O-linked N-acetylglucosamine modification on CCAAT enhancer-binding protein beta: role during adipocyte differentiation.
CCAAT enhancer-binding protein (C/EBP)beta is a basic leucine zipper transcription factor family member, and can be phosphorylated, acetylated, and sumoylated. C/EBPbeta undergoes sequential phosphorylation during 3T3-L1 adipocyte differentiation. Phosphorylation on Thr(188) by MAPK or cyclin A/cdk2 primes the phosphorylations on Ser(184)/Thr(179) by GSK3beta, and these phosphorylations are required for the acquisition of DNA binding activity of C/EBPbeta. Here we show that C/EBPbeta is modified by O-GlcNAc, a dynamic single sugar modification found on nucleocytoplasmic proteins. The GlcNAcylation sites are Ser(180) and Ser(181), which are in the regulation domain and are very close to the phosphorylation sites (Thr(188), Ser(184), and Thr(179)) required for the gain of DNA binding activity. Both in vitro and ex vivo experiments demonstrate that GlcNAcylation on Ser(180) and Ser(181) prevents phosphorylation on Thr(188), Ser(184), and Thr(179), as indicated by the decreased relative phosphorylation and DNA binding activity of C/EBPbeta delayed the adipocyte differentiation program. Mutation of both Ser(180) and Ser(181) to Ala significantly increase the transcriptional activity of C/EBPbeta. These data suggest that GlcNAcylation regulates both the phosphorylation and DNA binding activity of C/EBPbeta.
Li
Xi
X
Key Laboratory of Molecular Medicine, Ministry of Education, Fudan University Shanghai Medical College, Shanghai 200032, China.
Molina
Henrik
H
Huang
Haiyan
H
Zhang
You-You
YY
Liu
Mei
M
Qian
Shu-Wen
SW
Slawson
Chad
C
Dias
Wagner B
WB
Pandey
Akhilesh
A
Hart
Gerald W
GW
Lane
M Daniel
MD
Tang
Qi-Qun
QQ
D041721:3T3-L1 Cells; D000117:Acetylglucosamine / Q000378:metabolism*; D017667:Adipocytes / Q000166:cytology / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002454:Cell Differentiation; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D006031:Glycosylation; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D010766:Phosphorylation; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D022782:CCAAT-Enhancer-Binding Protein-beta; D009841:Oligonucleotides; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D008156:Luciferases; D000071679:Glycogen Synthase Kinase 3 beta; C000605507:Gsk3b protein, mouse; D020928:Mitogen-Activated Protein Kinases; D038362:Glycogen Synthase Kinase 3; D014357:Trypsin; D000117:Acetylglucosamine