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Tatsuji Haneji - One of the best experts on this subject based on the ideXlab platform.
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Calyculin a induces apoptosis and stimulates phosphorylation of p65nf κb in human osteoblastic osteosarcoma mg63 cells
International Journal of Oncology, 2007Co-Authors: Hiroaki Tanaka, Hirohiko Okamura, Kaya Yoshida, Hiroyuki Morimoto, Toshihiko Nagata, Tatsuji HanejiAbstract:: Previously, we reported that okadaic acid, a specific inhibitor of serine/threonine protein phosphatases, induced apoptosis in human osteoblastic cells. However, it is not clear whether Calyculin A, another inhibitor of protein phosphatases, would induce apoptosis in human osteoblastic cells and if so, which mechanisms are involved and whether the phosphorylation status of NF-kappaB could be affected by the treatment with Calyculin A. In this report, we demonstrate that Calyculin A induced apoptosis in MG63 cells, as judged by WST-8 assay, nuclear fragmentation, and DNA ladder formation. Expression of PTEN, FasL, and FasR mRNA was stimulated by Calyculin A treatment in MG63 cells. Calyculin A also enhanced the phosphorylation level of NF-kappaB, as judged from the results of Western blot analysis and an in vitro dephosphorylation assay. Western blot analysis with anti-phospho-p65NF-kappaB antibody disclosed that the NF-kappaB was phosphorylated on serine 536 in cytosol and translocated into nucleus with Calyculin A-treatment. The phosphorylation status of p65NF-kappaB was further confirmed by using the phosphorylation site-mutated p65NF-kappaB gene transfected into HEK293 cells. Unlike TNF-alpha, Calyculin A treatment did not degraded IkappaBalpha within 10 min, while it degraded IkappaBalpha at 2-h treatment. Our findings indicate that Calyculin A elicit phosphorylation of NF-kappaB on serine 536 in MG63 cells, resulting in the translocation of phospho-NF-kappaB to the nucleus, thereby promoting transcriptional activity of NF-kappaB-related genes.
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Calyculin a stimulates the expression of tnf α mrna via phosphorylation of akt in mouse osteoblastic mc3t3 e1 cells
Molecular and Cellular Endocrinology, 2007Co-Authors: Kaya Yoshida, Bruna Rabelo Amorim, Hirohiko Okamura, Tatsuji HanejiAbstract:Abstract Intracellular phosphatase activity has been recognized to play a central role in signal transduction. In the present study, we investigated the effects of Calyculin A, an inhibitor of protein phosphatases, on the expression of TNF-α mRNA and the possible signaling pathways in mouse osteoblastic MC3T3-E1 cells. The result of semiquantitative RT-PCR showed that Calyculin A increased the expression of TNF-α mRNA in MC3T3-E1 cells. Pre-treatment of LY294002 and Wortmannin, inhibitors of PI3K, inhibited the Calyculin A-stimulated TNF-α mRNA expression. Western blot result disclosed that Calyculin A increased the phosphorylation status of Akt at Ser473. However, U0126 and SB203580, specific inhibitor of MEK1/2 and p38MAPK, respectively, had no effect on Calyculin A-stimulated expression of TNF-α mRNA. BAY11-7085 and CAPE, inhibitors of NF-κB activity, did not alter the Calyculin A-stimulated TNF-α mRNA expression. Indirect immunofluorescent study confirmed that NF-κB was not translocated to the nucleus by Calyculin A treatment. Our present results suggest that inhibition of phosphatase activity by Calyculin A stimulate the phosphorylation of Akt at Ser473 by PI3K/Akt signaling pathway, resulting in the expression TNF-α mRNA.
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pten expression elicited by egr 1 transcription factor in Calyculin a induced apoptotic cells
Journal of Cellular Biochemistry, 2005Co-Authors: Hirohiko Okamura, Kaya Yoshida, Hiroyuki Morimoto, Tatsuji HanejiAbstract:PTEN is a tumor suppressor gene encoding a phosphatase that negatively regulates cell survival mediated by the PI3-kinase-Akt pathway. The gene for transcription factor EGR-1 is an early response gene essential for cellular growth, proliferation, and differentiation. Protein phosphatase inhibitors including Calyculin A and okadaic acid are potent inducers of apoptosis in several cell lines; however, the molecular mechanisms underlying their action are unknown. The purpose of this study was to examine the expression of PTEN and EGR-1 and the phosphorylation status of EGR-1 and Akt in Calyculin A-treated human squamous carcinoma cells (SCCTF). Phosphorylation of EGR-1 and upregulation of PTEN expression were observed to occur in SCCTF cells treated with Calyculin A in time- and dose-dependent fashions. The level of phosphorylated Akt decreased as the expression of PTEN protein increased in the Calyculin A-treated SCCTF cells. Calyculin A-stimulated expression of EGR-1 and PTEN might be p53 independent, because the expression of them was also detected in p53-null Saos-2 cells. RNA interference using double-stranded RNA specific for the EGR-1 gene inhibited not only EGR-1 expression but also PTEN expression in SCCTF cells treated or not with Calyculin A. Calyculin A induced nuclear fragmentation and chromatin condensation in SCCTF cells. The present results suggest that the level of PTEN expression and the phosphorylation status of Akt were associated with apoptosis induced by Calyculin A. These observations also support the view that EGR-1 regulates PTEN expression in the initial steps of the apoptotic pathway. © 2004 Wiley-Liss, Inc.
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pten expression elicited by egr 1 transcription factor in Calyculin a induced apoptotic cells
Journal of Cellular Biochemistry, 2005Co-Authors: Hirohiko Okamura, Kaya Yoshida, Hiroyuki Morimoto, Tatsuji HanejiAbstract:PTEN is a tumor suppressor gene encoding a phosphatase that negatively regulates cell survival mediated by the PI3-kinase-Akt pathway. The gene for transcription factor EGR-1 is an early response gene essential for cellular growth, proliferation, and differentiation. Protein phosphatase inhibitors including Calyculin A and okadaic acid are potent inducers of apoptosis in several cell lines; however, the molecular mechanisms underlying their action are unknown. The purpose of this study was to examine the expression of PTEN and EGR-1 and the phosphorylation status of EGR-1 and Akt in Calyculin A-treated human squamous carcinoma cells (SCCTF). Phosphorylation of EGR-1 and upregulation of PTEN expression were observed to occur in SCCTF cells treated with Calyculin A in time- and dose-dependent fashions. The level of phosphorylated Akt decreased as the expression of PTEN protein increased in the Calyculin A-treated SCCTF cells. Calyculin A-stimulated expression of EGR-1 and PTEN might be p53 independent, because the expression of them was also detected in p53-null Saos-2 cells. RNA interference using double-stranded RNA specific for the EGR-1 gene inhibited not only EGR-1 expression but also PTEN expression in SCCTF cells treated or not with Calyculin A. Calyculin A induced nuclear fragmentation and chromatin condensation in SCCTF cells. The present results suggest that the level of PTEN expression and the phosphorylation status of Akt were associated with apoptosis induced by Calyculin A. These observations also support the view that EGR-1 regulates PTEN expression in the initial steps of the apoptotic pathway.
Toshiyuki Wakimoto - One of the best experts on this subject based on the ideXlab platform.
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Calyculin: Nature's way of making the sponge-derived cytotoxin
Natural Product Reports, 2016Co-Authors: Toshiyuki Wakimoto, Yoko EgamiAbstract:Covering: up to 2015. Calyculin A is a major cytotoxic compound isolated from the Japanese marine sponge Discodermia calyx. Its potent cytotoxicity is attributable to the specific inhibition of protein phosphatases 1 and 2A, as in the case of okadaic acid and the microcystins. Its chemical structure is well-designed not only for enzyme inhibition but also for higher membrane permeability in order to impart its potent cytotoxicity. The biosynthetic gene cluster of this densely functionalized polyketide and nonribosomal peptide hybrid molecule was recently identified from the sponge–microbe association. The producer organism and the dynamic bioconversion process were also revealed. In this highlight, we focus on the recent studies addressing nature's design and biogenesis of the sponge-derived cytotoxin, Calyculin A.
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phosphoCalyculin c as a pyrophosphate protoxin of Calyculin c in the marine sponge discodermia calyx
Bioorganic & Medicinal Chemistry Letters, 2014Co-Authors: Yoko Egami, Toshiyuki WakimotoAbstract:Abstract Calyculin C, a minor derivative of the Calyculins, has an additional methyl group on C32 of Calyculin A. A recent biosynthetic study of Calyculins revealed that an end product of Calyculin biosynthesis is the pyrophosphate form, phosphoCalyculin A. However, the pyrophosphate counterpart derived from Calyculin C had not been reported. We isolated phosphoCalyculin C as a minor pyrophosphate derivative, by a detailed investigation of an extract from the sponge Discodermia calyx . The treatment of phosphoCalyculin C with the D. calyx cell-free extract significantly enhanced its cytotoxicity, providing molecular evidence for its role as the protoxin of Calyculin C.
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Calyculin biogenesis from a pyrophosphate protoxin produced by a sponge symbiont
Nature Chemical Biology, 2014Co-Authors: Toshiyuki Wakimoto, Yoko Egami, Yu Nakashima, Yukihiko Wakimoto, Takahiro Mori, Takayoshi Awakawa, Takuya Ito, Hiromichi Kenmoku, Yoshinori Asakawa, Jorn PielAbstract:The Japanese marine sponge Discodermia calyx contains a major cytotoxic compound, Calyculin A, which exhibits selective inhibition of protein phosphatases 1 and 2A. It has long been used as a chemical tool to evaluate intracellular signal transduction regulated by reversible protein phosphorylation. We describe the identification of the biosynthetic gene cluster of Calyculin A by a metagenome mining approach. Single-cell analysis revealed that the gene cluster originates in the symbiont bacterium 'Candidatus Entotheonella' sp. A phosphotransferase encoded in the gene cluster deactivated Calyculin A to produce a newly discovered diphosphate, which was actually the biosynthetic end product. The diphosphate had been previously overlooked because of the enzymatic dephosphorylation that occurred in response to sponge tissue disruption. Our work presents what is to our knowledge the first evidence for the biosynthetic process of Calyculin A along with a notable phosphorylation-dephosphorylation mechanism to regulate toxicity, suggesting activated chemical defense in the most primitive of all multicellular animals.
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allos hemiCalyculin a a photochemically converted Calyculin from the marine sponge discodermia calyx
Tetrahedron Letters, 2013Co-Authors: Miki Kimura, Toshiyuki WakimotoAbstract:Allos-hemiCalyculin A (1), a new derivative of Calyculin A, was isolated from the marine sponge Discodermia calyx collected off Shikine-jima Island, Japan. The structure of 1, including the absolute configurations, was elucidated by spectroscopic analyses and photochemical degradation experiments. Consequently, its structure was identical to the distal end of the peptide side chain of Calyculin A (2), previously isolated from D. calyx and generated by photochemical oxidative cleavage of the oxazole moiety. In stark contrast to the potent cytotoxicity of 2, 1 is no longer cytotoxic, in agreement with the previously reported structure–activity relationship data. Here we describe the isolation and structural elucidation of 1.
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crystal structure of the complex between Calyculin a and the catalytic subunit of protein phosphatase 1
Structure, 2002Co-Authors: Akiko Kita, Hirotaka Kataiwa, Nobuhiro Fusetani, Akira Takai, Shigeki Matsunaga, Toshiyuki Wakimoto, Minoru Isobe, Kunio MikiAbstract:Abstract The crystal structure of the catalytic subunit of the protein phosphatase 1 (PP1), PP1γ, in complex with a marine toxin, Calyculin A, was determined at 2.0 A resolution. The metal binding site contains the phosphate group of Calyculin A and forms a tight network via the hydrophilic interactions between PP1 and Calyculin A. Calyculin A is located in two of the three grooves, namely, in the hydrophobic groove and the acidic groove on the molecular surface. This is the first observation to note that the inhibitor adopts not a pseudocyclic conformation but an extended conformation in order to form a complex with the protein. The amino acid terminus of Calyculin A contributes, in a limited manner, to the binding to PP1γ, which is consistent with findings from the studies of dose-inhibition analysis.
Qing Tian - One of the best experts on this subject based on the ideXlab platform.
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melatonin ameliorates alzheimer like pathological changes and spatial memory retention impairment induced by Calyculin a
Journal of Psychopharmacology, 2011Co-Authors: Xifei Yang, Zhengqi Fu, Qiuyue Zhang, Yin Li, Jing Feng, Ying Yang, Qun Wang, Qing TianAbstract:We have reported recently that inhibition of protein phosphatase (PP)-2A and PP-1 by Calyculin A, a specific inhibitor of PP-2A and PP-1, induced Alzheimer-like hyperphosphorylation of tau and spatial memory retention impairment. In this study, we tested the in vivo effects of melatonin on these Alzheimer-like changes. We found that administration of melatonin intraperitoneally for 9 consecutive days before injection of Calyculin A could prevent Calyculin A-induced synaptophysin loss, memory retention deficits, as well as hyperphosphorylation of tau and neurofilaments. Furthermore, melatonin partially reversed the phosphorylation of the catalytic subunit of PP-2A at Tyrosine 307 (Y307), a crucial site negatively regulating the activity of PP-2A, and reduced the levels of malondialdehyde, a marker of oxidative stress, induced by Calyculin A. These results suggest that melatonin could serve as a potential therapeutic agent for preventing Alzheimer-like pathological changes and behavioral abnormality via mod...
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melatonin ameliorates alzheimer like pathological changes and spatial memory retention impairment induced by Calyculin a
Journal of Psychopharmacology, 2011Co-Authors: Xifei Yang, Qiuyue Zhang, Jing Feng, Ying Yang, Qun Wang, Jinzhuo Luo, Qing TianAbstract:We have reported recently that inhibition of protein phosphatase (PP)-2A and PP-1 by Calyculin A, a specific inhibitor of PP-2A and PP-1, induced Alzheimer-like hyperphosphorylation of tau and spatial memory retention impairment. In this study, we tested the in vivo effects of melatonin on these Alzheimer-like changes. We found that administration of melatonin intraperitoneally for 9 consecutive days before injection of Calyculin A could prevent Calyculin A-induced synaptophysin loss, memory retention deficits, as well as hyperphosphorylation of tau and neurofilaments. Furthermore, melatonin partially reversed the phosphorylation of the catalytic subunit of PP-2A at Tyrosine 307 (Y307), a crucial site negatively regulating the activity of PP-2A, and reduced the levels of malondialdehyde, a marker of oxidative stress, induced by Calyculin A. These results suggest that melatonin could serve as a potential therapeutic agent for preventing Alzheimer-like pathological changes and behavioral abnormality via modulating the activity of PP-2A and oxidative stress.
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berberine attenuates Calyculin a induced cytotoxicity and tau hyperphosphorylation in hek293 cells
Journal of Alzheimer's Disease, 2011Co-Authors: Guang Yu, Jianzhi Wang, Qing Tian, Qun Wang, Yi Li, Xiaochuan WangAbstract:The Chinese herb berberine has versatile health effects. Recent reports indicate that berberine has the potential to prevent and treat Alzheimer's disease (AD). In the present study, we employed tau-expressing HEK293 cells (HEK293/tau) treated with Calyculin-A as a cellular model to investigate the roles of berberine in cell viability, tau phosphorylation, and oxidative stress. We found a significant reduction of Calyculin A-induced tau hyperphosphorylation at Ser198/199/202, Ser396, Ser404, Thr205, and Thr231 24 h after treatment with 20g/ml berberine. Berberine also restored protein phosphatase 2A activity and reversed glycogen synthase kinase-3 (GSK-3) activation, as determined by phosphatase activity assay and GSK- 3 phosphorylation at Tyr216 and Ser9, respectively. Furthermore, berberine reversed both the increase of malondialdehyde and the decrease of superoxide dismutase activity induced by Calyculin A, indicating its role in anti-oxidative stress. Our findings suggest that berberine may be a potential therapeutic drug for AD.
Hiroshi Harayama - One of the best experts on this subject based on the ideXlab platform.
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identification of isoforms of Calyculin a sensitive protein phosphatases which suppress full type hyperactivation in bull ejaculated spermatozoa
Theriogenology, 2019Co-Authors: Yuka Arai, Mitsuhiro Sakase, Moriyuki Fukushima, Hiroshi HarayamaAbstract:Abstract In bull spermatozoa, extracellular Ca2+-dependent full-type hyperactivation, which is characterized by the asymmetrical beating in whole parts of the middle/principal pieces, is suppressed by Calyculin A-sensitive protein phosphatases. The aim of this study was to identify isoforms of these protein phosphatases. Ejaculated spermatozoa were used for the investigation on effects of protein phosphatase inhibitors (Calyculin A with high specificity for both of protein phosphatases 1 and 2A, and okadaic acid with relatively higher specificity for protein phosphatase 2A than protein phosphatase 1) on the induction of extracellular Ca2+-dependent full-type hyperactivation by incubation with CaCl2 and cAMP analog (cBiMPS). They were also used for the immunodetection of protein phosphatases 1α, 1β, 1γ, 2Aα and 2Aβ. Percentages of full-type hyperactivated spermatozoa significantly increased after incubation with Calyculin A (10 nM) in a concentration-dependent manner of CaCl2 (0–3.42 mM), though only minor increases in the percentages of full-type hyperactivated spermatozoa were observed after incubation with okadaic acid (10 nM). Moreover, the immunodetection of protein phosphatase isoforms showed sperm connecting piece and flagellum included protein phosphatases 1α and 1γ, but did not do the other isoforms. These results suggest that Calyculin A-sensitive and okadaic acid-less sensitive protein phosphatases (1α and 1γ) are suppressors for the extracellular Ca2+-dependent full-type hyperactivation in bull ejaculated spermatozoa.
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distinct segment specific functions of Calyculin a sensitive protein phosphatases in the regulation of camp triggered events in ejaculated bull spermatozoa
Molecular Reproduction and Development, 2015Co-Authors: Yohei Mizuno, Mitsuhiro Sakase, Ayane Isono, Aya Kojima, Miyuki M Arai, Taichi Noda, Moriyuki Fukushima, Hiroshi HarayamaAbstract:SUMMARY Livestock spermatozoa possess more tenacious suppressors of cAMP-triggered events—including capacitation-associated changes—than laboratory animal spermatozoa, leading to flagellar hyperactivation. In order to identify the suppressors, we examined effects of an inhibitor of serine/threonine protein phosphatases (Calyculin A) on cAMP-triggered changes in the protein phosphorylation state, and subsequent occurrence of hyperactivation and acrosome reaction in ejaculated bull spermatozoa. Ejaculated spermatozoa were incubated in cAMP-supplemented medium, then assessed for motility, acrosome morphology, and phosphorylated protein localization. The addition of Calyculin A greatly enhanced cAMP-triggered protein phosphorylation at serine/threonine and tyrosine residues in the connecting piece and induction of flagellar hyperactivation. Most hyperactivated spermatozoa exhibited extremely asymmetrical bends at the middle piece, which produced intensive twisting or figure-eight movements. In the sperm head, however, cAMP-triggered dephosphorylation of serine/threonine-phosphorylated proteins and subsequent acrosome reaction were abolished by the addition of Calyculin A. Based on these results, we suggest that Calyculin A-sensitive protein phosphatases in the connecting piece are suppressors of cAMP-triggered events leading to hyperactivation. By contrast, similar protein phosphatases in the sperm head accelerate cAMP-triggered events leading to the acrosome reaction. These findings are consistent with the indication that Calyculin A-sensitive protein phosphatases have distinct functions in the regulation of cAMP-triggered events in different regions of ejaculated bull spermatozoa. Mol. Reprod. Dev. 82: 232–250, 2015. © 2015 Wiley Periodicals, Inc.
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distinct segment specific functions of Calyculin a sensitive protein phosphatases in the regulation of camp triggered events in ejaculated bull spermatozoa
Molecular Reproduction and Development, 2015Co-Authors: Yohei Mizuno, Mitsuhiro Sakase, Ayane Isono, Aya Kojima, Miyuki M Arai, Taichi Noda, Moriyuki Fukushima, Hiroshi HarayamaAbstract:Livestock spermatozoa possess more tenacious suppressors of cAMP-triggered events-including capacitation-associated changes-than laboratory animal spermatozoa, leading to flagellar hyperactivation. In order to identify the suppressors, we examined effects of an inhibitor of serine/threonine protein phosphatases (Calyculin A) on cAMP-triggered changes in the protein phosphorylation state, and subsequent occurrence of hyperactivation and acrosome reaction in ejaculated bull spermatozoa. Ejaculated spermatozoa were incubated in cAMP-supplemented medium, then assessed for motility, acrosome morphology, and phosphorylated protein localization. The addition of Calyculin A greatly enhanced cAMP-triggered protein phosphorylation at serine/threonine and tyrosine residues in the connecting piece and induction of flagellar hyperactivation. Most hyperactivated spermatozoa exhibited extremely asymmetrical bends at the middle piece, which produced intensive twisting or figure-eight movements. In the sperm head, however, cAMP-triggered dephosphorylation of serine/threonine-phosphorylated proteins and subsequent acrosome reaction were abolished by the addition of Calyculin A. Based on these results, we suggest that Calyculin A-sensitive protein phosphatases in the connecting piece are suppressors of cAMP-triggered events leading to hyperactivation. By contrast, similar protein phosphatases in the sperm head accelerate cAMP-triggered events leading to the acrosome reaction. These findings are consistent with the indication that Calyculin A-sensitive protein phosphatases have distinct functions in the regulation of cAMP-triggered events in different regions of ejaculated bull spermatozoa.
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Calyculin a sensitive protein phosphatases are involved in maintenance of progressive movement in mouse spermatozoa in vitro by suppression of autophosphorylation of protein kinase a
Journal of Reproduction and Development, 2009Co-Authors: Namiko Goto, Hiroshi HarayamaAbstract:Protein serine/threonine phosphorylation in mammalian sperm flagella has been considered to play important roles in regulation of motility. Protein phosphorylation state reflects balance of enzymatic activities between protein phosphatases and protein kinases [predominantly protein kinase A (PKA)]. The aims of this study were to disclose roles of protein phosphatases in the regulation of sperm motility and to provide evidence for suppression of PKA full activation by protein phosphatases in sperm flagella. Mouse epididymal spermatozoa were incubated with a cell-permeable protein phosphatase 1 (PP1)/protein phosphatase 2A (PP2A) inhibitor (Calyculin A: 25-125 nM) at 37.5 C. After incubation, they were used for immunodetection of phosphorylated proteins, PKA and PP1 γ2, assessment for motility and co-immunoprecipitation of PP1γ2 with PKA. Incubation with Calyculin A enhanced the phosphorylation states of several proteins (>250 kDa, 170 kDa, 155 kDa, 140 kDa and 42 kDa for serine/threonine phosphorylation and 70 kDa for tyrosine phosphorylation) and PKA catalytic subunits [at the autophosphorylation residue (Thr-197) for its full enzymatic activation] in the flagella. Coincidently, this incubation induced changes of sperm flagellar movement from the progressive type to the hyperactivation-like type. Indirect immunofluorescence and co-immunoprecipitation showed that PKA was co-localized with PP1 γ2 in the principal pieces of sperm flagella. These findings suggest that Calyculin A-sensitive protein phosphatases (PP1/PP2A) suppress full activation of PKA as well as enhancement of the phosphorylation states of other flagellar proteins in sperm flagella in order to prevent precocious changes of flagellar movement from the progressive type to hyperactivation.
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effects of protein phosphatase inhibitor Calyculin a on the postacrosomal protein serine threonine phosphorylation state and acrosome reaction in boar spermatozoa incubated with a camp analog
Journal of Reproduction and Development, 2008Co-Authors: Jun Adachi, Masashi Miyake, Shunsuke Tate, Hiroshi HarayamaAbstract:In order to reveal the involvement of the sperm postacrosomal region in the acrosome reaction, we examined the effects of the protein phosphatase inhibitor Calyculin A on the postacrosomal protein serine/threonine phosphorylation state and acrosome morphology in boar spermatozoa incubated with a cAMP analog. Proteins were highly phosphorylated on the serine/threonine residues only in the postacrosomal region before incubation. After 90-min incubation without Calyculin A, the protein phosphorylation state declined in the postacrosomal region irrespective of the capacitation state while it remained under the detectable level in the other regions of the sperm head. However, addition of Calyculin A effectively suppressed the decline in protein phosphorylation state and increased an inactive form of protein phosphatase 1 in the postacrosomal region. On the other hand, this inhibitor had no influence on the protein phosphorylation state in the acrosome and equatorial segment. After incubation without Calyculin A for 180 or 360 min, many spermatozoa exhibited acrosomal changes and loss that indicated occurrence of the acrosome reaction. However, addition of Calyculin A significantly blocked these events. These results are consistent with our suggestion that postacrosomal serine/threonine-phosphorylated proteins are involved in suppression of the acrosome reaction in boar spermatozoa in vitro.
Nobuhiro Fusetani - One of the best experts on this subject based on the ideXlab platform.
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Calyculin a causes the activation of histone h1 kinase and condensation of chromosomes in unfertilized sea urchin eggs independently of the maturation promoting factor
Comparative Biochemistry and Physiology C-toxicology & Pharmacology, 2003Co-Authors: H Tosuji, Nobuhiro Fusetani, Yukari SekiAbstract:Abstract Calyculin A is known to inhibit the type-1 and type-2A phosphatases. We previously reported that Calyculin A induces contractile ring formation in unfertilized sea urchin eggs, an increase in histone H1 kinase activity, and chromosome condensation in the Calyculin A-treated unfertilized eggs, and the changes induced by Calyculin A are not affected by emetine, an inhibitor of protein synthesis. These observations suggest that the mechanism by which histone H1 kinases are activated by Calyculin A is different from that of maturation-promoting factor (MPF), which is activated by a molecular modification of existed cdc2 and newly synthesized cyclin B. We report here that no cyclin B was detected by immunoblotting of unfertilized Calyculin A-treated eggs. In addition, no DNA synthesis was induced by Calyculin A. As well, butyrolactone I (an inhibitor of cdc2 and cdk2 kinase) had no effect on the increase in histone H1 kinase activity nor the chromosome condensation, both of which were induced by Calyculin A. Thus, we conclude that Calyculin A induces histone H1 phosphorylation in an MPF-independent manner through inhibition of type-1 phosphatase, and that the chromosome condenses as a result of histone H1 phosphorylation.
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crystal structure of the complex between Calyculin a and the catalytic subunit of protein phosphatase 1
Structure, 2002Co-Authors: Akiko Kita, Hirotaka Kataiwa, Nobuhiro Fusetani, Akira Takai, Shigeki Matsunaga, Toshiyuki Wakimoto, Minoru Isobe, Kunio MikiAbstract:Abstract The crystal structure of the catalytic subunit of the protein phosphatase 1 (PP1), PP1γ, in complex with a marine toxin, Calyculin A, was determined at 2.0 A resolution. The metal binding site contains the phosphate group of Calyculin A and forms a tight network via the hydrophilic interactions between PP1 and Calyculin A. Calyculin A is located in two of the three grooves, namely, in the hydrophobic groove and the acidic groove on the molecular surface. This is the first observation to note that the inhibitor adopts not a pseudocyclic conformation but an extended conformation in order to form a complex with the protein. The amino acid terminus of Calyculin A contributes, in a limited manner, to the binding to PP1γ, which is consistent with findings from the studies of dose-inhibition analysis.
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insight into binding of Calyculin a to protein phosphatase 1 isolation of hemiCalyculin a and chemical transformation of Calyculin a
Chemistry & Biology, 2002Co-Authors: Toshiyuki Wakimoto, Shigeki Matsunaga, Akira Takai, Nobuhiro FusetaniAbstract:Calyculin A isolated from the marine sponge Discodermia calyx is a potent inhibitor of protein phosphatases 1 and 2A. We attempted to elucidate its mode of binding to the enzymes by examining the activity of natural and chemically transformed derivatives. Ten natural derivatives including a new compound, hemiCalyculin A, were provided. The structure of hemiCalyculin A, which comprises the southern hemisphere of Calyculin A, was firmly established by chemical methods. Six compounds were prepared by selective modifications of functional groups in Calyculin A. The enzyme inhibitory activity of these compounds indicated that 17-phosphate, 13-hydroxyl, and the hydrophobic tetraene moieties were all necessary for binding to the enzymes. The derivatives lacking the peptide portion were less cytotoxic even when they possessed full enzyme inhibitory activity.
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the solution structures of Calyculin a and dephosphonoCalyculin a by nmr
Bioorganic & Medicinal Chemistry Letters, 1999Co-Authors: Katie E Volter, Ronald J Quinn, Gregory K. Pierens, Shigeki Matsunaga, Toshiyuki Wakimoto, Nobuhiro FusetaniAbstract:Abstract The NMR solution structure of Calyculin A (1) in chloroform exhibits intramolecular interactions, resembling the original crystal structure1. In methanol, Calyculin A has the hydrogen bonding moieties solvent exposed. DephosphonoCalyculin A in chloroform resembles Calyculin A in chloroform and the crystal structure of Calyculin A. DephosphonoCalyculin A in methanol resembles Calyculin A in methanol.
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Calyculin a induces contractile ring like apparatus formation and condensation of chromosomes in unfertilized sea urchin eggs
Proceedings of the National Academy of Sciences of the United States of America, 1992Co-Authors: H Tosuji, Issei Mabuchi, Nobuhiro Fusetani, Tohru NakazawaAbstract:Abstract Calyculin A, a protein phosphatase inhibitor, induced cleavage-like morphological change in unfertilized sea urchin eggs. A contractile ring-like apparatus containing both filamentous actin and myosin was formed in the cleavage furrow. Wheat germ agglutinin receptors were also found in the same region. The eggs did not develop further after constriction of the ring. No aster-like microtubular structure was found in the Calyculin A-treated eggs. The cleavage was not inhibited by the antimicrotubule drug griseofulvin. Calyculin A also increased histone H1 kinase activity and induced chromosome condensation. These changes also occurred in the presence of emetine (an inhibitor of protein synthesis) and aphidicolin (an inhibitor of DNA synthesis). It is suggested that Calyculin A induced these changes in the sea urchin eggs by inhibiting the activity of protein phosphatase 1.
