In vivo putative O-GlcNAcylation of human SCP1 and evidence for possible role of its N-terminal disordered structure

Abstract

RNA polymerase II carboxyl-terminal domain (RNAPII CTD) phosphatases are responsible for the dephosphorylation of the C-terminal domain of the small subunit of RNAPII in eukaryotes. Recently, we demonstrated the identification of several interacting partners with human small CTD phosphatase1 (hSCP1) and the substrate specificity to delineate an appearance of the dephosphorylation catalyzed by SCP1. In this study, using the established cells for inducibly expressing hSCP1 proteins, we monitored the modification of β-O-linked N-acetylglucosamine (O-GlcNAc). O-GlcNAcylation is one of the most common post-translational modifications (PTMs). To gain 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 Ser41 residue of hSCP1 as the O-GlcNAc modification site. These results suggest that hSCP1 may be an O-GlcNAcylated protein in vivo, and its N-terminus may function a possible role in the PTM, providing a scaffold for binding the protein(s). [BMB Reports 2014; 47(10): 593-598]

A systematic study of nuclear interactome of C-terminal domain small phosphatase-like 2 using inducible expression system and shotgun proteomics

“…Identification of protein-protein interactions is the key to understand the specificity and fidelity of many physiological reactions. Immune-affinity purification combined with mass spectrometry has been successfully used to identify interacting molecules that are directly or indirectly associated with a protein of interest (12 , 13 , 16 , 17) . Large-scale immunoprecipitation can allow us identify protein partners of CTDSPL2 with high confidence.…”

“…Large-scale immunoprecipitation can allow us identify protein partners of CTDSPL2 with high confidence. The usefulness of Tet-On inducible system has been previously demonstrated (12 , 14 , 17 - 19) . A schematic schema of the strategy used to identify CTDSPL2 interacting proteins is shown in Fig.…”

“…To date, proteomics techniques have been used to investigate protein expression profile, protein quantification, protein-protein interactions, and post translational modification (PTM). Proteomics techniques have been increasing used in various biological studies (12 , 13) . Therefore, the aim of this study was to use proteomics approach (co-immunoprecipitation, 1D electrophoresis, mass spectrometry) to identify interacting partners of CTDSPL2 in the nucleus using a cell line that could specifically express CTDSPL2 protein in order to understand the physiological function and/or role of CTDSPL2 in the nucleus.…”

Induction of MAP kinase phosphatase 3 through Erk/MAP kinase activation in three oncogenic Ras (H-, K- and N-Ras)-expressing NIH/3T3 mouse embryonic fibroblast cell lines

“…Preparation of cytosolic and nuclear fractions from the cultured NIH/3T3/Ras cells with or without doxycycline was performed using a nuclear extraction kit from Cayman Chemical Co. (Ann Arbor, MI, USA) according to the manufacturer’s instructions (33) . The procedures for immunoblotting analysis were essentially performed as previously described (22 , 34 , 35) .…”

The O-GlcNAc Modification on Kinases

“…O-GlcNAcylation of protein tyrosine phosphatase 1B (PTP1B) at S104, S201, and S386 inhibits PTP1B activity, which leads to an increase in AKT and GSK3β activity and therefore insulin response in HepG2 cells . Human small CTD phosphatase 1 (hSCP1) was identified as O-GlcNAc modified by Western blot, and its glycosite at S41 was confirmed by Q-TOF MS and site-directed mutagenesis . Additionally, the phosphatase myosin phosphatase target subunit 1 (MYPT1) may regulate the substrate specificity of OGT .…”