NF-kappa B/Rel family members are physically associated phosphoproteins.
We performed radioimmunoprecipitation followed by serial immunoblots to show that, in the unstimulated Jurkat T cell line, the NF-kappa B/Rel family proteins, p80-c-Rel, p105-NF-kappa B, p65-NF-kappa B, p50-NF-kappa B and p36-I kappa B alpha, can be detected as complexes using antisera against c-Rel, p105-NF-kappa B or p65-NF-kappa B. p36-I kappa B alpha and p105, both known inhibitors of NF-kappa B function, can physically associate with NF-kappa B/Rel family members, but not with each other. In vivo and in vitro phosphorylation experiments demonstrated that NF-kappa B/Rel family members, including p105, c-Rel, p50, p65 (for the first time for p50 and p65) and p36-I kappa B alpha are also phosphoproteins. Phosphoserine and phosphothreonine residues were identified in these proteins isolated from unstimulated Jurkat cells. Both unphosphorylated and hyperphosphorylated forms of p36-I kappa B alpha were found in the complexes, suggesting that hyperphosphorylated I kappa B alpha is still capable of associating with the NF-kappa B/Rel family members. After stimulation with phorbol 12-myristate 13-acetate and phytohaemagglutinin for 10 min, p105-NF-kappa B and p50-NF-kappa B, but not p36-I kappa B, were highly phosphorylated. Phosphopeptide mapping of p105 showed that phorbol ester/phytohaemagglutinin stimulation may change p105 phosphorylation qualitatively.
Li
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Biological Carcinogenesis and Development Program, Program Resources, Inc./DynCorp., NCI-Frederick Cancer Research and Development Center 21702-1201.
Korner
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Ferris
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DK
Chen
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Dai
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RM
Longo
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DL
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D007106:Immune Sera / Q000276:immunology; D008970:Molecular Weight; D016328:NF-kappa B / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D010449:Peptide Mapping; D010766:Phosphorylation; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D015531:Radioimmunoprecipitation Assay; D013601:T-Lymphocytes / Q000187:drug effects; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D051841:Transcription Factor RelB; D014157:Transcription Factors
D007106:Immune Sera; D016328:NF-kappa B; D011518:Proto-Oncogene Proteins; D014157:Transcription Factors; D051841:Transcription Factor RelB; D013755:Tetradecanoylphorbol Acetate