Identification of OGT interacting proteins at M phase

Abstract

O‐GlcNAc transferase (OGT) modifies numerous proteins with O‐GlcNAc, a post‐translational modification consisting of the attachment of a single N‐acetylglucosamine residue to serine or threonine amino acids on nuclear and cytoplasmic proteins. During mitosis, OGT localizes at the mitotic spindle and midbody modifying over a hundred mitotic substrates. Interestingly, how OGT is able to target discrete mitotic substrates at specific times is largely unknown. We decided to address this issue by isolating mitotic OGT interacting proteins. First, we grew HeLa cells in SILAC (Stable Isotope Labeling of Amino Acids in Cell) media and synchronized the cells either in prophase, metaphase‐anaphase, or telophase. These lysates were purified for OGT interacting proteins by binding them to an OGT column. Interestingly, silver stains of proteins eluted from the OGT columns demonstrated an increase in binding in the telophase fraction. Furthermore, this pattern was evident in OGT co‐immunoprecipitation experiments from these same lysates. These data suggest that numerous proteins form interactions with OGT at the midbody. Next, the eluted proteins were combined, digested, and the resulting peptides were identified and quantified by mass spectrometry. We found numerous proteins that interact with OGT during M phase. These results suggest that OGT is targeted to mitotic structures by discrete proteins.