ABSTRACT
The M glycoprotein from the avian coronavirus, infectious bronchitis virus (IBV), contains information for localization to the cis-Golgi network in its first transmembrane domain. We hypothesize that localization to the Golgi complex may depend in part on specific interactions between protein transmembrane domains and membrane lipids. Because the site of sphingolipid synthesis overlaps the localization of IBV M, we asked whether perturbation of sphingolipids affected localization of IBV M. Short-term treatment with two inhibitors of sphingolipid synthesis had no effect on localization of IBV M or other Golgi markers. Thus, ongoing synthesis of these lipids was not required for proper localization. Surprisingly, a third inhibitor, d,l-threo-1-phenyl-2-decanoylamino-3-morpholino- 1-propanol (PDMP), shifted the steady-state distribution of IBV M from the Golgi complex to the ER. This effect was rapid and reversible and was also observed for ERGIC-53 but not for Golgi stack proteins. At the concentration of PDMP used, conversion of ceramide into both glucosylceramide and sphingomyelin was inhibited. Pretreatment with upstream inhibitors partially reversed the effects of PDMP, suggesting that ceramide accumulation mediates the PDMP-induced alterations. Indeed, an increase in cellular ceramide was measured in PDMP-treated cells. We propose that IBV M is at least in part localized by retrieval mechanisms. Further, ceramide accumulation reveals this cycle by upsetting the balance of anterograde and retrograde traffic and/ or disrupting retention by altering bilayer dynamics.
Subject(s)
Ceramides/metabolism , Golgi Apparatus/metabolism , Infectious bronchitis virus , Sphingolipids/biosynthesis , Viral Matrix Proteins/biosynthesis , Animals , Biomarkers , Cell Line , Cricetinae , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum/metabolism , Enzyme Inhibitors/pharmacology , Golgi Apparatus/drug effects , Golgi Apparatus/ultrastructure , Kidney , Kinetics , Models, Biological , Morpholines/pharmacology , Viral Matrix Proteins/analysisABSTRACT
Sphingosine-1-phosphate (SPP), formed by sphingosine kinase, is the ligand for EDG-1, a GPCR important for cell migration and vascular maturation. Here we show that cytoskeletal rearrangements, lamellipodia extensions, and cell motility induced by platelet-derived growth factor (PDGF) are abrogated in EDG-1 null fibroblasts. However, EDG-1 appears to be dispensable for mitogenicity and survival effects, even those induced by its ligand SPP and by PDGF. Furthermore, PDGF induced focal adhesion formation and activation of FAK, Src, and stress-activated protein kinase 2, p38, were dysregulated in the absence of EDG-1. In contrast, tyrosine phosphorylation of the PDGFR and activation of extracellular signal regulated kinase (ERK1/2), important for growth and survival, were unaltered. Our results suggest that EDG-1 functions as an integrator linking the PDGFR to lamellipodia extension and cell migration. PDGF, which stimulates sphingosine kinase, leading to increased SPP levels in many cell types, also induces translocation of sphingosine kinase to membrane ruffles. Hence, recruitment of sphingosine kinase to the cell's leading edge and localized formation of SPP may spatially and temporally stimulate EDG-1, resulting in activation and integration of downstream signals important for directional movement toward chemoattractants, such as PDGF. These results may also shed light on the vital role of EDG-1 in vascular maturation.
Subject(s)
Cell Movement/physiology , Immediate-Early Proteins/physiology , Lysophospholipids , Protein-Tyrosine Kinases/metabolism , Pseudopodia/physiology , Receptors, Cell Surface , Receptors, G-Protein-Coupled , Receptors, Platelet-Derived Growth Factor/metabolism , Sphingosine/analogs & derivatives , src-Family Kinases/metabolism , 3T3 Cells , Animals , Apoptosis/drug effects , Biological Transport/drug effects , Cell Division/drug effects , Cells, Cultured , Chemotaxis/drug effects , Cytoskeleton/drug effects , DNA/biosynthesis , DNA/drug effects , Embryo, Mammalian/cytology , Embryo, Mammalian/metabolism , Enzyme Activation , Fibroblasts/cytology , Fibroblasts/drug effects , Fibroblasts/metabolism , Focal Adhesion Kinase 1 , Focal Adhesion Protein-Tyrosine Kinases , Genotype , Green Fluorescent Proteins , Immediate-Early Proteins/genetics , Luminescent Proteins/drug effects , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Mice , Mice, Knockout , Microscopy, Confocal , Phosphorylation , Phosphotransferases (Alcohol Group Acceptor)/drug effects , Phosphotransferases (Alcohol Group Acceptor)/genetics , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Platelet-Derived Growth Factor/pharmacology , Receptors, Lysophospholipid , Receptors, Platelet-Derived Growth Factor/physiology , Recombinant Fusion Proteins/drug effects , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Signal Transduction , Sphingosine/pharmacology , Time FactorsABSTRACT
Subcellular fractionation revealed that a significant fraction of total sphingosine kinase, the enzyme that phosphorylates sphingosine to form the bioactive lipid metabolite sphingosine-1-phosphate, resides in the nuclei of Swiss 3T3 cells, localized to both the nuclear envelope and the nucleoplasm. Platelet-derived growth factor, in addition to rapidly stimulating cytosolic sphingosine kinase, also induced a large increase in nucleoplasm-associated activity after 12-24 h that correlated with progression of cells to the S-phase of the cell cycle and translocation of sphingosine kinase-green fluorescent protein fusion protein to the nuclear envelope. Our results add sphingosine kinase to the growing list of lipid-metabolizing enzymes associated with the nucleus, and suggest that sphingosine-1-phosphate may also play a role in signal transduction in the nucleus.