Specific phosphorylation of the acidic central region of the N-myc protein by casein kinase II.
The central region of the N-myc protein has a characteristic amino acid sequence EDTLSDSDDEDD, which is very similar to those of particular domains of adenovirus E1A, human papilloma virus E7, Simian virus 40 large T, c-myc and L-myc proteins. Domains of these three viral oncoproteins have recently been shown to be specific binding sites for the tumor-suppressor gene retinoblastoma protein. We have noted that the sequence of serine followed by a cluster of acidic amino acids is exactly the same as that of a typical substrate of casein kinase II (CKII). Therefore, we investigated whether these nuclear oncoproteins are phosphorylated by CKII. For this purpose, we fused the beta-galactosidase and N-myc genes including this domain and expressed it in Escherichia coli cells. Several mutant N-myc genes, containing single amino acid substitutions in this domain, were also used to produce fused proteins. Strong phosphorylation by CKII was detected with the fused protein of wild-type N-myc. However, no phosphorylation of beta-galactosidase itself was observed and the phosphorylations of fused mutant proteins were low. Another fused N-myc protein containing most of the C-terminal region downstream of this acidic region was not phosphorylated by CKII. Analysis of phosphorylation sites in synthetic peptides of this acidic region identified the major sites phosphorylated by CKII as Ser261 and Ser263. On two-dimensional tryptic mapping of phosphorylated N-myc proteins, major spots of in vitro-labeled and in-vivo-labeled N-myc proteins were detected in the same positions. These results suggest that two serine residues of the acidic central region of the N-myc protein are phosphorylated by CKII in vivo as well as in vitro. The functional significance of this acidic domain is discussed.
Hagiwara
T
T
Laboratory of Biological Chemistry, School of Pharmaceutical Sciences, Showa University, Tokyo, Japan.
Nakaya
K
K
Nakamura
Y
Y
Nakajima
H
H
Nishimura
S
S
Taya
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001345:Autoradiography; D001483:Base Sequence; D047390:Casein Kinase II; D002417:Cattle; D004247:DNA; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism; D010449:Peptide Mapping; D010766:Phosphorylation; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism*; D016160:Retinoblastoma Protein / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014357:Trypsin; D014407:Tumor Cells, Cultured
D010446:Peptide Fragments; D016271:Proto-Oncogene Proteins c-myc; D016160:Retinoblastoma Protein; D004247:DNA; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D014357:Trypsin