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ABSTRACT: Dr Tsuiki made three major contributions during his illustrious career as a biochemist. First, he developed the procedure for mucin isolation from bovine submaxillary glands. His work became the basis for mucin biochemistry. Second, he identified four distinct molecular species of mammalian sialidase. Subsequent studies based on his work led to the discovery that sialidase plays a unique role as an intracellular signalling factor involved in the regulation of a variety of cellular functions. Finally, he established the molecular basis for the diversity of mammalian protein phosphatases through protein purification and molecular cloning. His work prompted the functional studies of protein phosphatases.
Journal of biochemistry 11/2011; 150(5):483-90. · 1.95 Impact Factor
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ABSTRACT: MAPK phosphatase-7 (MKP-7) was identified as a JNK-specific phosphatase. However, despite its high specificity for JNK, MKP-7 interacts also with ERK. We previously showed that as a physiological consequence of their interaction, activated ERK phosphorylates MKP-7 at Ser-446, and stabilizing MKP-7. In the present study, we analyzed MKP-7 function in activation of ERK. A time-course experiment showed that both MKP-7 and its phosphatase-dead mutant prolonged mitogen-induced ERK phosphorylation, suggesting that MKP-7 functions as a scaffold for ERK. An important immunohistological finding was that nuclear translocation of phospho-ERK following PMA stimulation was blocked by co-expressed MKP-7 and, moreover, that phospho-ERK co-localized with MKP-7 in the cytoplasm. Reporter gene analysis indicated that MKP-7 blocks ERK-mediated transcription. Overall, our data indicate that MKP-7 down-regulates ERK-dependent gene expression by blocking nuclear accumulation of phospho-ERK.
Biochemical and Biophysical Research Communications 03/2010; 393(2):201-6. · 2.48 Impact Factor
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ABSTRACT: TNFalpha activated NF-kappaB and associated regulatory factors including IKK are strongly implicated in a variety of hematological and solid tumor malignancies. We show that tautomycetin (TC) specifically inhibits activation of NF-kappaB among the three TNFalpha effectors (NF-kappaB, JNK and caspase). TC inhibited T-loop phosphorylation of IKKalpha and IKKbeta, thereby preventing degradation of the NF-kappaB inhibitor, IkappaBalpha. Co-immunoprecipitation experiments revealed that the catalytic subunit of PP1 (PP1C) was involved in the IKK complex. Pull-down analysis using recombinant GST-TNFalpha, showed that PP1C was recruited to TNFR1 together with IKK complex, RIP and TAK1 upon stimulus. These results suggest that the PP1 positively regulates the TNFalpha-induced NF-kappaB pathway at the level of IKK activation. Thus, TC might be used therapeutically to suppress the TNFalpha/NF-kappaB pathway.
International Journal of Oncology 12/2008; 33(5):1027-35. · 2.40 Impact Factor
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Nobuhiro Tanuma,
Sei-Eun Kim,
Monique Beullens,
Yao Tsubaki,
Shinya Mitsuhashi,
Miyuki Nomura,
Takeshi Kawamura,
Kyoichi Isono,
Haruhiko Koseki,
Masami Sato,
Mathieu Bollen, Kunimi Kikuchi,
Hiroshi Shima
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ABSTRACT: Pre-mRNA splicing entails reversible phosphorylation of spliceosomal proteins. Recent work has revealed essential roles for Ser/Thr phosphatases, such as protein phosphatase-1 (PP1), in splicing, but how these phosphatases are regulated is largely unknown. We show that nuclear inhibitor of PP1 (NIPP1), a major PP1 interactor in the vertebrate nucleus, recruits PP1 to Sap155 (spliceosome-associated protein 155), an essential component of U2 small nuclear ribonucleoprotein particles, and promotes Sap155 dephosphorylation. C-terminally truncated NIPP1 (NIPP1-DeltaC) formed a hyper-active holoenzyme with PP1, rendering PP1 minimally phosphorylated on an inhibitory site. Forced expression of NIPP1-WT and -DeltaC resulted in slight and severe decreases in Sap155 hyperphosphorylation, respectively, and the latter was accompanied with inhibition of splicing. PP1 overexpression produced similar effects, whereas small interfering RNA-mediated NIPP1 knockdown enhanced Sap155 hyperphosphorylation upon okadaic acid treatment. NIPP1 did not inhibit but rather stimulated Sap155 dephosphorylation by PP1 in vitro through facilitating Sap155/PP1 interaction. Further analysis revealed that NIPP1 specifically recognizes hyperphosphorylated Sap155 thorough its Forkhead-associated domain and dissociates from Sap155 after dephosphorylation by associated PP1. Thus NIPP1 works as a molecular sensor for PP1 to recognize phosphorylated Sap155.
Journal of Biological Chemistry 11/2008; 283(51):35805-14. · 4.77 Impact Factor
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Journal of Biomolecular NMR 04/2008; 40(3):219-24. · 3.61 Impact Factor
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ABSTRACT: Dual-specificity phosphatases (DSPs), which dephosphorylate proteins at Ser/Thr as well as Tyr residues, are thought to be involved in critical signaling events such as control of MAP kinases (MAPKs). We have isolated the cDNA for a novel DSP and termed it low molecular mass DSP-4 (LDP-4). LDP-4 is composed of 211 amino acids with a predicted molecular mass of 23.9-kDa. Northern blot analysis using various mouse tissues showed that the LDP-4 transcript was expressed exclusively in brain. In situ hybridization showed that brain expression of LDP-4 was ubiquitous except for the hippocampus. When expressed in COS-7 cells, FLAG-tagged LDP-4 protein was present within the nucleus and Golgi apparatus. LDP-4 expression did not reduce phosphorylation levels of MAPKs, but rather evoked activation of JNK and p38.
Molecular and Cellular Biochemistry 03/2007; 296(1-2):177-84. · 2.06 Impact Factor
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ABSTRACT: In astrocytes, the PGF(2alpha) or ionomycin treatment induces the phosphorylation at Ser38 and Ser82 of vimentin, a type III intermediate filament, by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). We found here that vimentin phospho-Ser82 was dephosphorylated much slower than phospho-Ser38. Vimentin phospho-Ser38 was dephosphorylated quickly by purified PP1 catalytic subunit (PP1c) in vitro, whereas phospho-Ser82 was insensitive to PP1c. Because PP1c directly bound to vimentin through a VxF motif (Val83-Asp84-Phe85), the PP1c active site appeared to be unable to approach phospho-Ser82, leading to the prolongation of the phosphorylation at Ser-82. In astrocytes, PP1calpha was in vivo associated with vimentin filaments. The repetitive treatment by ionomycin at a short interval resulted in the sustained elevation of Ser82 phosphorylation, leading to the marked disassembly of vimentin filaments. Taken together, these results suggest that vimentin is a novel member of binding partner of PP1c in astrocytes, and vimentin-Ser82 may act as a memory phosphorylation site.
Genes to Cells 06/2006; 11(5):531-40. · 2.68 Impact Factor
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Kunimi Kikuchi
Seikagaku. The Journal of Japanese Biochemical Society 11/2005; 77(10):1247-54. · 0.04 Impact Factor
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ABSTRACT: In this study, we focus on the potential interaction of pp32/I-1(PP2A) (pp32) with the Raf-MEK-ERK signaling pathway. We show that overexpressed pp32 suppresses Raf-1 activation, thereby downregulating the level of ERK activation. This suppression of Raf-1 requires the C-terminal half of pp32. Conversely, knock-down of PP32 by siRNA enhances ERK and MEK activations. Taken together, we propose that tumor-suppression by pp32 is, at least in part, mediated by downregulation of the Raf-MEK-ERK signaling pathway.
Cancer Letters 09/2005; 226(2):155-60. · 4.24 Impact Factor
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ABSTRACT: The mRNA levels of three phosphoseryl/phosphothreonyl protein phosphatases, PP1, PP2A and PP2C, in rat liver have been determined by Northern blot analysis in various stages of rat chemical hepatocarcinogenesis using a Solt-Farber model. Five weeks after administration of diethyl-nitrosamine, the mRNA levels of PPlα, PP2A and PP2C were elevated 8, 29 and 11 times, respectively, as compared to those of the control livers. However, in primary hepatoma induced according to the Solt-Farber model, the mRNA levels of all three protein phosphatases were dramatically decreased to normal levels or even to much lower levels, whereas the mRNA level of glutathione S-transferase placental form, a tumor marker protein, was greatly elevated as compared with that of the control livers. In a poorly differentiated hepatoma AH13, a line of rat ascites hepatoma, the mRNA level of PP1α was 5.6 times higher than that of the control livers, whereas the mRNA level of PP2C was almost the same as that of the control livers and the level of PP2A mRNA was distinctly lower than that of the control livers. These data appear to suggest some involvement of protein phosphatases in hepatocarcinogenesis.
Cancer Science 08/2005; 83(1):66 - 71. · 3.33 Impact Factor
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ABSTRACT: The amounts of four isoforms of the catalytic subunit of type-1 protein phosphatases, PP1α, PP1γ1, PP1γ2, and PP1δ, have been determined in extracts of rat ascites hepatomas, AH131A, AH13, AH13NMOR, AH143A, and Yoshida sarcoma, and compared to those of rat liver by Western blot analysis. The amount of PP1α was increased over three times in all five hepatomas. The amount of PP1γ1 was increased over two times in AH13, AH13NMOR, and AH143A. The amount of PP1δ was selectively increased about 4 times in AH131A and AH143A. The PP1γ2 protein was undetectable in both liver and hepatomas. There was good parallelism between the general increase in only PP1α protein in the hepatomas and the previous data demonstrating the general increase in PP1α mRNA in numerous ascites hepatomas. These results suggest that PP1α plays important roles in the expression of malignant phenotype, that its amount is under strict control at the transcription level, and that PP1γ1 and PP1δ play different roles in the expression of some phenotype(s) of the ascites hepatomas.
Cancer Science 08/2005; 85(3):274 - 278. · 3.33 Impact Factor
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ABSTRACT: MAPK cascades can be negatively regulated by members of the MAPK phosphatase (MKP) family. However, how MKP activity is regulated is not well characterized. MKP-7, a JNK-specific phosphatase, possesses a unique COOH-terminal stretch (CTS) in addition to domains conserved among MKP family members. The CTS contains several motifs such as a nuclear localization signal, a nuclear export signal, PEST sequences, and a serine residue (Ser-446) that can be phosphorylated by activated ERK, suggesting an important regulatory role(s).(35)S-pulse labeling experiments indicate that the half-life of MKP-7 is 1.5 h, a period significantly elongated by deleting the CTS. We also show that overexpressed MKP-7 is polyubiquitinated when co-expressed with ubiquitin and that proteasome inhibitors markedly inhibit MKP-7 degradation. We also determined that MKP-7 phosphorylated at Ser-446 has a longer half-life than unphosphorylated form of the wild type protein, as does a phospho-mimic mutant of MKP-7. These results indicate that activation of the ERK pathway strongly blocks JNK activation through stabilization of MKP-7 mediated by phosphorylation.
Journal of Biological Chemistry 05/2005; 280(15):14716-22. · 4.77 Impact Factor
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ABSTRACT: I-2PP2A/SET, the translocation breakpoint-encoded protein expressed in acute undifferentiated leukemia, was identified as an inhibitor of protein phosphatase 2A (PP2A). Induction of exogenous I-2PP2A/SET at a ratio of 1:1 to the endogenous protein resulted in suppression of cell proliferation. In contrast, siRNA-mediated depletion of I-2PP2A/SET resulted in enhanced cell proliferation. Depletion of I-2PP2A/SET was accompanied with a decrease in the number of cells in G1 and an increase in cells in S phase. To examine the mode of action by which I-2PP2A/SET suppresses cell proliferation, we determined the effect of over-expressed I-2PP2A/SET on ERK activation. I-2PP2A/SET suppressed activation of ERK following EGF stimulation but did not affect activation levels of stress kinases, JNK and p38. By contrast, knocking down I-2PP2A/SET by siRNA resulted in enhancement of ERK and MEK activations, suggesting that I-2PP2A/SET negatively regulates MEK/ERK. These data suggest that I-2PP2A/SET negatively regulates cell growth by inhibiting the G1/S transition and inhibiting the MEK/ERK pathway stimulated by external stimuli. These data demonstrate that I-2PP2A/SET potentially functions as a tumor suppressor.
International Journal of Oncology 04/2005; 26(3):751-6. · 2.40 Impact Factor
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ABSTRACT: The interleukin-6 (IL-6) stimulates growth in cells such as multiple myeloma and B-cell plasmacytomas/hybridomas, while it inhibits growth in several myeloid leukemia cells. The IL-6 receptor has subunit called gp130. It was reported that Ser-782 of gp130 is phosphorylated by unidentified kinase(s) in cell extracts, and level of gp130 (S782A) transiently expressed on the cell surface of COS-7 is 6-times higher than that of the wild type. These results motivated us to analyze whether the phosphorylation of gp130 at Ser-782 is involved in its degradation or not. In this study, we demonstrated here that treatment of HepG2 cells with okadaic acid (OA), a potent inhibitor for PP2A, promotes phosphorylation of gp 130 at Ser-782 and degradation of gp 130. MG115, a proteasome inhibitor, suppressed this degradation. These effects of OA could not be replaced with tautomycetin (TC), an inhibitor for PP1. Purified PP2A dephosphorylated phospho-Ser-782 of gp130 in vitro. IL-6-induced activation of Stat3 was suppressed by preincubation of the cells with OA, suggesting that the IL-6 signaling pathway was blocked by OA through degradation of gp 130. Taken together, present results strongly suggest that degradation of gp 130 is regulated through a phosphorylation-dephosphorylation mechanism in which PP2A is crucially involved and that gp 130 is a potential therapeutic target in cancers.
Molecular and Cellular Biochemistry 02/2005; 269(1-2):183-7. · 2.06 Impact Factor
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ABSTRACT: Protein phosphatase type 1 (PP1) is involved in the regulation of numerous cell functions in mammalian cells. The major isoforms of PP1 catalytic subunit (PP1C)alpha, gamma1 and delta have nearly identical catalytic domain, but they vary in sequences at the amino and carboxyl termini. We previously showed that PP1Calpha is highly expressed in rat hepatoma cells. To examine isoform specific function of PP1C, each isoform was depleted from HeLa cells by RNA interference. The PP1Calpha-depleted cells rounded up and showed increased cell death, indicating that PP1Calpha is essential in cell proliferation. PP1Cgamma1-depleted cells slightly rounded up and have decreased G1 phase population and increased S phase population. The PP1Cdelta-depleted cells were enlarged, and appeared flat and rich in lamellipodia. These data suggested that each PP1C isoform has non-redundant function in vivo.
International Journal of Oncology 12/2004; 25(5):1383-8. · 2.40 Impact Factor
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ABSTRACT: We have isolated a mouse cDNA for a novel dual-specificity phosphatase designated LDP-3 (low-molecular-mass dual-specificity phosphatase 3). The 450 bp open reading frame encodes a protein of 150 amino acids with a predicted molecular mass of 16 kDa. Northern blot and reverse transcription-PCR analyses show that LDP-3 transcripts are expressed in almost all mouse tissues examined. In vitro analyses using several substrates and inhibitors indicate that LDP-3 possesses intrinsic dual-specificity phosphatase activity. When expressed in mammalian cells, LDP-3 protein is localized mainly to the apical submembrane area. Forced expression of LDP-3 does not alter activation of ERK (extracellular-signal-regulated kinase), but rather enhances activation of JNK (c-Jun N-terminal kinase) and p38 and their respective upstream kinases MKK4 (mitogen-activated protein kinase kinase 4) and MKK6 in cells treated with 0.4 M sorbitol. By screening with a variety of stimuli, we found that LDP-3 specifically enhances the osmotic stress-induced activation of JNK and p38.
Biochemical Journal 12/2004; 383(Pt. 3):447-55. · 4.90 Impact Factor
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ABSTRACT: Eight new dimeric bromopyrrole alkaloids, nagelamides A-H (1-8) and a monomeric one, 9,10-dihydrokeramadine (9), have been isolated from the Okinawan marine sponge Agelas sp., and the structures were elucidated from spectroscopic data. Nagelamides A-H (1-8) exhibited antibacterial activity against Gram-positive bacteria. Nagelamide G (7) inhibited protein phosphatase 2A activity.
Journal of Natural Products 09/2004; 67(8):1262-7. · 3.13 Impact Factor
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ABSTRACT: It is thought that the nuclear nonchromatin structures, such as the nuclear matrix and lamina, play regulatory roles in gene expression. In this study, we identified an insoluble protein that was associated with the chromatin-depleted nuclear structure of proliferating human leukemia HL-60 cells. Preparation of the chromatin-depleted nuclear structure, referred to as the nuclear matrix-intermediate filament scaffold (Fey, E., Krochmalnic, G., and Penman, S. (1986) J. Cell. Biol. 102, 1654-1665), involved cell extraction using a series of buffers containing Triton X-100, DNase I, and 2 M NaCl. A yeast two-hybrid assay revealed that this protein bound to the catalytic subunit of protein phosphatase-1 (PP1). Furthermore, it inhibited PP1 activity in vitro. We therefore named it scapinin (scaffold-associated PP1 inhibiting protein). cDNA cloning revealed that scapinin had two splicing variants of 448 amino acids (scapinin-S) and 518 amino acids (scapinin-L). Scapinin was down-regulated by differentiation in HL-60 cells. These results suggest that scapinin is a putative regulatory subunit of PP1 and is involved in transformed or immature phenotypes of HL-60 cells. We also describe the presence of scapinin family proteins from worm to human.
Journal of Biological Chemistry 12/2003; 278(46):45611-9. · 4.77 Impact Factor
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ABSTRACT: It has been shown that calcineurin (CN), a serine/threonine protein phosphatase type 2B (PP2B), plays an important role in the development and diseases of cardiac muscles. However, reports on CN activity in dilated cardiomyopathy (DCM) are inconsistent, since there are few good disease models and the measurement of the amount of CN is difficult. Previously, we developed a novel line of DCM hamster, J2N-k, and its healthy control counterpart, J2N-n, by crossbreeding cardiomyopathy (CM) hamsters, Bio 14.6, and Golden hamsters followed by consecutive sib mating. In this study, we identified the DCM-causative gene in J2N-k by analysis of F2 of these two lines, and then we analyzed the change in CN gene expression in the course of the disease, and the change in CN activity using a newly developed method. We show that: (i) the DCM gene of J2N-k hamster is the delta- sarcoglycan (SG) gene, (ii) CN expression and potential CN activities (CN activity fully activated with Ca(2+) and calmodulin) in the hearts of J2N-k and J2N-n hamsters are the same levels, (iii) transcription levels of natriuretic peptides, which are augmented by activation of Ca(2+)/calmodulin-dependent enzyme including CN, are significantly increased in the DCM stage in J2N-k hamster. J2N-k and J2N-n hamsters will be a useful tool for studying the pathogenesis, therapy, and prevention of human DCM. Although the total amount and potential activity of CN did not change in the cell extracts, targets of CN in vivo were activated in cardiomyocytes of DCM, suggesting that CN activity in the cells is activated by the raising of Ca(2+) concentration in cardiomyocytes of DCM, which is caused by the defect in the delta-SG gene. Our results reveal the complexity of CN regulation in the heart and indicate the need for additional experimentation.
Journal of Biochemistry 09/2003; 134(2):269-76. · 2.37 Impact Factor
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ABSTRACT: We previously showed that MKP-7 suppresses MAPK activation in COS-7 cells in the order of selectivity, JNK > p38 > ERK, but interacts with ERK as well as JNK and p38. In this study we found that, when expressed in COS-7 cells with HA-ERK2, the mobility of FLAG-MKP-7 was decreased on SDS-PAGE gels depending on several stimuli, including phorbol 12-myristate 13-acetate, fetal bovine serum, epidermal growth factor, H2O2, and ionomycin. By using U0126, a MEK inhibitor, and introducing several point mutations, we demonstrated that this upward mobility shift is because of phosphorylation and identified Ser-446 of MKP-7 as the phosphorylation site targeted by ERK activation. To determine how MKP-7 interacts with MAPKs, we identified three domains in MKP-7 required for interaction with MAPKs, namely, putative MAP kinase docking domains (D-domain) I and II and a long COOH-terminal stretch unique to MKP-7. The D-domain I is required for interaction with ERK and p38, whereas the D-domain II is required for interaction with JNK and p38, which is likely to be important for MKP-7 to suppress JNK and p38 activations. The COOH-terminal stretch of MKP-7 was shown to determine JNK preference for MKP-7 by masking MKP-7 activity toward p38 and is a domain bound by ERK. These data strongly suggested that Ser-446 of MKP-7 is phosphorylated by ERK.
Journal of Biological Chemistry 08/2003; 278(34):32448-56. · 4.77 Impact Factor
