A study of substrate specificity for a CTD phosphatase, SCP1, by proteomic screening of binding partners.
RNA polymerase II carboxyl-terminal domain (RNAPII CTD) phosphatases are a newly emerging family of phosphatases. Recently a CTD-specific phosphatase, small CTD phosphatase 1 (SCP1), has shown to act as an evolutionarily conserved transcriptional corepressor for inhibiting neuronal gene transcription in non-neuronal cells. In this study, using the established NIH/3T3 and HEK293T cells, which are expressing human SCP1 proteins under the tight control of expression by doxycycline, a proteomic screening was conducted to identify the binding partners for SCP1. Although the present findings provide the possibility for new avenues to provide to a better understanding of cellular physiology of SCP1, now these proteomic and some immunological approaches for SCP1 interactome might not represent the accurate physiological relevance in vivo. In this presentation, we focus the substrate specificity to delineate an appearance of the dephosphorylation reaction catalyzed by SCP1 phosphatase. We compared the phosphorylated sequences of the immunologically confirmed binding partners with SCP1 searched in HPRD. We found the similar sequences from CdcA3 and validated the efficiency of enzymatic catalysis for synthetic phosphopeptides the recombinant SCP1. This approach led to the identification of several interacting partners with SCP1. We suggest that CdcA3 could be an enzymatic substrate for SCP1 and that SCP1 might have the relationship with cell cycle regulation through enzymatic activity against CdcA3.
Dept. of Applied Biochemistry, Konkuk University, 268 Chungwon Dae-ro, Chungju-City, Chungbuk 380-701, Republic of Korea.
Young Jun
YJ
Kim
Dept. of Biotechnology, Konkuk University, 268 Chungwon Dae-ro, Chungju-City, Chungbuk 380-701, Republic of Korea. Electronic address: bahk12@empal.com.
Young Yil
YY
Bahk
D000595:Amino Acid Sequence; D000818:Animals; D018797:Cell Cycle Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D025941:Protein Interaction Mapping / Q000379:methods; D040901:Proteomics / Q000379:methods*; D013379:Substrate Specificity
C473796:CDCA3 protein, human; D018797:Cell Cycle Proteins; D009687:Nuclear Proteins; C413483:CTDSP1 protein, human; D010749:Phosphoprotein Phosphatases
CTD phosphatase SCP1; Inducible expression system; Interactome; Phosphopeptide; Substrate specificity