Extensive crosstalk between O-GlcNAcylation and phosphorylation regulates cytokinesis.
Like phosphorylation, the addition of O-linked beta-N-acetylglucosamine (O-GlcNAcylation) is a ubiquitous, reversible process that modifies serine and threonine residues on nuclear and cytoplasmic proteins. Overexpression of the enzyme that adds O-GlcNAc to target proteins, O-GlcNAc transferase (OGT), perturbs cytokinesis and promotes polyploidy, but the molecular targets of OGT that are important for its cell cycle functions are unknown. Here, we identify 141 previously unknown O-GlcNAc sites on proteins that function in spindle assembly and cytokinesis. Many of these O-GlcNAcylation sites are either identical to known phosphorylation sites or in close proximity to them. Furthermore, we found that O-GlcNAcylation altered the phosphorylation of key proteins associated with the mitotic spindle and midbody. Forced overexpression of OGT increased the inhibitory phosphorylation of cyclin-dependent kinase 1 (CDK1) and reduced the phosphorylation of CDK1 target proteins. The increased phosphorylation of CDK1 is explained by increased activation of its upstream kinase, MYT1, and by a concomitant reduction in the transcript for the CDK1 phosphatase, CDC25C. OGT overexpression also caused a reduction in both messenger RNA expression and protein abundance of Polo-like kinase 1, which is upstream of both MYT1 and CDC25C. The data not only illustrate the crosstalk between O-GlcNAcylation and phosphorylation of proteins that are regulators of crucial signaling pathways but also uncover a mechanism for the role of O-GlcNAcylation in regulation of cell division.
Wang
Zihao
Z
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Udeshi
Namrata D
ND
Slawson
Chad
C
Compton
Philip D
PD
Sakabe
Kaoru
K
Cheung
Win D
WD
Shabanowitz
Jeffrey
J
Hunt
Donald F
DF
Hart
Gerald W
GW
D000117:Acetylglucosamine / Q000378:metabolism*; D000595:Amino Acid Sequence; D034961:Antigens, Nuclear / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D016203:CDC2 Protein Kinase / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins; D018385:Centrosome / Q000378:metabolism; D048749:Cytokinesis / Q000235:genetics / Q000502:physiology*; D006031:Glycosylation; D006367:HeLa Cells; D006801:Humans; D008954:Models, Biological; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000378:metabolism; D034521:Nuclear Matrix-Associated Proteins / Q000378:metabolism; D028861:Nuclear Pore Complex Proteins / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D040901:Proteomics; D012097:Repressor Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D008941:Spindle Apparatus / Q000378:metabolism
D034961:Antigens, Nuclear; D018797:Cell Cycle Proteins; C480344:EMSY protein, human; C050045:NUMA1 protein, human; C079377:NUP153 protein, human; D009363:Neoplasm Proteins; D034521:Nuclear Matrix-Associated Proteins; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; D012097:Repressor Proteins; D017351:N-Acetylglucosaminyltransferases; C449880:O-GlcNAc transferase; D016203:CDC2 Protein Kinase; D000117:Acetylglucosamine