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Naohito Kobayashi,
Hiroji Uemura,
Kiyotaka Nagahama,
Koji Okudela,
Mitsuko Furuya,
Yoko Ino,
Yusuke Ito, Hisashi Hirano,
Yoshiaki Inayama,
Ichiro Aoki,
Yoji Nagashima,
Yoshinobu Kubota,
Hitoshi Ishiguro
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ABSTRACT: Prostate cancer (PCa) is the most common malignant carcinoma that develops in men in Western countries. MicroRNA (miRNA) have the potential to be used as biomarkers and therapeutic targets for the treatment of various cancers. We found significantly higher expression of miR-30d in 3 PCa cell lines (PC3, DU145 and LNCaP) compared with 2 normal prostate cell lines (RWPE-1 and PrSc) using miRNA microarrays and qPCR. Clinicopathological study revealed that miR-30d expression levels were significantly higher in cancer tissue samples than in the paired normal controls (P = 0.03). Furthermore, the miR-30d-high group had shorter time to biochemical recurrence (P = 0.026). MiR-30d overexpressed PCa cells promoted proliferation and invasion in vitro. Inoculation of miR-30d depleted PCa cells dramatically reduced tumor volumes in vivo. Using reporter gene assay, we identified miR-30d as a downregulator of SOCS1 expression by directly binding to 3'-UTR of SOCS1. MiR-30d regulated the expression of phospho-STAT3, MMP-2 and MMP-9 through the downregulation of SOCS1. The levels of SOCS1 mRNA and protein were significantly down-regulated in prostate cancer tissues. Consistently, miR-30d expression was inversely correlated with SOCS1 expression (P = 0.03). The miR-30d-high/SOCS1-low group was associated with an increased risk of early biochemical recurrence (P = 0.0057). Taken together, miR-30d appears to be a novel independent prognostic marker of PCa progression that allows clinicians to identify patients who need more intensive treatments.
Oncotarget 11/2012; 3(11):1455-71. · 4.78 Impact Factor
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ABSTRACT: The 26S proteasome is a large, complex multisubunit protease involved in protein quality control and other critical processes in eukaryotes. More than 110 post-translational modification (PTM) sites have been identified by a mass spectrometry of the 26S proteasome of Saccharomyces cerevisiae and are predicted to be implicated in the dynamic regulation of proteasomal functions. Here, we report that the N-myristoylation of the Rpt2 subunit controls the intracellular localization of the 26S proteasome. While proteasomes were mainly localized in the nucleus in normal cells, mutation of the N-myristoylation site of Rpt2 caused diffusion of the nuclear proteasome into the cytoplasm, where it formed aggregates. In mutant cells, the level of accumulation of cytoplasmic proteasomes was significantly increased in the nonproliferating state. Although the molecular assembly and peptidase activity of the 26S proteasome were totally unchanged in the nonmyristoylated mutants of Rpt2, an increased level of accumulation of polyubiquitinated proteins and a severe growth defect were observed in mutant cells induced for protein misfolding. In addition, polyubiquitinated protein and the nuclear protein Gcn4 tended not to colocalize with the proteasome in normal and mutant cells. Our results suggest that N-myristoylation is involved in regulating the proper intracellular distribution of proteasome activity by controlling the nuclear localization of the 26S proteasome.
Biochemistry 10/2012; · 3.42 Impact Factor
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ABSTRACT: Microorganisms such as plant pathogens secrete glycoside hydrolases (GHs) to digest the polysaccharide chains of plant cell walls. The degradation of cell walls by these enzymes is a crucial step for nutrition and invasion. To protect the cell wall from these enzymes, plants secrete glycoside hydrolase inhibitor proteins (GHIPs). Xyloglucan-specific endo-β-1,4-glucanase (XEG), a member of GH family 12 (GH12), could be a great threat to many plants because xyloglucan is a major component of the cell wall in most plants. Understanding the inhibition mechanism of XEG by GHIP is therefore of great importance in the field of plant defense, but to date the mechanism and specificity of GHIPs remain unclear. We have determined the crystal structure of XEG in complex with extracellular dermal glycoprotein (EDGP), a carrot GHIP that inhibits XEG. The structure reveals that the conserved arginines of EDGP intrude into the active site of XEG and interact with the catalytic glutamates of the enzyme. We have also determined the crystal structure of the XEG-xyloglucan complex. These structures show that EDGP closely mimics the XEG-xyloglucan interaction. Although EDGP shares structural similarity to a wheat GHIP (Triticum aestivum xylanase inhibitor-IA (TAXI-IA)) that inhibits GH11 family xylanases, the arrangement of GH and GHIP in the XEG-EDGP complex is distinct from that in the xylanase-TAXI-IA complex. Our findings imply that plants have evolved structures of GHIPs to inhibit different GH family members that attack their cell walls.
Journal of Biological Chemistry 04/2012; 287(22):18710-6. · 4.77 Impact Factor
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Yoichi Kurata,
Yayoi Kimura,
Yuko Yamanaka,
Akiyo Ishikawa,
Hiroyuki Okamoto,
Tetsuji Masaoka,
Hiroyuki Nagoya,
Kazuo Araki,
Shunsuke Moriyama, Hisashi Hirano,
Tsukasa Mori
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ABSTRACT: Growth hormone 1 (GH1), a pituitary hormone, plays a key role in the regulation of growth. Both excess GH1 treatment and overexpression of a GH1 transgene promote growth of salmon, but these animals exhibit physiological abnormalities in viability, fertility and metabolism, which might be related to pituitary function. However, the molecular dynamics induced in the pituitary by excess GH1 remain unknown. In this study, we performed iTRAQ proteome analysis of the amago salmon pituitary, with and without excess GH1 treatment, and found that the expression levels of proteins related to endocrine systems, metabolism, cell growth and proliferation were altered in the GH1-treated pituitary. Specifically, pituitary hormone prolactin (2.29 fold), and somatolactin α (0.14 fold) changed significantly. This result was confirmed by proteome and transcriptome analyses of pituitary from the GH1-transgenic (GH1-Tg) amago salmon. The dynamics of protein and gene expression in the pituitary of GH1-Tg amago salmon were similar to those of pituitary treated with excess GH1. Our findings suggest that not only excess GH1 hormone, but also the quantitative changes in other pituitary hormones, might be essential for the abnormal growth of amago salmon. These data will be useful in future attempts to increase the productivity of fish farming.
Journal of proteomics 12/2011; 75(6):1718-31. · 5.07 Impact Factor
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ABSTRACT: Heat shock protein 90 (Hsp90), a conserved molecular chaperone for a specific set of proteins critical for signal transduction including several oncogenic proteins, has been recognized as a promising target for anticancer therapy. Hsp90 inhibition also sensitizes cancer cells to DNA damage. However, the underlying mechanisms are not fully understood. Here, we provide evidence that Hsp90 is a general regulator of phosphatidylinositol 3-kinase-related protein kinase (PIKK) family proteins, central regulators of stress responses including DNA damage. Inhibition of Hsp90 causes a reduction of all PIKK and suppresses PIKK-mediated signaling. In addition, Hsp90 forms complexes with RUVBL1/2 complex and Tel2 complex, both of which have been shown to interact with all PIKK and control their abundance and functions. These results suggest that Hsp90 can form multiple complexes with the RUVBL1/2 complex and Tel2 complex and function in the regulation of PIKK, providing additional rationale for the effectiveness of Hsp90 inhibition for anticancer therapy, including sensitization to DNA damage.
Cancer Science 09/2011; 103(1):50-7. · 3.33 Impact Factor
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ABSTRACT: In the 1990s, a technique was developed to transfer proteins from electrophoresis gels onto poly(vinylidene difluoride) (PVDF) membranes, digest the proteins on the membranes with proteases such as trypsin and analyze the resulting peptides on the membranes directly by mass spectrometry to identify the proteins. This technique, based on gel electrophoresis, is particularly useful for analyzing protein isoforms, splicing variants and post-translationally modified proteins. Previously, the low ionization efficiency of peptides immobilized on the membranes often rendered this technique useless. However, this technique has been improved by the use of PVDF membranes with a small pore size, which has enabled highly efficient and effective electroblotting and mass spectrometric analyses. Here, the advantage of this technique is discussed.
FEBS Journal 08/2011; 278(20):3807-14. · 3.79 Impact Factor
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ABSTRACT: The peptidyl-prolyl isomerase Pin1 regulates a subset of phosphorylated proteins by catalyzing the cis-trans isomerization of their specific phosphorylated Ser/Thr-Pro motifs. Although Pin1 has been shown to be involved in cell transformation and the maintenance of the malignant phenotype in prostate cancer, its specific substrates during these processes have not yet been determined.
Cancer-specific phosphorylated proteins were isolated from two human prostate cancer cell lines (PC-3, LNCaP) and the Dunning rat prostate cancer cell lines by GST-pull down analysis with recombinant GST-Pin1 protein. These proteins were then identified by the LC-MS/MS analysis using a Q-Tof micro mass spectrometer and processed for further functional analysis.
We newly identified five prostate cancer-specific Pin1 binding proteins (PINBPs) in this screen. Among these, TRK-fused gene (TFG) was found to be preferentially up-regulated in prostate cancer cell lines and tissues. The targeted inhibition of TFG by specific siRNA resulted in the reduced cell proliferation and the induction of premature senescence in PC3 prostate cancer cells. We further found that TFG can facilitate the cell signaling mediated by NF-kappaB and androgen receptor (AR). Tissue micro-dissection based quantitative RT-PCR analysis of prostate cancer tissues following radical prostatectomy further revealed that TFG expression is closely associated with both a higher probability and shorter period of tumor recurrence following surgery.
Pin1-based proteomics analysis is a useful tool for the identification of prostate cancer-specific phosphorylated proteins. TFG could be a potential diagnostic and/or prognostic marker and therapeutic target in prostate cancer.
The Prostate 08/2011; 72(6):626-37. · 3.48 Impact Factor
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ABSTRACT: Extracellular dermal glycoprotein (EDGP) may play an important role in the plant defence system of the carrot (Daucus carota) as it has inhibitory activity against endo-β-glucanase produced by invading pathogens. Here, EDGP and the inhibition complex that it forms with FI-CMCase, a carboxyl methyl cellulase from Aspergillus aculeatus, were successfully crystallized. The hexagonal crystal of EDGP belonged to space group P6(2), with unit-cell parameters a=b=130.4, c=44.5 Å, γ=120°. The monoclinic crystal of the complex of EDGP with FI-CMCase belonged to space group C2, with unit-cell parameters a=169.5, b=143.0, c=63.0 Å, β=110.9°.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 07/2011; 67(Pt 7):830-2. · 0.51 Impact Factor
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ABSTRACT: In diploid Saccharomyces cerevisiae cells, bud-site selection is determined by two cortical landmarks, Bud8p and Bud9p, at the distal and proximal poles, respectively. Their localizations depend on the multigenerational proteins Rax1p/Rax2p. Many genes involved in bud-site selection were identified previously by genome-wide screening of deletion mutants, which identified BUD32 that causes a random budding in diploid cells. Bud32p is an atypical kinase involved in a signaling cascade of Sch9p kinase, the yeast homolog of Akt/PKB, and a component of the EKC/KEOPS (endopeptidase-like, kinase, chromatin-associated/kinase, putative endopeptidase, and other proteins of small size) complex that functions in telomere maintenance and transcriptional regulation. However, its role in bipolar budding has remained unclear. In this report, we show that the Sch9p kinase cascade does not affect bipolar budding but that the EKC/KEOPS complex regulates the localization of Bud9p. The kinase activity of Bud32p, which is essential for the functions of the EKC/KEOPS complex but is not necessary for the Sch9p signaling cascade, is required for bipolar bud-site selection. BUD9 is necessary for random budding in each deletion mutant of EKC/KEOPS components, and RAX2 is genetically upstream of EKC/KEOPS genes for the regulation of bipolar budding. The asymmetric localization of Bud9p was dependent on the complex, but Bud8p and Rax2p were not. We concluded that the EKC/KEOPS complex is specifically involved in the regulation of Bud9p localization downstream of Rax1p/Rax2p.
Genetics 05/2011; 188(4):871-82. · 4.01 Impact Factor
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ABSTRACT: β-Linked glucans such as cellulose and xyloglucan are important components of the cell walls of most dicotyledonous plants. These β-linked glucans are constantly exposed to degradation by various endo-β-glucanases from pathogenic bacteria and fungi. To protect the cell wall from degradation by such enzymes, plants secrete proteinaceous endo-β-glucanases inhibitors, such as xyloglucan-specific endo-β-1,4-glucanase inhibitor protein (XEGIP) in tomato. XEGIPs typically inhibit xyloglucanase, a member of the glycoside hydrolase (GH)12 family. XEGIPs are also found in legumes, including soybean and lupin. To date, tomato XEGIP has been well studied, whereas XEGIPs from legumes are less well understood. Here, we determined the crystal structure of basic 7S globulin (Bg7S), a XEGIP from soybean, which represents the first three-dimensional structure of XEGIP. Bg7S formed a tetramer with pseudo-222 symmetry. Analytical centrifugation and size exclusion chromatography experiments revealed that the assembly of Bg7S in solution depended on pH. The structure of Bg7S was similar to that of a xylanase inhibitor protein from wheat (Tritinum aestivum xylanase inhibitor) that inhibits GH11 xylanase. Surprisingly, Bg7S lacked inhibitory activity against not only GH11 but also GH12 enzymes. In addition, we found that XEGIPs from azukibean, yardlongbean and mungbean also had no impact on the activity of either GH12 or GH11 enzymes, indicating that legume XEGIPs generally do not inhibit these enzymes. We reveal the structural basis of why legume XEGIPs lack this inhibitory activity. This study will provide significant clues for understanding the physiological role of Bg7S.
FEBS Journal 04/2011; 278(11):1944-54. · 3.79 Impact Factor
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Mayuko Nishi,
Hidenori Akutsu,
Shinji Masui,
Asami Kondo,
Yoji Nagashima,
Hirokazu Kimura,
Kilian Perrem,
Yasushi Shigeri,
Masashi Toyoda,
Akiko Okayama, Hisashi Hirano,
Akihiro Umezawa,
Naoki Yamamoto,
Sam W. Lee,
Akihide Ryo
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ABSTRACT: The prominent characteristics of pluripotent stem cells are their unique capacity to self-renew and pluripotency. Although
pluripotent stem cell proliferation is maintained by specific intracellular phosphorylation signaling events, it has not been
well characterized how the resulting phosphorylated proteins are subsequently regulated. We here report that the peptidylprolyl
isomerase Pin1 is indispensable for the self-renewal and maintenance of pluripotent stem cells via the regulation of phosphorylated
Oct4 and other substrates. Pin1 expression was found to be up-regulated upon the induction of induced pluripotent stem (iPS)
cells, and the forced expression of Pin1 with defined reprogramming factors was observed to further enhance the frequency
of iPS cell generation. The inhibition of Pin1 activity significantly suppressed colony formation and induced the aberrant
differentiation of human iPS cells as well as murine ES cells. We further found that Pin1 interacts with the phosphorylated
Ser12-Pro motif of Oct4 and that this in turn facilitates the stability and transcriptional activity functions of Oct4. Our current
findings thus uncover an atypical role for Pin1 as a putative regulator of the induction and maintenance of pluripotency via
the control of phosphorylation signaling. These data suggest that the manipulation of Pin1 function could be a potential strategy
for the stable induction and proliferation of human iPS cells.
Journal of Biological Chemistry 03/2011; 286(13):11593-11603. · 4.77 Impact Factor
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ABSTRACT: The protein annexin IV (ANX4) is elevated specifically and characteristically in ovarian clear cell adenocarcinoma (CCA), a highly malignant histological subtype of epithelial ovarian cancer. On the basis of the hypothesis that the expression of ANX4 in CCA is regulated by a unique transcription mechanism, we explored the cis-elements involved in CCA-specific ANX4 expression using a luciferase reporter. We compared the transcriptional activities of the region from -1534 to +1010 relative to the ANX4 transcription start site in CCA and non-CCA-type cell lines, and found that two repeated binding motifs for the tumor suppressor protein, p53, in the first intron of ANX4 were involved in CCA-specific transcriptional activity. Furthermore, chromatin immunoprecipitation showed that endogenous p53 bound to this site in CCA cell lines. Moreover, the use of short interference RNA to silence the p53 gene decreased the transcriptional activity and mRNA expression of ANX4 in CCA cell lines. Thus, the ANX4 gene is, at least in part, regulated by p53 in CCA cells. Mutations in the p53 gene were absent and levels of p53 target genes were higher in several CCA-derived cell lines. Although the expression of ANX4 is typically low in these non-CCA cell lines, ANX4 levels were elevated more than three-fold by the overexpression of wild-type but not mutant p53. Therefore, we conclude that the ANX4 gene is a direct transcriptional target of p53, and its expression is enhanced by wild-type p53 in CCA cells.
FEBS Journal 02/2011; 278(9):1470-83. · 3.79 Impact Factor
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Mayuko Nishi,
Hidenori Akutsu,
Shinji Masui,
Asami Kondo,
Yoji Nagashima,
Hirokazu Kimura,
Kilian Perrem,
Yasushi Shigeri,
Masashi Toyoda,
Akiko Okayama, Hisashi Hirano,
Akihiro Umezawa,
Naoki Yamamoto,
Sam W Lee,
Akihide Ryo
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ABSTRACT: The prominent characteristics of pluripotent stem cells are their unique capacity to self-renew and pluripotency. Although pluripotent stem cell proliferation is maintained by specific intracellular phosphorylation signaling events, it has not been well characterized how the resulting phosphorylated proteins are subsequently regulated. We here report that the peptidylprolyl isomerase Pin1 is indispensable for the self-renewal and maintenance of pluripotent stem cells via the regulation of phosphorylated Oct4 and other substrates. Pin1 expression was found to be up-regulated upon the induction of induced pluripotent stem (iPS) cells, and the forced expression of Pin1 with defined reprogramming factors was observed to further enhance the frequency of iPS cell generation. The inhibition of Pin1 activity significantly suppressed colony formation and induced the aberrant differentiation of human iPS cells as well as murine ES cells. We further found that Pin1 interacts with the phosphorylated Ser(12)-Pro motif of Oct4 and that this in turn facilitates the stability and transcriptional activity functions of Oct4. Our current findings thus uncover an atypical role for Pin1 as a putative regulator of the induction and maintenance of pluripotency via the control of phosphorylation signaling. These data suggest that the manipulation of Pin1 function could be a potential strategy for the stable induction and proliferation of human iPS cells.
Journal of Biological Chemistry 02/2011; 286(13):11593-603. · 4.77 Impact Factor
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ABSTRACT: Basic 7S globulin (Bg7S) is expressed by soybeans in response to biotic or abiotic stress. Bg7S is capable of binding to a 4 kDa protein which is supposedly involved in cell proliferation. Bg7S is widely found not only in legumes, but also in other plants; however, its function is still unclear. Here, Bg7S was successfully crystallized. Orthorhombic and monoclinic crystals of Bg7S were obtained under different conditions and belonged to space groups P2(1)2(1)2, with unit-cell parameters a=111.9, b=130.1, c=287.8 Å, and P2(1), with unit-cell parameters a=85.3, b=137.6, c=162.1 Å, β=91.2°, respectively.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 01/2011; 67(Pt 1):87-9. · 0.51 Impact Factor
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Yoshinari Ando,
Yasuhiro Tomaru,
Ayako Morinaga,
Alexander Maxwell Burroughs,
Hideya Kawaji,
Atsutaka Kubosaki,
Ryuichiro Kimura,
Maiko Tagata,
Yoko Ino, Hisashi Hirano,
Joe Chiba,
Harukazu Suzuki,
Piero Carninci,
Yoshihide Hayashizaki
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ABSTRACT: Human DICER1 protein cleaves double-stranded RNA into small sizes, a crucial step in production of single-stranded RNAs which are mediating factors of cytoplasmic RNA interference. Here, we clearly demonstrate that human DICER1 protein localizes not only to the cytoplasm but also to the nucleoplasm. We also find that human DICER1 protein associates with the NUP153 protein, one component of the nuclear pore complex. This association is detected predominantly in the cytoplasm but is also clearly distinguishable at the nuclear periphery. Additional characterization of the NUP153-DICER1 association suggests NUP153 plays a crucial role in the nuclear localization of the DICER1 protein.
PLoS ONE 01/2011; 6(8):e23385. · 4.09 Impact Factor
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ABSTRACT: N(α)-Acetyltransferases (NATs) cause the N(α)-acetylation of the majority of eukaryotic proteins during their translation, although the functions of this modification have been largely unexplored. In yeast (Saccharomyces cerevisiae), four NATs have been identified: NatA, NatB, NatC, and NatD. In this study, the N(α)-acetylation status of ribosomal protein was analyzed using NAT mutants combined with two-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry (MS). A total of 60 ribosomal proteins were identified, of which 17 were N(α)-acetylated by NatA, and two by NatB. The N(α)-acetylation of two of these, S17 and L23, by NatA was not previously observed. Furthermore, we tested the effect of ribosomal protein N(α)-acetylation on protein synthesis using the purified ribosomes from each NAT mutant. It was found that the protein synthesis activities of ribosomes from NatA and NatB mutants were decreased by 27% and 23%, respectively, as compared to that of the normal strain. Furthermore, we have shown that ribosomal protein N(α)-acetylation by NatA influences translational fidelity in the presence of paromomycin. These results suggest that ribosomal protein N(α)-acetylation is necessary to maintain the ribosome's protein synthesis function.
Journal of proteomics 12/2010; 74(4):431-41. · 5.07 Impact Factor
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ABSTRACT: The polyubiquitin chain is generated by the sequential addition of ubiquitin moieties to target molecules, a reaction between specific lysine residues that is catalyzed by E3 ubiquitin ligase. The Lys(48)-linked and Lys(63)-linked polyubiquitin chains are well established inducers of proteasome-dependent degradation and signal transduction, respectively. The concept has recently emerged that polyubiquitin chain-mediated regulation is even more complex because various types of atypical polyubiquitin chains have been discovered in vivo. Here, we demonstrate that a novel complex ubiquitin chain functions as an internalization signal for major histocompatibility complex class I (MHC I) membrane proteins in vivo. Using a tetracycline-inducible expression system and quantitative mass spectrometry, we show that the polyubiquitin chain generated by the viral E3 ubiquitin ligase of Kaposi sarcoma-associated herpesvirus, MIR2, is a Lys(11) and Lys(63) mixed-linkage chain. This novel ubiquitin chain can function as an internalization signal for MHC I through its association with epsin1, an adaptor molecule containing ubiquitin-interacting motifs.
Journal of Biological Chemistry 11/2010; 285(46):35311-9. · 4.77 Impact Factor
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ABSTRACT: The polyubiquitin chain is generated by the sequential addition of ubiquitin moieties to target molecules, a reaction between
specific lysine residues that is catalyzed by E3 ubiquitin ligase. The K48-linked and K63-linked polyubiquitin chains are
well-established inducers of proteasome-dependent degradation and signal transduction, respectively. The concept has recently
emerged that polyubiquitin chain-mediated regulation is even more complex, because various types of atypical polyubiquitin
chains have been discovered in vivo. Here, we demonstrate that a novel complex ubiquitin chain functions as an internalization
signal for major histocompatibility complex class I (MHC I) membrane proteins in vivo. Using a Tet-on expression system and
quantitative mass spectrometry, we show that the polyubiquitin chain generated by the viral E3 ubiquitin ligase of Kaposi
sarcoma-associated herpes virus, MIR2, is a K11 and K63 mixed linkage chain. This novel ubiquitin chain can function as an
internalization signal for MHC I through its association with epsin 1, an adaptor molecule containing ubiquitin-interacting
motifs.
Journal of Biological Chemistry 09/2010; · 4.77 Impact Factor
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ABSTRACT: In Saccharomyces cerevisiae, the bud site selection of diploid cells is regulated by at least four persistent landmarks, Bud8p, Bud9p, Rax1p, and Rax2p. Bud8p and Bud9p are essential for the establishment of bipolar budding and localize mainly to the distal and the proximal poles, respectively. Their subcellular localizations are regulated through interaction with Rax1p/Rax2p. We investigated when and where Bud8p and Bud9p physically interact with Rax2p in vivo using a split-GFP method. GFP fluorescence showed that Bud8p physically interacted with Rax2p at the proximal or distal pole in unbudded cells; a physical interaction was also observed at the opposite pole to the growing bud in mother cells with a large-size bud. Bud9p physically interacted with Rax2p at the birth scar in budded mother cells. These observations suggest that the interaction of Rax2p with Bud8p and Bud9p may contribute to the translocation of bipolar landmarks to the correct sites.
Biochemical and Biophysical Research Communications 09/2010; 399(4):525-30. · 2.48 Impact Factor
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ABSTRACT: The phosphorylation of heterogeneous nuclear ribonucleoprotein K (hnRNP K) is thought to play an important role in cell regulation and signal transduction. However, the relationship between hnRNP K phosphorylation and cellular events has only been indirectly examined, and the phosphorylated forms of endogenous hnRNP K have not been biochemically characterized in detail. In this study, we extensively examined the phosphorylated forms of endogenous hnRNP K by direct protein-chemical characterization using phosphate-affinity electrophoresis followed by immunoblotting and MS. Phosphate-affinity electrophoresis enabled us to sensitively detect and separate the phosphorylated forms of hnRNP K. When we used 2-DE with phosphate-affinity SDS-PAGE in the second dimension, the nuclear fraction contained more than 20 spots of endogenous hnRNP K on the 2-D map. We determined that the multiple forms of hnRNP K were produced mainly by alternative splicing of the single hnRNP K gene and phosphorylation of Ser116 and/or Ser284. Furthermore, the subcellular localization of these proteins revealed by the 2-D gel correlated with their phosphorylation states and alternative splicing patterns. The results also indicated that the multiple forms of hnRNP K were differentially modulated in response to external stimulation with bacterial lipopolysaccharide or serum.
Proteomics 09/2010; 10(21):3884-95. · 4.43 Impact Factor
