Proteomic Mapping of Mitotic O‐GlcNAc Sites

• DOI: 10.1096/fasebj.29.1_supplement.570.20
• Publication Date: 2021-06-21T18:21:38Z
• Authors: Stephen Whelan; Ee Phie Tan; Chris Lanza; Catherine Costello; Mark McComb; Chad Slawson
• Keywords: 0301 basic medicine; 03 medical and health sciences

Regulated mitotic progression coupled with high‐fidelity chromosome segregation is crucial for normal cellular division. Phosphorylation. Although, phosphorylation plays a critical role regulating mitosis, it alone cannot account for all the complexities of mitosis. We have demonstrated that O‐GlcNAcylation is critical for proper mitotic regulation and disruptions in O‐GlcNAc signaling lead to aberrant cell division. O‐GlcNAc (b‐N‐acetylglucosamine) is an ubiquitous protein modification consisting of a single N‐acetylglucosamine residue attached to Ser or Thr in cytoplasmic and nuclear proteins in response to changes in the cellular environment. Alterations to the proper rate of O‐GlcNAc cycling lead to mitotic defects. The need for detailed O‐GlcNAc site‐mapping information is paramount for our understanding of the role of O‐GlcNAc in regulating mitotic progression. We have developed a combination of tools and methods to site map O‐GlcNAc using a Q Exactive hybrid Quadrupole‐Orbitrap Mass Spectrometer. Interestingly, we have found the Repo‐Man protein to be modified by O‐GlcNAc. Repo‐man is a Protein Phosphatase 1γ binding protein that recruits the phosphatase to the spindle in order to antagonize the function of the spindle kinase Aurora B (AurB). Repo‐man is critical for proper spindle development; loss of Repo‐man expression is lethal. Together, our data demonstrate an efficient and robust method to map O‐GlcNAc sites. The identification of O‐GlcNAc modified mitotic proteins will provide new context as to how O‐GlcNAc cycling regulates mitotic progression and spindle development. NIH‐NHLBI Contract No. HHSN268201000031C, NIH‐GM grant P41 GM104603 and DK100595