In vivo putative O-GlcNAcylation of human SCP1 and evidence for possible role of its N-terminal disordered structure
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RNA polymerase II carboxyl-terminal domain (RNAPII CTD)phosphatases are responsible for the dephosphorylation of theC-terminal domain of the small subunit of RNAPII ineukaryotes. Recently, we demonstrated the identification ofseveral interacting partners with human small CTD phosphatase1(hSCP1) and the substrate specificity to delineate anappearance of the dephosphorylation catalyzed by SCP1. Inthis study, using the established cells for inducibly expressinghSCP1 proteins, we monitored the modification of β-O-linkedN-acetylglucosamine (O-GlcNAc). O-GlcNAcylation is one ofthe most common post-translational modifications (PTMs). Togain insight into the PTM of hSCP1, we used the Western blot,immunoprecipitation, succinylayed wheat germ agglutininprecipitation,liquid chromatography-mass spectrometry analyses,and site-directed mutagenesis and identified the Ser41residue of hSCP1 as the O-GlcNAc modification site. Theseresults suggest that hSCP1 may be an O-GlcNAcylated proteinin vivo, and its N-terminus may function a possible role in thePTM, providing a scaffold for binding the protein(s).
Abstract
RNA polymerase II carboxyl-terminal domain (RNAPII CTD)phosphatases are responsible for the dephosphorylation of theC-terminal domain of the small subunit of RNAPII ineukaryotes. Recently, we demonstrated the identification ofseveral interacting partners with human small CTD phosphatase1(hSCP1) and the substrate specificity to delineate anappearance of the dephosphorylation catalyzed by SCP1. Inthis study, using the established cells for inducibly expressinghSCP1 proteins, we monitored the modification of β-O-linkedN-acetylglucosamine (O-GlcNAc). O-GlcNAcylation is one ofthe most common post-translational modifications (PTMs). Togain insight into the PTM of hSCP1, we used the Western blot,immunoprecipitation, succinylayed wheat germ agglutininprecipitation,liquid chromatography-mass spectrometry analyses,and site-directed mutagenesis and identified the Ser41residue of hSCP1 as the O-GlcNAc modification site. Theseresults suggest that hSCP1 may be an O-GlcNAcylated proteinin vivo, and its N-terminus may function a possible role in thePTM, providing a scaffold for binding the protein(s).
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