Biomedical subjects

Takamitsu Sano

Publications and source records attributed to Takamitsu Sano.

10 recordsLinked to original sources

PubMed2006

Changes in S1P1 and S1P2 expression during embryonal development and primitive endoderm differentiation of F9 cells.

Sphingosine 1-phosphate (S1P) is a ligand for S1P family receptors (S1P(1)-S1P(5)). Of these receptors, S1P(1), S1P(2), and S1P(3) are ubiquitously expressed in adult mice, while S1P(4) and S1P(5) are tissue specific. However, little is known of their expression during embryonal development. We performed Northern blot analyses in mouse embryonal tissue and found that such expression is developmentally regulated. We also examined the expression of these receptors during primitive endoderm (PrE) differentiation of mouse F9 embryonal carcinoma (EC) cells, a well-known in vitro endoderm differentiation system. S1P(2) mRNA was abundantly expressed in F9 EC cells, but little S1P(1) and no S1P(3), S1P(4), or S1P(5) mRNA was detectable. However, S1P(1) mRNA expression was induced during EC-to-PrE differentiation. Studies using small interference RNA of S1P(1) indicated that increased S1P(1) expression is required for PrE differentiation. Thus, S1P(1) may play an important function in PrE differentiation that is not substituted for by S1P(2).

Animals↗

PubMed2006

Intracellular localization and tissue-specific distribution of human and yeast DHHC cysteine-rich domain-containing proteins.

Increasing evidence indicates that DHHC cysteine-rich domain-containing proteins (DHHC proteins) are protein acyltransferases. Although multiple DHHC proteins are found in eukaryotes, characterization has been examined for only a few. Here, we have cloned all the yeast and human DHHC genes and investigated their intracellular localization and tissue-specific expression. Most DHHC proteins are localized in the ER and/or Golgi, with a few localized in the plasma membrane and one in the yeast vacuole. Human DHHC mRNAs also differ in their tissue-specific expression. These results may provide clues to aid in discovering the specific function(s) of each DHHC protein.

Acyltransferases↗

PubMed2005

Sphingosine 1-phosphate is released from the cytosol of rat platelets in a carrier-mediated manner.

Sphingosine 1-phosphate (S1P) is accumulated in platelets and released on stimulation by thrombin or Ca(2+). Thrombin-stimulated S1P release was inhibited by staurosporin, whereas Ca(2+)-stimulated release was not. When the platelet plasma membrane was permeabilized with streptolysin O (SLO), S1P leaked out with cytosol markers, whereas granular markers remained in the platelets. The SLO-induced S1P leakage required BSA, probably for solubilization of S1P in the medium. These results indicate that S1P is localized in the inner leaflet of the plasma membrane and that its release is a carrier-mediated process. We also used alpha-toxin (ATX), which makes smaller pores in the plasma membrane than SLO and depletes cytosolic ATP without BSA-dependent S1P leakage. The addition of ATP drove S1P release from ATX platelets. The ATP-driven S1P release from ATX platelets was greatly enhanced by thrombin. An ATP binding cassette transporter inhibitor, glyburide, prevents ATP- and thrombin-induced S1P release from platelets. Ca(2+) also stimulated S1P release from ATX platelets without ATP, whereas the Ca(2+)-induced release was not inhibited by glyburide. Our results indicate that two independent S1P release systems might exist in the platelet plasma membrane, an ATP-dependent system stimulated by thrombin and an ATP-independent system stimulated by Ca(2+).

Animals↗

PubMed2005

Regulation of the sphingoid long-chain base kinase Lcb4p by ergosterol and heme: studies in phytosphingosine-resistant mutants.

Sphingoid long-chain base 1-phosphates (LCBPs) are widely conserved, bioactive lipid molecules. In yeast, LCBPs are known to be involved in several cellular responses such as heat shock resistance and Ca(2+) mobilization, although their target molecules and signaling pathways remain unclear. To identify genes involved in LCBP signaling and in regulation of LCBP synthesis, we performed transposon mutagenesis in cells lacking the LCBP lyase DPL1 and LCBP phosphatase LCB3 genes and screened for phytosphingosine-resistant clones. Further isolation and identification revealed eight genes (PBP1, HEM14, UFD4, HMG1, TPS1, KES1, WHI2, and ERG5), in addition to the previously characterized LCB4 and PDR5 genes, that are involved in phytosphingosine resistance. Of these eight, four are ergosterol-related genes (HEM14, HMG1, KES1, and ERG5). We also demonstrated that protein expression of the long-chain base kinase Lcb4p was reduced in Deltahem14 and Deltahmg1 cells, likely as a consequence of decreased activity of the heme-dependent transcription factor Hap1p. In addition, phosphorylation of Lcb4p was decreased in all the ergosterol-related mutants isolated and other ergosterol mutants constructed (Deltaerg2, Deltaerg3, and Deltaerg6). Finally, plasma membrane localization of Lcb4p was found to be reduced in Deltaerg6 cells. These results suggest that changes in sterol composition affect the phosphorylation of Lcb4p because of the altered localization. The other genes isolated (PBP1, UFD4, TPS1, and WHI2) may be involved in LCBP signaling.

Biomarkers↗

PubMed2005

Mechanisms of sphingosine and sphingosine 1-phosphate generation in human platelets.

The bioactive molecule sphingosine 1-phosphate (S1P) is abundantly stored in platelets and can be released extracellularly. However, although they have high sphingosine (Sph) kinase activity, platelets lack the de novo sphingolipid biosynthesis necessary to provide the substrates. Here, we reveal a generation pathway for Sph, the precursor of S1P, in human platelets. Platelets incorporated extracellular 3H-labeled Sph much faster than human megakaryoblastic cells and rapidly converted it to S1P. Furthermore, Sph formed from plasma sphingomyelin (SM) by bacterial sphingomyelinase (SMase) and neutral ceramidase (CDase) was rapidly incorporated into platelets and converted to S1P, suggesting that platelets use extracellular Sph as a source of S1P. Platelets abundantly express SM, possibly supplied from plasma lipoproteins, at the cell surface. Treating platelets with bacterial SMase resulted in Sph generation at the cell surface, conceivably by the action of membrane-bound neutral CDase. Simultaneously, a time-dependent increase in S1P levels was observed. Finally, we demonstrated that secretory acid SMase also induces S1P increases in platelets. In conclusion, our results suggest that in platelets, Sph is supplied from at least two sources: generation in the plasma followed by incorporation, and generation at the outer leaflet of the plasma membrane, initiated by cell surface SM degradation.

Acyltransferases↗

PubMed2005

Long-chain base kinase Lcb4 Is anchored to the membrane through its palmitoylation by Akr1.

Sphingoid long-chain base kinase Lcb4 catalyzes the production of the bioactive lipid molecules the long-chain base 1-phosphates. Although Lcb4 has no apparent transmembrane-spanning domain, it is tightly associated with the membrane. Here, we demonstrate that Lcb4 is modified by palmitoylation. This modification was greatly reduced in mutants for AKR1, which was recently identified as encoding a protein acyltransferase. In vitro experiments revealed that Akr1 indeed acts as a protein acyltransferase for Lcb4. Studies using site-directed mutagenesis indicated that Cys-43 and Cys-46 are palmitoylated. The loss of palmitoylation on Lcb4 caused several effects, including mislocalization of the protein to the cytosol, reduced phosphorylation, and loss of downregulation during the stationary phase. Although Akr2 is highly homologous to Akr1, the deletion of AKR2 did not result in any remarkable phenotypes. However, overproduction of Akr2 resulted in reduced amounts of Lcb4. We demonstrated that Akr2 is an unstable protein and is degraded in the vacuole. Akr2 exhibits high affinity for Lcb4, and in Akr2-overproducing cells this interaction caused unusual delivery of Lcb4 to the vacuole and degradation.

Acyltransferases↗

PubMed2004

Phosphorylation by Pho85 cyclin-dependent kinase acts as a signal for the down-regulation of the yeast sphingoid long-chain base kinase Lcb4 during the stationary phase.

Sphingoid long-chain base 1-phosphates (LCBPs) act as bioactive lipid molecules in eukaryotic cells. In yeast, LCBPs are synthesized mainly by the long-chain base kinase Lcb4p. Until now, the regulatory mechanism for Lcb4p has been unclear. In the present study, we found that Lcb4p is post-translationally modified by phosphorylation. Using a protein kinase mutant yeast collection, we further demonstrated that the cyclin-dependent kinase Pho85p is involved in this phosphorylation. Pho85p functions in a number of cellular processes, especially in response to environmental changes. Two of 10 Pho85p cyclins, Pcl1p and Pcl2p had overlapping functions in the phosphorylation of Lcb4p. Site-directed mutagenesis identified the phosphorylation sites in Lcb4p as Ser(451) and Ser(455). Additionally, pulse-chase experiments revealed that Lcb4p is degraded via the ubiquitin-dependent pathway. The protein was stabilized in Deltapho85 cells, suggesting that phosphorylation acts as a signal for the degradation. Lcb4p is down-regulated in the stationary phase of cell growth, and both phosphorylation and ubiquitination appear to be important for this process. Moreover, we demonstrated that Lcb4p is delivered to the vacuole for degradation via the multivesicular body. Since forced accumulation of LCBPs results in prolonged growth during the stationary phase, down-regulation of Lcb4p may be physiologically important for proper cellular responses to nutrient deprivation.

Binding Sites↗

PubMed2003

Synthesis of fluorescence-labeled sphingosine and sphingosine 1-phosphate; effective tools for sphingosine and sphingosine 1-phosphate behavior.

A fluorescence-labeled sphingosine and sphingosine 1-phosphate have been successfully synthesized from the oxazolidinone methyl ester derived from glycidol via monoalkylation and the stereoselective reduction of the resulting ketone. The labeled sphingosine was converted into its phosphate by treatment with sphingosine kinase 1 (SPHK1) from mouse, and in platelets, and it was incorporated into the Chinese Hamster Ovarian (CHO) cells. In addition, MAPK was activated by NBD-Sph-1-P through Edg-1, Sph-1-P receptor.

4-Chloro-7-nitrobenzofurazan↗

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

Multiple mechanisms linked to platelet activation result in lysophosphatidic acid and sphingosine 1-phosphate generation in blood.

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (Sph1P) production was examined in vitro under conditions that simulated blood clotting. Several approaches were utilized to elucidate the metabolic pathways. 1) Platelet phospholipids were labeled using [32P]orthophosphate, and the production of [32P]Sph1P and LPA was examined. Thrombin stimulation of platelets resulted in rapid secretion of Sph1P stored within the platelet. In contrast, LPA was neither stored within nor secreted from platelets. Nonetheless, extracellular levels of LPA gradually increased following stimulation. 2) Stable-isotope dilution mass spectrometry was used to quantify the molecular species of LPA generated from platelets in vitro. Only 10% of the LPA generated following thrombin stimulation was associated with platelets, the remaining 90% was contained within the extracellular medium. The acyl composition of LPA produced by platelets differed depending on the presence or absence of plasma in the incubation. 3) The fate of exogenously added fluorescent phospholipid analogs was determined. Incubation of [(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl-(NBD)-labeled phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine with the supernatant fractions from thrombin-stimulated platelets yielded no LPA production. However, these lipids were converted to the corresponding lysolipids by released PLA1 and PLA2 activities. When incubated with plasma or serum the NBD-labeled lysophospholipids were readily converted to LPA. Inhibitors of lysophospholipase D and the biological activity of LPA were detected in plasma. These results suggest that the bulk of LPA produced through platelet activation results from the sequential cleavage of phospholipids to lysophospholipids by released phospholipases A1 and A2 and then to LPA by plasma lysophospholipase D.

Animals↗