Phosphorylation of CCAAT-enhancer binding protein by protein kinase C attenuates site-selective DNA binding.
Four DNA-recombinant proteins, corresponding to the DNA-binding domain of CCAAT/enhancer binding protein (C/EBP), were phosphorylated in vitro by protein kinase C (PKC). High-performance liquid chromatography-peptide mapping of 32P-labeled C/EBP indicated the presence of three major 32P-labeled peptides: S299 (P)RDK, AKKS277 (P)VDK, and GAAGLPGPGGS248 (P)LK. Phosphorylation of C/EBP by PKC or M-kinase resulted in an attenuation of binding to a 32P-labeled CCAAT oligodeoxynucleotide. Three other truncated forms of C/EBP, C/EBP87, C/EBP87S-C, and C/EBP60, were studied to define the sites of phosphorylation affecting DNA binding. Phosphorylation of the C/EBP87, containing sites Ser299 and Ser277, and C/EBP60, containing only site Ser299, by PKC also resulted in attenuation of DNA binding. In contrast, phosphorylation of C/EBP87S-C, which retained Ser277 but had a Cys in place of Ser299, had no effect on DNA binding. Ser299 could not be phosphorylated by PKC if the protein is already bound to specific DNA. Phosphorylation of intact C/EBP from liver nuclear extract by PKC or M-kinase occurred at Ser299 and Ser277 and at an additional site, as demonstrated by immunoprecipitation and peptide mapping.
Mahoney
C W
CW
Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
Shuman
J
J
McKnight
S L
SL
Chen
H C
HC
Huang
K P
KP
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins / Q000378:metabolism*; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D010449:Peptide Mapping; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000378:metabolism
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D010768:Phosphoserine; D011493:Protein Kinase C