PRODUCTION AND CHARACTERIZATION OF ANTIBODIES AGAINST C-TERMINAL PEPTIDE OF PROTEIN F1 - A NOVEL PHOSPHORYLATION AT SERINE-209 OF THE PEPTIDE BY PROTEIN-KINASE-C

Protein F1 (GAP-43, B-50, neuromodulin, P-57), a neural tissue-specifi c phosphoprotein enriched in the growth cones of elongating neurites, is suggested to be involved in synaptic plasticity, neuronal developme nt, and neurotransmitter release. In this study, a 21 amino acid polyp eptide (AKPKESARQDEGKEDPEADQE) that corresponds to the C-terminus seq uence of protein F1 (from position 204-224) was synthesized and used t o produce anti-protein Fl antibodies. Immunoblot analysis has demonstr ated that the prepared antibodies recognized intact protein F1. Protei n Fl and the synthesized Fl peptide were phosphorylated in vitro by PK C. Furthermore, phosphorylated protein Fl was immunoprecipitated by an ti-Fl peptide antibodies demonstrating that these antibodies recognize d both native, non-phosphorylated and phosphorylated protein. The anti -protein F1 antibodies also stained the plasma membranes of cell bodie s and neurites of mouse neuronal cultures obtained from l l-day old sp inal embryonic tissue. By contrast, no glial cells were stained. These data suggest that serine 209 at the C-terminus of protein F1 may be a substrate for PKC phosphorylation in vivo. In addition, antibodies ra ised against FI peptide revealed protein Fl immunoreactivity that outl ined all neurites of cultured mouse spinal neurons.