Covalent linkage between nucleotides and platelet-derived endothelial cell growth factor.

K Usuki,K Miyazono,C.H Heldin

doi:10.1016/s0021-9258(18)54956-5

Abstract

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.

Highlights

ECGF) is a 46-kDa peptide mitogen which is present to be produced by different cell types in culture, including in platelets and placenta and produced by certain cul-human foreskin fibroblasts, human vascular smooth muscle tured cell lines
The cell lysates were incubated with Platelet-derived endothelial cell growth factor (PD-ECGF) antiserum[18],in the presence or absence of unlabeled pure PD-ECGF, and theprecipitates were analyzed by SDS-gel electrophoresis and autoradiography
Labeling of a 45-kDa component was noticed in the precipitate with the immune serum, but notwith nonimmune serum (Fig. 1).This component was not seen when an excess amount of PD-ECGF was present during immunoprecipitation, indicating that 32P radioactivity is incorporated in vivo into PD-ECGF

Summary

Introduction

ECGF) is a 46-kDa peptide mitogen which is present to be produced by different cell types in culture, including in platelets and placenta and produced by certain cul-human foreskin fibroblasts, human vascular smooth muscle tured cell lines. The cell lysates were incubated with PD-ECGF antiserum[18],in the presence or absence of unlabeled pure PD-ECGF, and theprecipitates were analyzed by SDS-gel electrophoresis and autoradiography. Incubations were terminated by the addition of 100 mM EDTA; samples were analyzed by SDS-gel electrophoresis and autoradiography.

Results

Conclusion