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 [P-32]orthophosphate revealed the incorporation of P-32 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-P-32]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-H-3]ATP, [2,5'-8-H-3] ATP, or [alpha-P-32]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-[P-32]phosphoserine from PD-ECGF labeled in vitro with [gamma-P-32] ATP. Furthermore, P-32-radiolabeled nucleotides were released after snake venom phosphodiesterase or piperidine treatment from PD-ECGF labeled in vitro with [alpha-P-32]ATP or [gamma-P-32]ATP, as well as from PD-ECGF labeled in vivo with [P-32]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.