The MYCN protein of human neuroblastoma cells is phosphorylated by casein kinase II in the central region and at serine 367.
The MYCN gene has been implicated in certain neuronal tumours, such as neuroblastomas and retinoblastomas. These tumours express high levels of mRNA and protein of MYCN as a result of amplification. MYCN encodes a short-lived nuclear phosphoprotein whose function has not yet been elucidated. This study was undertaken to determine the pattern of MYCN protein (pMYCN) phosphorylation in human neuroblastoma cells. We report that pMYCN is phosphorylated in vitro by purified casein kinase II (CK-II). Two-dimensional phosphopeptide maps showed that most of the phosphopeptides of pMYCN phosphorylated in vitro by CK-II correspond to those phosphorylated in vivo. Fine mapping of the phosphorylation sites was performed using two synthetic MYCN peptides corresponding to pMYCN CK-II consensus sequences. Both peptides were found to be phosphorylated by CK-II and competitively inhibited CK-II phosphorylation of pMYCN in vitro. Thus, we have localized two major CK-II phosphorylation sites in pMYCN, one to the highly acidic central region and the second to serine 367 proximal to the C-terminus. Our data demonstrate that pMYCN is a physiological substrate for CK-II and, since CK-II activity is stimulated in response to mitogens, CK-II phosphorylation of pMYCN may, therefore, represent one signal transduction pathway used by neuroblastoma cells.
Institut für Experimentelle Pathologie, Deutsches Krebsforschungszentrum, Heidelberg, Germany.
U
U
Hamann
A
A
Wenzel
R
R
Frank
M
M
Schwab
D000595:Amino Acid Sequence; D047388:Casein Kinases; D006801:Humans; D008969:Molecular Sequence Data; D009447:Neuroblastoma / Q000235:genetics*; D010449:Peptide Mapping; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism*; D016271:Proto-Oncogene Proteins c-myc / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured / Q000378:metabolism
D047428:Protein Kinase Inhibitors; D016271:Proto-Oncogene Proteins c-myc; D011494:Protein Kinases; D047388:Casein Kinases