Transcriptional repression activity of N-MYC protein requires phosphorylation by MAP kinase.
The transrepressing function of the N-myc protein is due to the distinct domains located at the N-terminus. In this report we introduced various point mutations around the myc boxes of the N-myc protein to examine whether the phosphorylation of the protein affected its transrepressing function. Serine (Ser) residues located at amino acid numbers 12, 31, 51, and 65 were changed to leucine or arginine, and the expression vectors of the mutant proteins were transfected to HeLa cells together with the luciferase gene linked to the MHC class I gene. Among the mutants, only the N(51)-myc carrying mutation at Ser(51), a target for mitogen-activated protein kinase (MAP kinase), lost the repression activity, while the other mutant proteins preserved it. Formation in vitro of the specific nucleoprotein complexes on the H2TF1/NFkappaB element, a major target for transrepression by N-myc protein, was interfered by the wild-type N-myc protein, but not by the Ser(51)-mutated protein. The results suggest that the phosphorylation of the N-myc protein at Ser(51) by MAP kinase is required for the transcriptional repression activity of the protein.
Manabe
A
A
Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Iguchi-Ariga
S M
SM
Iizuka
H
H
Ariga
H
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D005805:Genes, MHC Class I; D006367:HeLa Cells; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016328:NF-kappa B / Q000378:metabolism; D009838:Oligodeoxyribonucleotides; D010766:Phosphorylation; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D016271:Proto-Oncogene Proteins c-myc / Q000096:biosynthesis / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D012097:Repressor Proteins / Q000096:biosynthesis / Q000378:metabolism*; D012694:Serine; D014158:Transcription, Genetic; D014162:Transfection
D007074:Immunoglobulin G; D016328:NF-kappa B; D009838:Oligodeoxyribonucleotides; D016271:Proto-Oncogene Proteins c-myc; D011994:Recombinant Proteins; D012097:Repressor Proteins; D012694:Serine; D017871:Calcium-Calmodulin-Dependent Protein Kinases