Advanced
Identification of Undifferentiated Embryonic Cell Transcription Factor 1 as a Potential Substrate of Carboxyl-Terminal Domain Small Phosphatases
Identification of Undifferentiated Embryonic Cell Transcription Factor 1 as a Potential Substrate of Carboxyl-Terminal Domain Small Phosphatases
Journal of the Korean Chemical Society. 2015. Apr, 59(2): 188-193
Copyright © 2015, Korean Chemical Society
  • Received : January 24, 2015
  • Accepted : February 26, 2015
  • Published : April 20, 2015
Download
PDF
e-PUB
PubReader
PPT
Export by style
Article
Author
Metrics
Cited by
TagCloud
About the Authors
Jimoo Hong
Department of Applied Biochemistry, Konkuk University, 268 Chungwondaero, Chungju 380-701, Korea. *E-mail:ykim@kku.ac.kr
Hackyoung Kim
Department of Applied Biochemistry, Konkuk University, 268 Chungwondaero, Chungju 380-701, Korea. *E-mail:ykim@kku.ac.kr
Chanin Park
Division of Applied Life Science, Systems and Synthetic Agrobiotech Center, Plant Molecular Biology and Biotechnology Research Center, Research Institute of Natural Science, Gyeongsang National University, 501 Jinju-daero, Jinju 660-701, Korea
Minky Son
Division of Applied Life Science, Systems and Synthetic Agrobiotech Center, Plant Molecular Biology and Biotechnology Research Center, Research Institute of Natural Science, Gyeongsang National University, 501 Jinju-daero, Jinju 660-701, Korea
Keun Woo Lee
Division of Applied Life Science, Systems and Synthetic Agrobiotech Center, Plant Molecular Biology and Biotechnology Research Center, Research Institute of Natural Science, Gyeongsang National University, 501 Jinju-daero, Jinju 660-701, Korea
Young Jun Kim
Department of Applied Biochemistry, Konkuk University, 268 Chungwondaero, Chungju 380-701, Korea. *E-mail:ykim@kku.ac.kr

Abstract
Keywords
PPT Slide
Lager Image
PPT Slide
Lager Image
PPT Slide
Lager Image
PPT Slide
Lager Image
EXPERIMENTAL
- Preparation and Purification of Human Recombinant CTDSP1
An E. coli plasmid containing the human CTDSP1 gene spanning residues 76–261 was subcloned into the E. coli expression vector pET 21a(+). The pET 21a(+)/ΔN CTDSP1 76− 261 /His vector was introduced into the E. coli Rosetta 2 (DE3) strain (EMD Bioscience, Darmstadt, Germany). After an OD 600 value of 0.6 was reached, the E. coli culture was transferred to a pre-cooled incubator at 16 °C, and expression of the recombinant protein was induced with 0.4mM isopropyl-β-D-thiogalactopyranoside (IPTG; Sigma Korea, Seoul, Korea). The human recombinant CTDSP1 was expressed and purified by following a previously described method. 3
- Synthesis of Phosphopeptides
The phosphopeptides generated from human Utf1 based on the phosphorylation position reported in the Human Protein Reference Database (HPRD) were synthesized by FMOC solid phase peptide synthesis with ASP48S and purified by reverse-phase high-performance liquid chromatography (HPLC) on the Vydac Everest C 18 Column (Peptron Inc., Daejeon, Korea). The sequences of the synthetic phosphopeptides are summarized in 1B .
- Malachite Green Assay
CTDSP1-catalyzed dephosphorylation of the phosphorylated substrate was performed as previously described 3 with slight modifications. The assays were conducted at 37 °C in a buffer composed of 50 mM sodium acetate (pH 5.5), 20 mM MgCl 2 , 5 μM–1 mM phosphopeptides, and 5–50 ng of the wild-type CTDSP1. Phosphate release was quantified by a malachite green-based colorimetric assay for inorganic phosphate by measuring the absorbance at 620 nm. The malachite green solution and inorganic phosphate standards were prepared as previously described. 20 To derive the KM and kcat values, the data were fitted by a nonlinear regression to the Michaelis-Menten equation by using PRISM software.
- Molecular Docking
Molecular docking calculations were performed to predict the binding modes of the six phosphopeptides CTD, S18, T35, S42, S45, and S245. We selected a structure of the holo form containing a Mg 2+ ion (PDB ID: 3PGL, B chain), 21 which is one of the seven X-ray crystal structures for human CTDSP1 available in the Protein Data Bank (PDB). The structures of the phosphopeptides were drawn and then subjected to energy minimization with the CHARMm force field by using the Discovery Studio 3.5 (Accelrys Software Inc., San Diego, CA). The phosphopeptides were docked into the active site of the CTDSP1 by using GOLD 5.2 software 22 and SwissDock web-server. 23,24 GOLD uses a genetic algorithm (GA) to explore the ligand conformational space in the protein binding site. The residues for docking calculation were selected within a radius of 15 Å from a coordinate that is defined from the center of mass of the cocrystal ligand in the CTDSP1-phosphopeptide complex structure (PDB ID: 2GHQ). 3 The number of GA runs was set to 150. All other parameters were set as their default values. An additional docking was also performed using the webbased docking server SwissDock, which is based on the docking algorithm EADock DSS.
- Immunoprecipitation Pull-Down Assay
A plasmid containing the human CTDSP1 gene was subcloned into the mammalian expression vector pCDNAV5- His (Invitrogen, San Diego, CA, USA), and a pCMVMyc- DDK vector containing the human Utf1 gene was purchased from Origene (Rockville, MD, USA). The pulldown assay was performed using immunocomplexes that were immunoprecipitated from the total lysates of co-transfected HeLa cells with pCDNA-CTDSP1-V5-His and pCMVUtf1- Myc-DDK. Co-transfected cells were lysed with a lysis buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), and 1% TritonX- 100 containing protease and phosphatase inhibitor cocktails (Roche, Manheim, Germany)] or radioimmunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitors. The procedures for immunoprecipitation assays were essentially performed as previously described. 25 Primary antibodies used were as follows: mouse anti-V5 (Invitrogen) and mouse anti-DDK (Origene).
Supporting Information . Figure S1 showing sequence alignment and accession numbers of Utf1 proteins in Euarchontoglires, Figure S2 of kinetic characterization of CTDSP1, and the sequences of synthesized phosphopeptides.
Acknowledgements
This work was supported by the Basic Science Research Program (NRF-2013R1A1A2007315 to Y.J.K.) by the Management of Climate Change Program (2010-0029084 to K.L.) through the National Research Foundation of Korea (NRF) funded by the Ministry of Education and by the Next-Generation BioGreen 21 Program (PJ009486 to K.L.) from Rural Development Administration (RDA) in Republic of Korea. This paper was written as part of Konkuk University’s research support program for its faculty on sabbatical leave in 2014.
References
Alonso A. , Sasin J. , Bottini N. , Friedberg I. , Osterman A. , Godzik A. , Hunter T. , Dixon J. , Mustelin T. 2004 Cell 117 699 -    DOI : 10.1016/j.cell.2004.05.018
Yeo M. , Lin P. S. , Dahmus M. E. , Gill G. N. 2003 J. Biol. Chem. 278 26078 -    DOI : 10.1074/jbc.M301791200
Zhang Y. , Kim Y. , Genoud N. , Gao J. , Kelly J. W. , Pfaff S. L. , Gill G. N. , Dixon J. E. , Noel J. P. 2006 Mol. Cell 24 759 -    DOI : 10.1016/j.molcel.2006.10.027
Yeo M. , Lin P. S. 2007 Methods Mol. Biol. 365 335 -
Yeo M. , Lee S. K. , Lee B. , Ruiz E. C. , Pfaff S. L. , Gill G. N. 2005 Science 307 596 -    DOI : 10.1126/science.1100801
Kim Y. J. , Bahk Y. Y. 2014 Biochem. Biophys. Res. Commun. 448 189 -    DOI : 10.1016/j.bbrc.2014.04.089
Wrighton K. H. , Willis D. , Long J. , Liu F. , Lin X. , Feng X. H. 2006 J. Biol. Chem. 281 38365 -    DOI : 10.1074/jbc.M607246200
R H. R. , Kim H. , Noh K. , Kim Y. J. 2014 BMB. Rep. 47 192 -    DOI : 10.5483/BMBRep.2014.47.4.060
Okuda A. , Fukushima A. , Nishimoto M. , Orimo A. , Yamagishi T. , Nabeshima Y. , Kuro-o M. , Nabeshima Y. , Boon K. , Keaveney M. , Stunnenberg H. G. , Muramatsu M. 1998 EMBO. J. 17 2019 -    DOI : 10.1093/emboj/17.7.2019
Nishimoto M. , Katano M. , Yamagishi T. , Hishida T. , Kamon M. , Suzuki A. , Hirasaki M. , Nabeshima Y. , Katsura Y. , Satta Y. , Deakin J. E. , Graves J. A. , Kuroki Y. , Ono R. , Ishino F. , Ema M. , Takahashi S. , Kato H. , Okuda A. 2013 PLoS. One 8 e68119 -    DOI : 10.1371/journal.pone.0068119
Kooistra S. M. , Thummer R. P. , Eggen B. J. 2009 Stem Cell Res. 2 211 -    DOI : 10.1016/j.scr.2009.02.001
Gifford C. A. , Ziller M. J. , Gu H. , Trapnell C. , Donaghey J. , Tsankov A. , Shalek A. K. , Kelley D. R. , Shishkin A.A. , Issner R. , Zhang X. , Coyne M. , Fostel J. L. , Holmes L. , Meldrim J. , Guttman M. , Epstein C. , Park H. , Kohlbacher O. , Rinn J. , Gnirke A. , Lander E. S. , Bernstein B. E. , Meissner A. 2013 Cell 153 1149 -    DOI : 10.1016/j.cell.2013.04.037
Wu X. L. , Zheng P. S. 2013 Carcinogenesis 34 1660 -    DOI : 10.1093/carcin/bgt102
Williamson A. J. , Smith D. L. , Blinco D. , Unwin R. D. , Pearson S. , Wilson C. , Miller C. , Lancashire L. , Lacaud G. , Kouskoff V. , Whetton A. D. 2008 Mol. Cell. Proteomics 7 459 -
Van Hoof D. , Munoz J. , Braam S. R. , Pinkse M. W. , Linding R. , Heck A. J. , Mummery C. L. , Krijgsveld J. 2009 Cell Stem Cell 5 214 -    DOI : 10.1016/j.stem.2009.05.021
Kamenski T. , Heilmeier S. , Meinhart A. , Cramer P. 2004 Mol. Cell 15 399 -    DOI : 10.1016/j.molcel.2004.06.035
Almo S. C. , Bonanno J. B. , Sauder J. M. , Emtage S. , Dilorenzo T. P. , Malashkevich V. , Wasserman S. R. , Swaminathan S. , Eswaramoorthy S. , Agarwal R. , Kumaran D. , Madegowda M. , Ragumani S. , Patskovsky Y. , Alvarado J. , Ramagopal U. A. , Faber-Barata J. , Chance M. R. , Sali A. , Fiser A. , Zhang Z. Y. , Lawrence D. S. , Burley S. K. 2007 J. Struct. Funct. Genomics 8 121 -    DOI : 10.1007/s10969-007-9036-1
Laskowski A. I. , Knoepfler P. S. 2013 Biochem. Biophys. Res. Commun. 435 551 -    DOI : 10.1016/j.bbrc.2013.04.100
Laskowski A. I. , Knoepfler P. S. 2012 Cell Stem Cell 11 732 -    DOI : 10.1016/j.stem.2012.11.012
Taylor G. S. , Dixon J. E. 2004 Methods Mol. Biol. 284 217 -
Zhang M. , Cho E. J. , Burstein G. , Siegel D. , Zhang Y. 2012 ACS Chemical Biology 6 511 -
Verdonk M. L. , Cole J. C. , Hartshorn M. J. , Murray C. W. , Taylor R. D. 2003 Proteins 52 609 -    DOI : 10.1002/prot.10465
Grosdidier A. , Zoete V. , Michielin O. 2011 J. Comput. Chem.    DOI : 10.1002/jcc.21797
Grosdidier A. , Zoete V. , Michielin O. 2011 Nucleic Acids Res. 39 270 -    DOI : 10.1093/nar/gkr366
Kim Y. , Gentry M. S. , Harris T. E. , Wiley S. E. , Lawrence J. C. , Dixon J. E. 2007 Proc. Natl. Acad. Sci. 104 6596 -    DOI : 10.1073/pnas.0702099104