Covalent linkage between nucleotides and platelet-derived endothelial cell growth factor.
Platelet-derived endothelial cell growth factor (PD-ECGF) is a 45-kDa peptide mitogen which is present in platelets and placenta and produced by certain cultured cell lines. Immunoprecipitation of A431 cells metabolically labeled with [32P]orthophosphate revealed the incorporation of 32P radioactivity into PD-ECGF. Phosphoamino acid analysis showed that serine residues of PD-ECGF were phosphorylated in vivo. Forskolin, 12-O-tetradecanoylphorbol-13-acetate, and epidermal growth factor had no effect on the in vivo phosphorylation of PD-ECGF. Moreover, incubation of pure PD-ECGF with [gamma-32P]ATP led to labeling of PD-ECGF. Optimal labeling was achieved by incubation at 95 degrees C for 5 min in the presence of sodium dodecyl sulfate, dithiothreitol, and Mg2+ or Mn2+. PD-ECGF was also labeled with [2,8-3H]ATP, [2,5',8-3H]ATP, or [alpha-32P]ATP. ATP and GTP were the preferred nucleotide substrates by comparison with other nucleotides and related components. Partial amino acid hydrolysis liberated a significant amount of O-[32P]phosphoserine from PD-ECGF labeled in vitro with [gamma-32P] ATP. Furthermore, 32P-radiolabeled nucleotides were released after snake venom phosphodiesterase or piperidine treatment from PD-ECGF labeled in vitro with [alpha-32P]ATP or [gamma-32P]ATP, as well as from PD-ECGF labeled in vivo with [32P]orthophosphate. These data indicate that serine residues of PD-ECGF can be covalently linked to phosphate groups of nucleotides, resulting in a nucleotidylated protein. The functional significance of this post-translational modification remains to be determined.
Usuki
K
K
Ludwig Institute for Cancer Research, Biomedical Center, Uppsala, Sweden.
Miyazono
K
K
Heldin
C H
CH
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D005576:Colforsin / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004815:Epidermal Growth Factor / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013939:Thymidine Phosphorylase / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D009711:Nucleotides; D005576:Colforsin; D004815:Epidermal Growth Factor; D000255:Adenosine Triphosphate; D013939:Thymidine Phosphorylase; D013755:Tetradecanoylphorbol Acetate