Involvement of sphingosine 1-phosphate signaling in insulin-like growth factor-II/mannose 6-phosphate receptor trafficking from endosome to the trans-Golgi network.

The insulin-like growth factor II/mannose 6-phosphate (IGF-II/M6P) receptor is a multifunctional glycoprotein not only play roles in IGF-II degradation and pro-TGFβ activation but binding to and transport M6P-bearing lysosomal enzymes from the trans-Golgi network (TGN) or the cell surface to lysosomes. At present, information regarding a retrograde transport of IGF-II/M6P receptor from endosomes to the TGN is still limited. We show here that a continuous ligand-dependent activation of sphingosine 1-phosphate receptor type 3 (S1PR) on the endosomal membranes is required for subsequent recycling back of cargo-unloaded IGF-II/M6P receptors to the TGN.

Extracellular α-synuclein impairs sphingosine 1-phosphate receptor type 3 (S1PR3)-regulated lysosomal delivery of cathepsin D in HeLa cells.

α-Synuclein (α-Syn)-positive intracellular fibrillar protein deposits, known as Lewy bodies, are thought to be involved in the pathogenesis of Parkinson's disease (PD). Although recent lines of evidence suggested that extracellular α-Syn secreted from pathogenic neurons contributes to the propagation of PD pathology, the precise mechanism of action remains unclear. We have reported that extracellular α-Syn caused sphingosine 1-phosphate (S1P) receptor type 1 (S1PR1) uncoupled from Gi and inhibited downstream G-protein signaling in SH-SY5Y cells, although its patho/physiological role remains to be clarified.

Features and mechanisms of propofol-induced protein kinase C (PKC) translocation and activation in living cells.

In this study, we aimed to elucidate the action mechanisms of propofol, particularly those underlying propofol-induced protein kinase C (PKC) translocation. Various PKCs fused with green fluorescent protein (PKC-GFP) or other GFP-fused proteins were expressed in HeLa cells, and their propofol-induced dynamics were observed using confocal laser scanning microscopy. Propofol-induced PKC activation in cells was estimated using the C kinase activity receptor (CKAR), an indicator of intracellular PKC activation.

Constitutive activation of S1P receptors at the -Golgi network is required for surface transport carrier formation.

The importance of the G-protein βγ subunits in the regulation of cargo transport from the -Golgi network (TGN) to the plasma membrane (PM) is well accepted; however, the molecular mechanism underlying the G-protein activation at the TGN remains unclear. We show here that sphingosine 1-phosphate (S1P) receptors at the PM were trafficked to the TGN in response to a surface transport cargo, temperature-sensitive vesicular stomatitis virus glycoprotein tagged with green fluorescent protein accumulation in the Golgi. The receptor internalization occurred in an S1P-independent manner but required phosphorylation by G-protein receptor kinase 2 and β-arrestin association before internalization.

Identification of novel OCRL isoforms associated with phenotypic differences between Dent disease-2 and Lowe syndrome.

Background: Although Lowe syndrome and Dent disease-2 are caused by Oculocerebrorenal syndrome of Lowe (OCRL) mutations, their clinical severities differ substantially and their molecular mechanisms remain unclear. Truncating mutations in OCRL exons 1-7 lead to Dent disease-2, whereas those in exons 8-24 lead to Lowe syndrome. Herein we identified the mechanism underlying the action of novel OCRL protein isoforms.

Involvement of Receptor-Mediated S1P Signaling in EGF-Induced Macropinocytosis in COS7 Cells.

Macropinocytosis is a highly conserved cellular process of endocytosis by which extracellular fluid and nutrients are taken up into cells through large, heterogeneous vesicles known as macropinosomes. Growth factors such as epidermal growth factor (EGF) can induce macropinocytosis in many types of cells, although precise mechanism underlying EGF-induced macropinocytosis remains unclear. In the present studies we have shown the involvement of S1P signaling in EGF-induced macropinocytosis in COS7 cells.

Activation of atypical protein kinase C by sphingosine 1-phosphate revealed by an aPKC-specific activity reporter.

Atypical protein kinase C (aPKC) isozymes are unique in the PKC superfamily in that they are not regulated by the lipid second messenger diacylglycerol, which has led to speculation about whether a different second messenger acutely controls their function. Here, using a genetically encoded reporter that we designed, aPKC-specific C kinase activity reporter (aCKAR), we found that the lipid mediator sphingosine 1-phosphate (S1P) promoted the cellular activity of aPKC. Intracellular S1P directly bound to the purified kinase domain of aPKC and relieved autoinhibitory constraints, thereby activating the kinase.

Quantitative Analysis of Cargo Density in Single-extracellular Vesicles by Imaging.

Function of extracellular vesicles such as exosomes and microvesicles is determined by their wide ranges of cargoes inside them. Even in the pure exosomes or microvesicles the cargo contents are very heterogeneous. To understand this heterogeneous nature of extracellular vesicles, we need information of the vesicles, which will give us some parameters including vesicle size, number and cargo content of each vesicle.

Essential Role of Sphingosine Kinase 2 in the Regulation of Cargo Contents in the Exosomes from K562 Cells.

Sphingosine 1-phosphate (S1P) is a bioactive phosphorylated product of sphingosine catalyzed by sphingosine kinase (SphK) and implicated in diverse cellular functions including vesicular trafficking. In the present study we have shown the importance of one of the subtypes of SphK, SphK2, in the regulation of cargo content in exosomes released from human myeloid leukemia K562 cells. First, SphK2 has been shown to localize with N-Rh-PE-positive late endosomes in the cells.

Extracellular α-synuclein drives sphingosine 1-phosphate receptor subtype 1 out of lipid rafts, leading to impaired inhibitory G-protein signaling.

α-Synuclein (α-Syn)-positive intracytoplasmic inclusions, known as Lewy bodies, are thought to be involved in the pathogenesis of Lewy body diseases, such as Parkinson's disease (PD). Although growing evidence suggests that cell-to-cell transmission of α-Syn is associated with the progression of PD and that extracellular α-Syn promotes formation of inclusion bodies, its precise mechanism of action in the extracellular space remains unclear. Here, as indicated by both conventional fractionation techniques and FRET-based protein-protein interaction analysis, we demonstrate that extracellular α-Syn causes expulsion of sphingosine 1-phosphate receptor subtype 1 (S1PR) from the lipid raft fractions.

Erratum: Extracellular α-synuclein induces sphingosine 1-phosphate receptor subtype 1 uncoupled from inhibitory G-protein leaving β-arrestin signal intact.

This corrects the article DOI: 10.1038/srep44248.

Erratum: Impairment of PDGF-induced chemotaxis by extracellular α-synuclein through selective inhibition of Rac1 activation.

This corrects the article DOI: 10.1038/srep37810.

DHHC5-mediated palmitoylation of S1P receptor subtype 1 determines G-protein coupling.

Sphingosine 1-phosphate (S1P) is a pleiotropic lipid mediator involved in the regulation of immune cell trafficking and vascular permeability acting mainly through G-protein-coupled S1P receptors (S1PRs). However, mechanism underlying how S1PRs are coupled with G-proteins remains unknown. Here we have uncovered that palmitoylation of a prototypical subtype S1PR is prerequisite for subsequent inhibitory G-protein (Gi) coupling.

Involvement of Gβγ subunits of G protein coupled with S1P receptor on multivesicular endosomes in F-actin formation and cargo sorting into exosomes.

Exosomes play a critical role in cell-to-cell communication by delivering cargo molecules to recipient cells. However, the mechanism underlying the generation of the exosomal multivesicular endosome (MVE) is one of the mysteries in the field of endosome research. Although sphingolipid metabolites such as ceramide and sphingosine 1-phosphate (S1P) are known to play important roles in MVE formation and maturation, the detailed molecular mechanisms are still unclear.

Extracellular α-synuclein induces sphingosine 1-phosphate receptor subtype 1 uncoupled from inhibitory G-protein leaving β-arrestin signal intact.

Parkinson's disease (PD) is the second most common neurodegenerative disorder. The presence of α-synuclein (α-Syn)-positive intracytoplasmic inclusions, known as Lewy bodies, is the cytopathological hallmark of PD. Increasing bodies of evidence suggest that cell-to-cell transmission of α-Syn plays a role in the progression of PD.

Impairment of PDGF-induced chemotaxis by extracellular α-synuclein through selective inhibition of Rac1 activation.

Parkinson's disease (PD) is characterized by α-synuclein (α-Syn)-positive intracytoplasmic inclusions, known as Lewy bodies. Although it is known that extracellular α-Syn is detected in the plasma and cerebrospinal fluid, its physiological significance remains unclear. Here, we show that extracellular α-Syn suppresses platelet-derived growth factor (PDGF)-induced chemotaxis in human neuroblastoma SH-SY5Y cells.

MicroRNA Regulation of Human Breast Cancer Stem Cells.

MicroRNAs (miRNAs) are involved in virtually all biological processes, including stem cell maintenance, differentiation, and development. The dysregulation of miRNAs is associated with many human diseases including cancer. We have identified a set of miRNAs differentially expressed between human breast cancer stem cells (CSCs) and non-tumorigenic cancer cells.

Sphingosine kinases modulate the secretion of amyloid β precursor protein from SH-SY5Y neuroblastoma cells: the role of α-synuclein.

Sphingosine kinases (SphK 1&2) are involved in the regulation of cell survival, differentiation and neurotransmitter secretion. Current data suggest potential links between sphingolipid signalling, α-synuclein (ASN) and Alzheimer's disease (AD). Our aim was to investigate the possible role of SphKs and ASN in the regulation of the production and secretion of the amyloid β precursor protein (APP).

Ongoing activation of sphingosine 1-phosphate receptors mediates maturation of exosomal multivesicular endosomes.

During late endosome maturation, cargo molecules are sorted into intralumenal vesicles (ILVs) of multivesicular endosomes (MVEs), and are either delivered to lysosomes for degradation or fused with the plasma membranes for exosome release. The mechanism underlying formation of exosomal ILVs and cargo sorting into ILVs destined for exosome release is still unclear. Here we show that inhibitory G protein (Gi)-coupled sphingosine 1-phosphate (S1P) receptors regulate exosomal MVE maturation.

Yasutomi Nishizuka: father of protein kinase C.

It is 6 years since we received the tragic news that our great mentor Dr Yasutomi Nishizuka passed away suddenly at the age of 72 years. Dr Nishizuka made several epoch-making discoveries in his life, e.g.

Sphingosine kinase 1 regulates mucin production via ERK phosphorylation.

Our previous report showed that inhibition of sphingosine kinase (SphK) ameliorates eosinophilic inflammation and mucin production in a mouse asthmatic model. To clarify the role of SphK in airway mucin production, we utilized the mouse asthmatic model and found that both SphK and MUC5AC expression were increased and co-localized in airway epithelium. Next we cultured normal human bronchial epithelial cells in an air-liquid interface and treated with IL-13 to induce their differentiation into goblet cells.

Roles of extracellular and intracellular sphingosine 1-phosphate in cell migration.

Sphingosine 1-phosphate (S1P) is an important factor for the regulation of cell motility acting both inside and outside the cells. The precise role of S1P in the control of cell motility, however, remains unclear. Here we describe the roles of S1P in the regulation of cell motility by dissecting them into intracellular and extracellular actions using a liposomal S1P transfer technique.

CtBP1/BARS is an activator of phospholipase D1 necessary for agonist-induced macropinocytosis.

Vesicular trafficking such as macropinocytosis is a dynamic process that requires coordinated interactions between specialized proteins and lipids. A recent report suggests the involvement of CtBP1/BARS in epidermal growth factor (EGF)-induced macropinocytosis. Detailed mechanisms as to how lipid remodelling is regulated during macropinocytosis are still undefined.