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Biomedical subjects

Akio Kihara

Publications and source records attributed to Akio Kihara.

29 records · Page 2Linked to original sources
PubMed2004

Asp177 in C4 domain of mouse sphingosine kinase 1a is important for the sphingosine recognition.

Sphingosine kinase (SK) is the enzyme that catalyzes the formation of sphingosine 1-phosphate (S1P). Although diverse biological functions have been reported for SK, its recognition site for its substrate sphingosine (Sph) is still unclear. We constructed various mutants of mouse sphingosine kinase 1a (mSK1a), carrying mutations in the C4 domain, which we had expected to encompass the Sph-binding site. We analyzed the influence of these mutations on the SK activity and substrate kinetics. One mutation, Asp177-->Asn177, caused a dramatic decrease in SK activity (to approximately 6% of wild type) and an increase in the Km value for Sph (10.1-->108 microM), with no change in the affinity for ATP. This result suggests that the C4 domain, especially the Asp177, is involved in the specific recognition of Sph. In this report, we are able, for the first time, to provide an account of the Sph-binding site of SK.

Adenosine Triphosphate↗
PubMed2004

Cross talk between sphingolipids and glycerophospholipids in the establishment of plasma membrane asymmetry.

Glycerophospholipids and sphingolipids are distributed asymmetrically between the two leaflets of the lipid bilayer. Recent studies revealed that certain P-type ATPases and ATP-binding cassette (ABC) transporters are involved in the inward movement (flip) and outward movement (flop) of glycerophospholipids, respectively. In this study of phytosphingosine (PHS)-resistant yeast mutants, we isolated mutants for PDR5, an ABC transporter involved in drug efflux as well as in the flop of phosphatidylethanolamine. The pdr5 mutants exhibited an increase in the efflux of sphingoid long-chain bases (LCBs). Genetic analysis revealed that the PHS-resistant phenotypes exhibited by the pdr5 mutants were dependent on Rsb1p, a putative LCB-specific transporter/translocase. We found that the expression of Rsb1p was increased in the pdr5 mutants. We also demonstrated that expression of RSB1 is under the control of the transcriptional factor Pdr1p. Expression of Rsb1p also was enhanced in mutants for the genes involved in the flip of glycerophospholipids, including ROS3, DNF1, and DNF2. These results suggest that altered glycerophospholipid asymmetry induces the expression of Rsb1p. Conversely, overexpression of Rsb1p resulted in increased flip and decreased flop of fluorescence-labeled glycerophospholipids. Thus, there seems to be cross talk between sphingolipids and glycerophospholipids in maintaining the functional lipid asymmetry of the plasma membrane.

4-Chloro-7-nitrobenzofurazan↗
PubMed2004

FVT-1 is a mammalian 3-ketodihydrosphingosine reductase with an active site that faces the cytosolic side of the endoplasmic reticulum membrane.

Sphingolipids are essential membrane components of eukaryotic cells. Their synthesis is initiated with the condensation of l-serine with palmitoyl-CoA, producing 3-ketodihydrosphingosine (KDS), followed by a reduction to dihydrosphingosine by KDS reductase. Until now, only yeast TSC10 has been identified as a KDS reductase gene. Here, we provide evidence that the human FVT-1 (hFVT-1) and mouse FVT-1 (mFVT-1) are functional mammalian KDS reductases. The forced expression of hFVT-1 or mFVT-1 in TSC10-null yeast cells suppressed growth defects, and hFVT-1 overproduced in cultured cells exhibited KDS reductase activity in vitro. Moreover, purified recombinant hFVT-1 protein exhibited NADPH-dependent KDS reductase activity. The identification of the FVT-1 genes enabled us to characterize the mammalian KDS reductase at the molecular level. Northern blot analyses demonstrated that both hFVT-1 and mFVT-1 mRNAs are ubiquitously expressed, suggesting that FVT-1 is a major KDS reductase. We also found the presence of hFVT-1 variants, which were differentially expressed among tissues. Immunofluorescence microscopic analysis revealed that hFVT-1 is localized at the endoplasmic reticulum. Moreover, a proteinase K digestion assay revealed that the large hydrophilic domain of hFVT-1, which contains putative active site residues, faces the cytosol. These results suggest that KDS is converted to dihydrosphingosine in the cytosolic side of the endoplasmic reticulum membrane. Moreover, the topology studies provide insight into the spatial organization of the sphingolipid biosynthetic pathway.

Alcohol Oxidoreductases↗
PubMed2004

Identification of the human sphingolipid C4-hydroxylase, hDES2, and its up-regulation during keratinocyte differentiation.

The C4-hydroxylation of dihydrosphingosine or dihydroceramide is a key reaction in the biosynthesis of phytosphingolipids, both in yeasts and in mammalian cells. Mouse DES2 (mDES2) was recently cloned and shown to work as a Delta4-desaturase/C4-hydroxylase, when expressed in yeast cells. Here, we cloned a human homologue of mDES2, hDES2, by homology search utilizing a BLAST program. When expressed in HEK 293 cells, hDES2 exhibited hydroxylase activity for dihydroceramide. Northern blot analyses of hDES2 revealed high expression in skin, intestines, and kidney, sites reportedly possessing high levels of phytosphingolipids. Furthermore, up-regulation of hDES2 mRNA expression and subsequent phytoceramide production were observed during vitamin C/serum-induced differentiation of human keratinocytes. These results suggest that the newly cloned hDES2 plays an essential role in phytosphingolipid synthesis in human skin and other phytosphingolipid-containing tissues.

Amino Acid Motifs↗
PubMed2003

Distribution of sphingosine kinase activity in mouse tissues: contribution of SPHK1.

Sphingosine kinase (SPHK) phosphorylates sphingosine to form a bioactive lipid mediator, sphingosine 1-phosphate (S1P). S1P mediates such diverse biological processes as regulation of cell differentiation, motility, and apoptosis both extracellularly, via S1P (Edg) family receptors, and intracellularly, through unidentified targets. In cells S1P is short-lived, so the synthetic process catalyzed by sphingosine kinase may be important in maintaining the cellular levels of the compound. Thus far, two sphingosine kinases have been reported, with SPHK1 exhibiting the higher activity. However, several studies suggest the existence of unidentified sphingosine kinases. Therefore, to further elucidate the role of SPHK1 in the formation of S1P, we investigated its contribution to the total sphingosine kinase activity in mouse tissues. We found that SPHK1 is a major sphingosine kinase in many tissues, especially in brain, heart, and colon. However, some tissues such as spleen, small intestine, and lung contained sphingosine kinase activity that was not attributable to SPHK1 or SPHK2, as determined by immunodepletion assays. Furthermore, the presence of other sphingosine kinases with different properties, i.e., higher activity toward phytosphingosine and a different subcellular distribution, is suggested.

Animals↗
PubMed2003

Csg1p and newly identified Csh1p function in mannosylinositol phosphorylceramide synthesis by interacting with Csg2p.

Csg1p and Csg2p have been shown to be involved in the synthesis of mannosylinositol phosphorylceramide (MIPC) from inositol phosphorylceramide. YBR161w, termed CSH1 here, encodes a protein that exhibits a strong similarity to Csg1p. To examine whether Csh1p also functions in MIPC synthesis, we performed a [3H]dihydrosphingosine labeling experiment. Deltacsg1 cells exhibited only a reduction in the synthesis of mannosylated sphingolipids compared with wild-type cells, whereas the Deltacsg1 Deltacsh1 double deletion mutant exhibited a total loss. These results indicated that Csg1p and Csh1p have redundant functions in MIPC synthesis. Analyses using Deltacsg1 and Deltacsh1 cells in the Deltaipt1, Deltasur2, or Deltascs7 genetic background demonstrated that Csh1p has a different substrate specificity from Csg1p. We also revealed that Csg2p interacts with both Csg1p and Csh1p. Deletion of the CSG2 gene reduced the Csg1p activity and abolished the Csh1p activity. These results suggested that two distinct inositol phosphorylceramide mannosyltransferase complexes, Csg1p-Csg2p and Csh1p-Csg2p, exist.

Amino Acid Sequence↗
PubMed2003

Sphingosine-1-phosphate lyase is involved in the differentiation of F9 embryonal carcinoma cells to primitive endoderm.

Sphingosine 1-phosphate (S1P) is a bioactive lipid molecule that acts both extracellularly and intracellularly. The SPL gene encodes a mammalian S1P lyase that degrades S1P. Here, we have disrupted the SPL gene in mouse F9 embryonal carcinoma cells by gene targeting. This is the first report of gene disruption of mammalian S1P lyase. The SPL-null cells exhibited no S1P lyase activity, and intracellular S1P was increased approximately 2-fold, compared with wild-type cells. Treatment of F9 embryonal carcinoma cells with retinoic acid induces differentiation to primitive endoderm (PrE). An acceleration in this PrE differentiation was observed in the SPL-null cells. This effect was apparently caused by the accumulated S1P, since N,N-dimethylsphingosine, a S1P synthesis inhibitor, had an inhibitory effect on the PrE differentiation. Moreover, F9 cells stably expressing sphingosine kinase also exhibited an acceleration in the differentiation. Exogenous S1P had no effect on differentiation, indicating that intracellular but not extracellular S1P is involved. Moreover, we determined that expression of the SPL protein is up-regulated during the progression to PrE. We also showed that sphingosine kinase activity is increased in PrE-differentiated cells. These results suggest that intracellular S1P has a role in the PrE differentiation and that SPL may be involved in the regulation of intracellular S1P levels during this differentiation.

Aldehyde-Lyases↗
PubMed2003

Transmembrane topology of sphingoid long-chain base-1-phosphate phosphatase, Lcb3p.

BACKGROUND: Sphingoid long-chain base-1-phosphates (LCBPs) are thought to act as intracellular signalling molecules in yeast. Lcb3p is a member of the LCBPs-specific phosphatase family (SPP family). Other yeast phosphatases, Lpp1p and Dpp1p, are members of a different lipid phosphatase family (LPP family) known to exhibit broader substrate specificities. Until now, only the membrane topology of mammalian LPP family members has been reported, whereas that of the SPP family has remained unclear. RESULTS: In our in vitro system, Lcb3p displayed major phosphatase activity against dihydrosphingosine-1-phosphate, while Dpp1p and Lpp1p also exhibited activities. Here, we determined that Lpp1p and Dpp1p exhibit the topology common to the LPP family. Moreover, we examined the transmembrane topology of Lcb3p using a C-terminal reporter approach. From our results we deduced a structural model illustrating that Lcb3p has eight membrane-spanning domains with its highly conserved phosphatase motifs positioned within the endoplasmic reticulum (ER) lumen. Consistent with this result, Lcb3p collected in low speed pellet fractions was highly resistant to exogenous proteinase K unless the membrane was disrupted. CONCLUSION: Our results suggest that the active site of Lcb3p is located in the ER lumen and, thus, the phosphate group of the LCBP is hydrolysed on the lumenal side.

Cell Membrane↗
PubMed2002

Identification and characterization of a novel human sphingosine-1-phosphate phosphohydrolase, hSPP2.

Sphingosine 1-phosphate (S1P) is a bioactive lipid molecule that acts as both an extracellular signaling mediator and an intracellular second messenger. S1P is synthesized from sphingosine by sphingosine kinase and is degraded either by S1P lyase or by S1P phosphohydrolase. Recently, mammalian S1P phosphohydrolase (SPP1) was identified and shown to constitute a novel lipid phosphohydrolase family, the SPP family. In this study we have identified a second human S1P phosphohydrolase, SPP2, based on sequence homology to human SPP1. SPP2 exhibited high phosphohydrolase activity against S1P and dihydrosphingosine 1-phosphate. The dihydrosphingosine-1-phosphate phosphohydrolase activity was efficiently inhibited by excess S1P but not by lysophosphatidic acid, phosphatidic acid, or glycerol 3-phosphate, indicating that SPP2 is highly specific to sphingoid base 1-phosphates. Immunofluorescence microscopic analysis demonstrated that SPP2 is localized to the endoplasmic reticulum. Although the enzymatic properties and localization of SPP2 were similar to those of SPP1, the tissue-specific expression pattern of SPP2 was different from that of SPP1. Thus, SPP2 is another member of the SPP family that may play a role in attenuating intracellular S1P signaling.

Amino Acid Sequence↗
PubMed2002

Identification and characterization of a Saccharomyces cerevisiae gene, RSB1, involved in sphingoid long-chain base release.

Sphingoid long-chain bases (LCBs) and long-chain base phosphates (LCBPs) act as signaling molecules in eukaryotic cells. Accumulation of LCBPs results in cell growth inhibition in yeast, although the mechanism is unknown. Here, we identified a novel yeast gene, RSB1 (resistance to sphingoid long-chain base), by screening a multicopy suppressor of the LCB-sensitive phenotype of the LCBP lyase mutant. RSB1 encodes a polypeptide of 354 amino acids with a molecular mass of 40.4 kDa. Rsb1p is predicted to be an integral membrane protein with seven transmembrane-spanning domains. We demonstrated that cells overproducing Rsb1p showed a decrease in accumulation of exogenously added sphingosine and dihydrosphingosine because of their increased release. This release was ATP-dependent, and a mutant of the predicted ATP binding motif had no activity. Substrate specificity analysis of Rsb1p demonstrated that it is active on LCBs but not on LCBPs or other hydrophobic compounds. These results suggest that Rsb1p is a transporter or flippase that translocates LCBs from the cytoplasmic side toward the extracytoplasmic side of the membrane.

Amino Acid Sequence↗