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Takayoshi Kuno

Publications and source records attributed to Takayoshi Kuno.

At least 19 recordsLinked to original sources
PubMed2006

Rho2 is a target of the farnesyltransferase Cpp1 and acts upstream of Pmk1 mitogen-activated protein kinase signaling in fission yeast.

We have previously demonstrated that knockout of the calcineurin gene or inhibition of calcineurin activity by immunosuppressants resulted in hypersensitivity to Cl- in fission yeast. We also demonstrated that knockout of the components of the Pmk1 mitogen-activated protein kinase (MAPK) pathway, such as Pmk1 or Pek1 complemented the hypersensitivity to Cl-. Using this interaction between calcineurin and Pmk1 MAPK, here we developed a genetic screen that aims to identify new regulators of the Pmk1 signaling and isolated vic (viable in the presence of immunosuppressant and chloride ion) mutants. One of the mutants, vic1-1, carried a missense mutation in the cpp1+ gene encoding a beta subunit of the protein farnesyltransferase, which caused an amino acid substitution of aspartate 155 of Cpp1 to asparagine (Cpp1(D155N)). Analysis of the mutant strain revealed that Rho2 is a novel target of Cpp1. Moreover, Cpp1 and Rho2 act upstream of Pck2-Pmk1 MAPK signaling pathway, thereby resulting in the vic phenotype upon their mutations. Interestingly, compared with other substrates of Cpp1, defects of Rho2 function were more phenotypically manifested by the Cpp1(D155N) mutation. Together, our results demonstrate that Cpp1 is a key component of the Pck2-Pmk1 signaling through the spatial control of the small GTPase Rho2.

Alleles↗
PubMed2006

Genetic evidence for phospholipid-mediated regulation of the Rab GDP-dissociation inhibitor in fission yeast.

We have previously identified mutant alleles of genes encoding two Rab proteins, Ypt3 and Ryh1, through a genetic screen using the immunosuppressant drug FK506 in fission yeast. In the same screen, we isolated gdi1-i11, a mutant allele of the essential gdi1+ gene encoding Rab GDP-dissociation inhibitor. In gdi1-i11, a conserved Gly267 was substituted by Asp. The Gdi1G267D protein failed to extract Rabs from membrane and Rabs were depleted from the cytosolic fraction in the gdi1-i11 mutant cells. Consistently, the Gdi1G267D protein was found mostly in the membrane fraction, whereas wild-type Gdi1 was found in both the cytosolic and the membrane fraction. Notably, overexpression of spo20+, encoding a phosphatidylcholine/phosphatidylinositol transfer protein, rescued gdi1-i11 mutation, but not ypt3-i5 or ryh1-i6. The gdi1-i11 and spo20-KC104 mutations are synthetically lethal, and the wild-type Gdi1 failed to extract Rabs from the membrane in the spo20-KC104 mutant. The phosphatidylinositol-transfer activity of Spo20 is dispensable for the suppression of the gdi1-i11 mutation, suggesting that the phosphatidylcholine-transfer activity is important for the suppression. Furthermore, knockout of the pct1+ gene encoding a choline phosphate cytidyltransferase rescued the gdi1-i11 mutation. Together, our findings suggest that Spo20 modulates Gdi1 function via regulation of phospholipid metabolism of the membranes.

Alleles↗
PubMed2006

Real-time monitoring of calcineurin activity in living cells: evidence for two distinct Ca2+-dependent pathways in fission yeast.

In fission yeast, calcineurin dephosphorylates and activates the Prz1 transcription factor. Here, we identified the calcineurin-dependent response element (CDRE) in the promoter region of prz1(+) gene and monitored the calcineurin activity in living cells using a destabilized luciferase reporter gene fused to three tandem repeats of CDRE. Elevated extracellular CaCl(2) caused an increase in calcineurin activity with an initial peak and then approached a sustained constant level in a concentration-dependent manner. In CaCl(2)-sensitive mutants such as Deltapmc1, the response was markedly enhanced, reflecting its high intracellular Ca(2+). Agents expected to induce Ca(2+) influx showed distinct patterns of the CDRE-reporter activity, suggesting different mechanisms of calcineurin activation. Knockout of yam8(+) or cch1(+) encoding putative subunits of a Ca(2+) channel abolished the activation of calcineurin upon exposure to various stimuli, including high extracellular NaCl and cell wall-damaging agents. However, knockout of yam8(+) or cch1(+) did not affect the activation of calcineurin upon stimulation by elevated extracellular Ca(2+). The Pck2 protein kinase C-Pmk1 mitogen-activate protein kinase pathway was required for the stimulation of calcineurin via Yam8/Cch1-mediated Ca(2+) influx, but it was not required for the stimulation by elevated extracellular Ca(2+), suggesting two distinct pathways for calcineurin activation.

Calcineurin↗
PubMed2006

Genetic and functional interaction between Ryh1 and Ypt3: two Rab GTPases that function in S. pombe secretory pathway.

We have previously isolated ypt3-i5 mutant and showed that Ypt3 GTPase functions in the fission yeast secretory pathway. Here, the same genetic screen led to the isolation of ryh1-i6, a mutant allele of the ryh1+ gene encoding a homolog of Rab6. The ryh1-i6 mutant showed phenotypes that support its role in retrograde traffic from endosome to the Golgi. Interestingly, ryh1+ gene deletion was synthetically lethal with ypt3-i5 mutation. Consistently, the over-expression of the GDP-conformational mutant, Ryh1T25 N, inhibited the growth of ypt3-i5 mutant but had no effect on that of wild-type cells. Furthermore, the over-expression of the Ryh1T25N mutant inhibited the acid phosphatase glycosylation and exacerbated the cell wall integrity of ypt3-i5 mutant, but had no effect on those of wild-type cells. GFP-Ryh1 and GFP-Ypt3 both localized at the Golgi/endosome, but showed distinct subcellular localizations. The localization of GFP-Ryh1 in ypt3-i5 mutant and that of GFP-Ypt3 in ryh1-i6 mutant were distinct from those in wild-type cells. In addition, Ryh1 as well as Ypt3 were shown to be involved in acid phosphatase secretion. These results suggest that Ryh1 is involved in the secretory pathway and may have a potential overlapping function with Ypt3 in addition to its role in recycling.

Acid Phosphatase↗
PubMed2005

Phosphatidylinositol-4-phosphate 5-kinase regulates fission yeast cell integrity through a phospholipase C-mediated protein kinase C-independent pathway.

Fission yeast its3-1 mutant is an allele of the essential gene its3+ that encodes a phosphatidylinositol-4-phosphate 5-kinase (PIP5K) that produces phosphatidylinositol 4,5-bisphosphate. We found that the its3-1 mutant is sensitive to micafungin, a (1,3)-beta-D-glucan synthase inhibitor, suggesting a cell wall integrity defect. Consistently, its3-1 mutation caused synthetic lethality with a (1,3)-beta-D-glucan synthase mutant, bgs1-i2, and its3-1 mutant cells showed aberrant localization of green fluorescent protein-Bgs1. Similar aberrant localization of green fluorescent protein-tagged Rgf1, a putative phosphatidylinositol 4,5-bisphosphate-binding guanine nucleotide exchange factor for Rho protein, in its3-1 mutants was observed, suggesting a defective Rgf1/Rho pathway. To unravel the molecular mechanism(s), putative downstream components of PIP5K signaling were analyzed. Unexpectedly, overexpression of phospholipase C (Plc1), but not that of protein kinase C (PKC; Pck1 and Pck2), suppressed the phenotypes of the its3-1 mutant. These findings indicate that PKCs are not involved in the suppression, and further analysis revealed that PKCs are not downstream of Plc1 in fission yeast. Also, the enzymatic activity of Plc1 is essential for the suppression of the phenotypes and for the viability of the its3-1 mutant. These findings suggest that Its3 PIP5K regulates cell integrity through a Plc1-mediated PKC-independent pathway, in addition to the Rho/PKC pathway.

Alleles↗
PubMed2005

Comparison of toxicity and toxicokinetics/pharmacokinetics of an alpha 1L-adrenoceptor agonist in rats and rhesus monkeys.

We have investigated the toxicity of an alpha(1L)-adrenoceptor agonist, ESR 1150 CL, and compared the toxicokinetics and pharmacokinetics in rats and monkeys. In rats, this compound induced death with remarkable sacculated aneurysms of the aorta in groups given more than 3 mg/kg per day in a 4-week repeated oral administration study. On the other hand, these findings were not observed in monkeys during a 2-week repeated oral administration study at doses up to 30 mg/kg per day. Orally administered ESR 1150 CL raised blood pressure transiently and dose-dependently during the 4-week repeated study in rats, whereas no increase of blood pressure was observed during the 2-week oral toxicity study in monkeys. Contrary to our expectation, the exposure level of ESR 1150 CL in rats was not higher than that in monkeys in the toxicokinetic evaluation. Pharmacokinetic evaluation indicated good absorption of the compound, but the bioavailability was very low in both rats and monkeys. These findings suggest that the potent species difference in toxicity of ESR 1150 CL between rats and monkeys does not depend on differences of toxicokinetics/pharmacokinetics. Rather, we suggest that the reason is likely to be species difference in the biological susceptibility of the alpha(1L)-adrenoceptor subtypes between rats and monkeys, which would be closely related with the effect on blood pressure by alpha(1L)-adrenoceptor agonist.

Adrenergic alpha-1 Receptor Agonists↗
PubMed2005

The role of the regulatory subunit of fission yeast calcineurin for in vivo activity and its relevance to FK506 sensitivity.

Calcineurin, a protein phosphatase required for Ca2+ signaling in many cell types, is a heterodimer composed of catalytic and regulatory subunits. The fission yeast genome encodes a single set of catalytic (Ppb1) and regulatory (Cnb1) subunits, providing an ideal model system to study the functions of these subunits in vivo. Here, we cloned the cnb1+ gene and showed that the cnb1 knock-out (Deltacnb1) exhibits identical phenotypes with Deltappb1 and that overexpression of Ppb1 failed to suppress the phenotypes of Deltacnb1. Interestingly, overexpression of the C-terminal-deleted Ppb1 (Ppb1DeltaC), the constitutively active form of Ppb1, also failed to suppress the phenotypes of Deltacnb1. FK506 caused MgCl2 sensitivity to the wild-type cells in an FKBP12-dependent manner. Co-overexpression of Ppb1 and Cnb1 suppressed the FK506-induced MgCl2 sensitivity, but the suppression was only partial, suggesting that an excess amount of the Ppb1-Cnb1 complex cannot compete out the FKBP12-FK506 complex. Although overexpression of Ppb1DeltaC alone had little effect on cell growth, co-overexpression of Ppb1DeltaC and Cnb1 caused a distinct growth defect. FK506 suppressed the growth defect when Cnb1 was co-expressed using the attenuated nmt1 promoter, but it failed to suppress the defect when Cnb1 was co-expressed using the wild-type nmt1 promoter. Knock-out of the prz1+ gene, encoding a downstream target transcription factor of calcineurin, suppressed the growth defect irrespective of the promoter potency. These results suggest that Cnb1 is essential for the activation of calcineurin and that the activated calcineurin is the pharmacological target of the FKBP12-FK506 complex in vivo.

Actins↗
PubMed2004

Loss of Apm1, the micro1 subunit of the clathrin-associated adaptor-protein-1 complex, causes distinct phenotypes and synthetic lethality with calcineurin deletion in fission yeast.

Calcineurin is a highly conserved regulator of Ca(2+) signaling in eukaryotes. In fission yeast, calcineurin is not essential for viability but is required for cytokinesis and Cl(-) homeostasis. In a genetic screen for mutations that are synthetically lethal with calcineurin deletion, we isolated a mutant, cis1-1/apm1-1, an allele of the apm1(+) gene that encodes a homolog of the mammalian micro1A subunit of the clathrin-associated adaptor protein-1 (AP-1) complex. The cis1-1/apm1-1 mutant as well as the apm1-deleted (Deltaapm1) cells showed distinct phenotypes: temperature sensitivity; tacrolimus (FK506) sensitivity; and pleiotropic defects in cytokinesis, cell integrity, and vacuole fusion. Electron micrographs revealed that Deltaapm1 cells showed large vesicular structures associated with Golgi stacks and accumulated post-Golgi secretory vesicles. Deltaapm1 cells also showed the massive accumulation of the exocytic v-SNARE Syb1 in the Golgi/endosomes and a reduced secretion of acid phosphatase. These phenotypes observed in apm1 mutations were accentuated upon temperature up-shift and FK506 treatment. Notably, Apm1-GFP localized to the Golgi/endosomes, the spindle pole bodies, and the medial region. These findings suggest a role for Apm1 associated with the Golgi/endosome function, thereby affecting various cellular processes, including secretion, cytokinesis, vacuole fusion, and cell integrity and also suggest that calcineurin is involved in these events.

Acid Phosphatase↗
PubMed2004

Upregulation of mRNA in MAPK signaling: transcriptional activation or mRNA stabilization?

Mitogen-activated protein kinases (MAPKs), found in all eukaryotes, are signal-transducing enzymes that play a central role in a variety of biological processes. MAPK phosphatase has dual catalytic activity toward phosphotyrosine- and phosphothreonine-containing proteins, and is known to inactivate ERKs and JNKs/SAPKs, thus playing a crucial role in MAPK regulation. Although MAPK phosphatase has been implicated in a feedback loop that inactivates MAPKs after stimulation by mitogens and during the cellular response to stress, signaling pathways leading to MAPK phosphatase gene expression have not been fully elucidated. Recently, we have shown that a novel RNA-binding protein Rnc1 plays a crucial role in negative feedback regulation of MAPK signaling by stabilizing the mRNA of a MAPK phosphatase at the post-transcriptional level. One important aspect of our findings is that the increase in mRNA levels involves not only the transcriptional upregulation, but also the post-transcriptional gene regulation, especially the regulation of mRNA stability. Our discovery highlights a potential role and an emerging view of RNA-binding protein as a regulator of cell signaling and as a future target of drug discovery.

Animals↗
PubMed2004

Pmr1, a P-type ATPase, and Pdt1, an Nramp homologue, cooperatively regulate cell morphogenesis in fission yeast: the importance of Mn2+ homeostasis.

Schizosaccharomyces pombe pmr1+ gene is homologous to Saccharomyces cerevisiae PMR1 gene, which encodes the P-type Ca2+/Mn2+-ATPase. Addition of Mn2+, as well as Ca2+, to the medium induced pmr1+ gene expression in a calcineurin-dependent manner. The pmr1 knockout (Deltapmr1) cells exhibited hypersensitivity to EGTA. A screen for high gene dosage-suppressors of the EGTA-hypersensitive phenotype of Deltapmr1 led to the identification of pdt1+ gene, which encodes an Nramp-related metal transporter. The Deltapmr1 cells showed round cell morphology. Although Deltapdt1 cells appeared normal in the regular medium, it showed round cell morphology similar to that of the Deltapmr1 cells when Mn2+ was removed from the medium. The removal of Mn2+ also exacerbated the round morphology of the Deltapmr1 cells. The Deltapmr1Deltapdt1 double mutants grew very slowly and showed extremely aberrant cell morphology with round, enlarged and depolarized shape. The addition of Mn2+, but not Ca2+, to the medium completely suppressed the morphological defects, while both Mn2+ and Ca2+ markedly improved the slow growth of the double mutants. These results suggest that Pmr1 and Pdt1 cooperatively regulate cell morphogenesis through the control of Mn2+ homeostasis, and that calcineurin functions as a Mn2+ sensor as well as a Mn2+ homeostasis regulator.

Calcineurin↗
PubMed2004

Lub1 participates in ubiquitin homeostasis and stress response via maintenance of cellular ubiquitin contents in fission yeast.

Ubiquitin-dependent proteolysis plays a pivotal role in stress responses. To investigate the mechanisms of these cellular processes, we have been studying Schizosaccharomyces pombe mutants that have altered sensitivities to various stress conditions. Here, we showed that Lub1, a homologue of Ufd3p/Zzz4p/Doa1p in budding yeast, is involved in the regulation of ubiquitin contents. Disruption of the lub1+ gene resulted in monoubiquitin as well as multiubiquitin depletion without change in mRNA level and in hypersensitivity to various stress conditions. Consistently, overexpression of genes encoding ubiquitin suppressed the defects associated with lub1 mutation, indicating that the phenotypes of the lub1 mutants under stress conditions were due to cellular ubiquitin shortage at the posttranscriptional level. In addition, the lub1-deleted cells showed aberrant functions in ubiquitin/proteasome-dependent proteolysis, with accelerated degradation of ubiquitin. Also Cdc48, a stress-induced chaperon-like essential ATPase, was found to interact with Lub1, and this association might contribute to the stabilization of Lub1. Our results indicated that Lub1 is responsible for ubiquitin homeostasis at the protein level through a negative regulation of ubiquitin degradation.

Cysteine Endopeptidases↗
PubMed2003

Feedback regulation of MAPK signalling by an RNA-binding protein.

Mitogen-activated protein kinases (MAPKs) are evolutionarily conserved enzymes that convert extracellular signals into various outputs such as cell growth, differentiation and cell death. MAPK phosphatases selectively inactivate MAPKs by dephosphorylating critical phosphothreonine and phosphotyrosine residues. The transcriptional induction of MAPK phosphatase expression by various stimuli, including MAPK activation, has been well documented as a negative-feedback mechanism of MAPK signalling. Here we show that Rnc1, a novel K-homology-type RNA-binding protein in fission yeast, binds and stabilizes Pmp1 messenger RNA, the MAPK phosphatase for Pmk1 (refs 10, 11). Rnc1 therefore acts as a negative regulator of Pmk1 signalling. Notably, Pmk1 phosphorylates Rnc1, causing enhancement of the RNA-binding activity of Rnc1. Thus, Rnc1 is a component of a new negative-feedback loop that regulates the Pmk1 pathway through its binding to Pmp1 mRNA. Our findings--the post-transcriptional mRNA stabilization of a MAPK phosphatase mediated by an RNA-binding protein--provide an additional regulatory mechanism for fine-tuning of MAPK signalling pathways.

Deoxyribonucleases↗
PubMed2003

Zinc finger protein Prz1 regulates Ca2+ but not Cl- homeostasis in fission yeast. Identification of distinct branches of calcineurin signaling pathway in fission yeast.

Calcineurin is an important mediator that connects the Ca(2+)-dependent signaling to various cellular responses in a wide variety of cell types and organisms. In budding yeast, activated calcineurin exerts its function mainly by regulating the Crz1p/Tcn1 transcription factor. Here, we cloned the fission yeast prz1(+) gene, which encodes a zinc finger transcription factor highly homologous to Crz1/Tcn1. Similar to the results in budding yeast, calcineurin dephosphorylated Prz1 and resulted in the trans-location of Prz1 from the cytoplasm to the nucleus. Prz1 expression was stimulated by high extracellular Ca(2+) in a calcineurin-dependent fashion. However, unlike in budding yeast, the prz1-null cells did not show any phenotype similar to those previously reported in calcineurin deletion such as aberrant cell morphology, mating defect, or hypersensitivity to Cl(-). Instead, the prz1-null cells showed hypersensitivity to Ca(2+), consistent with a dramatic decrease in transcription of Pmc1 Ca(2+) pump. Interestingly, overexpression of Prz1 did not suppress the Cl(-) hypersensitivity of calcineurin deletion, and overexpression of Pmp1 MAPK phosphatase suppressed the Cl(-) hypersensitivity of calcineurin deletion but not the Ca(2+) hypersensitivity of prz1 deletion. In addition, mutations in the its2(+)/cps1(+), its8(+), and its10(+)/cdc7(+) genes that showed synthetic lethal genetic interaction with calcineurin deletion did not exhibit synthetic lethality with the prz1 deletion. Our results suggest that calcineurin activates at least two distinct signaling branches, i.e. the Prz1-dependent transcriptional regulation and an unknown mechanism, which functions antagonistically with the Pmk1 MAPK pathway.

Amino Acid Sequence↗
PubMed2003

Expression of mPOU protein in the human pituitary adenomas.

OBJECTIVE: mPOU is a POU protein classified as class VI. It is present in the pituitary gland as well as the brain, heart muscle, skeletal muscle, lung, and lymphocytes. In our previous investigation, mPOU bound to the Pit-1-binding DNA elements of the rat PRL gene, and promoted transcription of the GH and PRL genes. In this study, we immunohistologically investigated the expression of mPOU in pituitary adenomas. METHODS: 17 patients with pituitary adenoma underwent tumor excision by transsphenoidal approach at our hospital (PRL: 5, GH: 4, FSH: 1, non-functioning: 7). The expression in the tissue sections was investigated using immunostaining (ABC method). RESULTS: In all GH-producing and PRL-producing adenomas, mPOU protein was specifically expressed, particularly in the nuclei. DISCUSSION: Pit-1 has been considered to be a factor determining the expression of GH and PRL genes, but mPOU may also be involved in the expression.

Adenoma↗
PubMed2002

Estrogen levels influence beta-3-adrenoceptor-mediated relaxation of the female rat detrusor muscle.

OBJECTIVES: To observe the expression of beta-3-adrenoceptor in the detrusor muscle in female rats and investigate the relaxant effect of beta-adrenoceptor agonists on detrusor muscle in ovariectomized rats with or without estrogen replacement therapy. METHODS: We first performed reverse transcriptase-polymerase chain reaction to demonstrate mRNA encoding the beta-3-adrenoceptor in the detrusor muscle from female rats. We then performed pharmacologic and physiologic studies to determine the effect of estrogen replacement therapy on the beta-adrenoceptor-mediated relaxation of the detrusor muscle of the ovariectomized rats. RESULTS: Beta-3-adrenoceptor was expressed in the detrusor muscle in female rats with or without ovariectomy. A nonselective beta-adrenoceptor agonist relaxed precontracted detrusor muscle irrespective of ovariectomy or estrogen replacement in a dose-dependent manner; and a selective beta-3-adrenoceptor agonist relaxed the detrusor muscle more in ovariectomized rats than in ovariectomized rats with estrogen replacement or in control rats. CONCLUSIONS: Selective beta-3-adrenoceptor agonists relaxed the detrusor muscle of female rats with low estrogen levels. This result may give a clue to the treatment of frequent urination or incontinence in postmenopausal women who are not receiving hormonal replacement therapy.

Adrenergic beta-Agonists↗
PubMed2002

Calcineurin phosphatase in signal transduction: lessons from fission yeast.

Calcineurin (protein phosphatase 2B), the only serine/threonine phosphatase under the control of Ca2+/calmodulin, is an important mediator in signal transmission, connecting the Ca2+-dependent signalling to a wide variety of cellular responses. Furthermore, calcineurin is specifically inhibited by the immunosuppressant drugs cyclosporin A and tacrolimus (FK506), and these drugs have been a powerful tool for identifying many of the roles of calcineurin. Calcineurin is enriched in the neural tissues, and also distributes broadly in other tissues. The structure of the protein is highly conserved from yeast to man. The combined use of powerful genetics and of specific calcineurin inhibitors in fission yeast Schizosaccharomyces pombe (S. pombe) identified new components of the calcineurin pathway, and defined new roles of calcineurin in the regulation of the many cellular processes. Recent data has revealed functional interactions in which calcineurin phosphatase is involved, such as the cross-talk between the Pmk1 MAP kinase signalling, or the PI signalling. Calcineurin also participates in membrane traffic and cytokinesis of fission yeast through its functional connection with members of the small GTPase Rab/Ypt family, and Type II myosin, respectively. These findings highlight the potential of fission yeast genetic studies to elucidate conserved elements of signal transduction cascades.

Calcineurin↗