Identification of Undifferentiated Embryonic Cell Transcription Factor 1 as a Potential Substrate of Carboxyl-Terminal Domain Small Phosphatases

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 CTDSP176− 261/His vector was introduced into the E. coli Rosetta 2 (DE3) strain (EMD Bioscience, Darmstadt, Germany). After an OD600 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 C18 Column (Peptron Inc., Daejeon, Korea). The sequences of the synthetic phosphopeptides are summarized in Fig. 1B.

Malachite Green Assay

CTDSP1-catalyzed dephosphorylation of the phosphorylated substrate was performed as previously described3 with slight modifications. The assays were conducted at 37 °C in a buffer composed of 50 mM sodium acetate (pH 5.5), 20 mM MgCl2, 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 Mg2+ 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 software22 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.