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 pept...

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Bibliographic Details
Published in:The Journal of biological chemistry 1992-09, Vol.267 (27), p.19396-19403
Main Authors: Mahoney, C W, Shuman, J, McKnight, S L, Chen, H C, Huang, K P
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Binding Sites
Biological and medical sciences
CCAAT-Enhancer-Binding Proteins
DNA-Binding Proteins - metabolism
Fundamental and applied biological sciences. Psychology
In Vitro Techniques
Molecular and cellular biology
Molecular genetics
Molecular Sequence Data
Nuclear Proteins - metabolism
Peptide Mapping
Phosphorylation
Phosphoserine - metabolism
Protein Kinase C - metabolism
Rats
Recombinant Proteins - metabolism
Structure-Activity Relationship
Transcription Factors - metabolism
Transcription. Transcription factor. Splicing. Rna processing
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Summary: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.
ISSN:0021-9258
1083-351X
DOI:10.1016/s0021-9258(18)41789-9