Two N-myc polypeptides with distinct amino termini encoded by the second and third exons of the gene.

Abstract

The N-myc and c-myc genes encode closely related nuclear phosphoproteins. We found that the N-myc protein from human tumor cell lines appears as four closely migrating polypeptide bands (p58 to p64) in sodium dodecyl sulfate-polyacrylamide gels. This and the recent finding that the c-myc protein is synthesized from two translational initiation sites located in the first and second exons of the gene (S. R. Hann, M. W. King, D. L. Bentley, C. W. Anderson, and R. N. Eisenman, Cell 52:185-195, 1988) prompted us to study the molecular basis of the N-myc protein heterogeneity. Dephosphorylation by alkaline phosphatase reduced the four polypeptide bands to a doublet with an electrophoretic mobility corresponding to the two faster-migrating N-myc polypeptides (p58 and p60). When expressed transiently in COS cells, an N-myc deletion construct lacking the first exon produced polypeptides similar to the wild-type N-myc protein, indicating that the first exon of the N-myc gene is noncoding. Furthermore, mutants deleted of up to two thirds of C-terminal coding domains still retained the capacity to produce a doublet of polypeptides, suggesting distinct amino termini for the two N-myc polypeptides. The amino-terminal primary structure of the N-myc protein was studied by site-specific point mutagenesis of the 5' end of the long open reading frame and by N-terminal radiosequencing of the two polypeptides. Our results show that the N-myc polypeptides are initiated from two alternative in-phase AUG codons located 24 base pairs apart at the 5' end of the second exon. Both of these polypeptides are phosphorylated and localized to the nucleus even when expressed separately. Interestingly, DNA rearrangements activating the c-myc gene are often found in the 1.7-kilobase-pair region between the two c-myc translational initiation sites and correlate with the loss of the longer c-myc polypeptide. Thus the close spacing of the two N-myc initiation codons could explain the relative resistance of the N-myc gene to similar modes of oncogenic activation.The N-myc gene was originally identified as an amplified DNA fragment from human neuroblastomas on the basis of its homology to the v-myc oncogene (43). The N-myc gene is closely related to the more extensively investigated c-myc gene: both contain three exons, with long 5' and 3' untranslated regions, and a predicted single long open reading frame beginning with an ATG codon in the second exon. Overall homology of the deduced amino acid sequences is 32%, and distinct regions throughout the proteins are nearly identical (6,43,50). Interestingly, even the nonhomologous coding regions are highly conserved between the human and mouse N-myc genes, which show an overall homology of over 85% (13,23,50,51). This suggests that the two genes perform similar but not identical functions. In line with such

Distinct patterns of alteration of myc genes associated with integration of human papillomavirus type 16 or type 45 DNA in two genital tumours

“…In human tumour cell lines expressing amplified N- myc sequences, the N-myc protein is detected as two doublets of closely migrating phosphorylated (p62 and p64) and non-phosphorylated (p58 and p60) polypeptides that are translated from two in-frame AUG codons located 25 bp apart in the second exon of the N- myc gene (Mäkelä et al , 1989 ). In order to determine whether high levels of N- myc mRNA in IC4 cells were associated with N-myc protein expression, Western blot experiments were performed using a protein extract from IC4 tumour cells.…”

“…Protein extracts from IC4 cells and from neuroblastoma-derived SKNBE cells as a positive control and from IC2 cells as a negative control were analysed by Western blotting with an anti-human N- myc antibody. The migration of the N-myc protein in the IC4 and SKNBE cell extracts (p58-64) corresponds to apparent molecular masses in the range 58–64 kDa (Mäkelä et al , 1989 ; Wenzel et al , 1991 ). ).…”

“…Comparable large amounts of N-myc protein were detected in IC4 cells and in SKNBE neuroblastoma-derived cells, which show a 50-fold amplification of the N- myc gene (de Crémoux et al , 1997 ). The slightly faster migration of IC4 N-myc protein may indicate that this protein corresponds to the non-phosphorylated form (Mäkelä et al , 1989 ). The effects of phosphorylation on the functions of myc proteins (transactivation, DNA binding, interaction with Max protein) are far from understood (Lüscher & Larsson, 1999 ).…”

Amplified MYCN in Human Neuroblastoma

“…The nucleotide sequence of MYCN shows a high degree of similarity to that of MYC 55. MYCN encodes two polypeptides of relative molecular masses of 62 and 64 kD that result from alternative use of two translational start codons 56. The two proteins are localized in the nucleus of the cell, and they are phosphorylated.…”

The N‐myc Proto‐oncogene: Developmental Expression and in vivo Site‐Directed Mutagenesis

“…N-myc codes for a nuclear phosphoprotein, approximately 69 Kd in size (16,17). Multiple isoforms are observed in immunoprecipitates from human cells that differ both in their amino terminus and in their degree of phosphorylation (18,19). (The 5' coding sequences of mouse and human N-myc contain two initiator AUG codons separated by six nucleotides.)…”