cytokine/enzyme, neuroleukin/glucose‐6‐phosphate isomerase. Neuroleukin is a neurotrophic factor that promotes neuronal survival and sprouting at the neuromuscular junction. The AMF receptor (AMF‐R) has been identified and shown to be highly expressed in malignant tumors with minimal expression in adjacent normal tissue. Neuroleukin mRNA is highly expressed in the cerebellum and we therefore undertook a developmental study of AMF‐R expression in rat cerebellum. As determined by immunoblot, AMF‐R is expressed at equivalent high levels in brain and cerebellum of postnatal day 5 (P5) and 12 (P12) rats and at significantly reduced levels in the adult. Coimmunofluorescence studies with MAP‐2 and γ‐actin revealed that at P12, AMF‐R was mainly localized to Purkinje and granule cells. Moreover, the premigratory cells of the external granular layer were also immunoreactive for AMF‐R suggesting a role for AMF‐R in granule cell migration during cerebellar development in the first two weeks after birth. In the adult, AMF‐R distribution was similar to P12, although weaker, and was localized to Purkinje and granule cells. AMF‐R labeling of GFAP positive glial processes could not be detected in cerebellar sections although in cerebellar primary cultures, both neurons and glial cells were labeled for AMF‐R. In neurons, AMF‐R labeling was present in the cell body, neurites and growth cones. These data indicate that regulation of the neurotrophic function of neuroleukin might be regulated spatially and temporally by expression of its receptor, AMF‐R, in developing and adult cerebellum. J. Neurosci. Res. 60:602–612, 2000

AMF-R Tubules Concentrate in a Pericentriolar Microtubule Domain After MSV Transformation of Epithelial MDCK Cells

Autocrine motility factor receptor (AMF-R) is localized to an intracellular microtubule-associated membranous organelle, the AMF-R tubule. In well-spread untrans-formed MDCK epithelial cells, the microtubules originate from a broad perinuclear region and AMF-R tubules extend throughout the cytoplasm of the cells. In Moloney sarcoma virus (mos)-transformed MDCK (MSV-MDCK) cells, microtubules accumulate around the centrosome, forming a microtubule domain rich in stabilized detyrosinated microtubules. AMF-R tubules are quantitatively associated with this pericentriolar microtubule domain and the rough endoplasmic reticulum and lysosomes also co-distribute with the pericentriolar mass of microtubules. The Golgi apparatus is closely associated with the microtubule organizing center (MTOC) within the juxtanuclear mass of AMF-R tubules, and no co-localization of AMF-R tubules with the Golgi marker β-COP could be detected by confocal microscopy. After nocodazole treatment and washout, microtubule nucleation occurs exclusively at the centrosome of MSV-MDCK cells, and only after microtubule extension to the cell periphery does the microtubule cytoskeleton reorganize to generate the pericentriolar microtubule domain after 30–60 min. AMF-R tubules dispersed by nocodazole treatment concentrate in the pericentriolar region in parallel with the reorganization of the microtubule cytoskeleton. MSV transformation of epithelial MDCK cells results in the stabilization of a pericentriolar microtubule domain responsible for the concentration and polarized distribution of AMF-R tubules.