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-specific phosphoprotein enriched in the growth cones of elongating neurites, is suggested to be involved in synaptic plasticity, neuronal development, and neurotransmitter release. In this study, a 21 amino acid polypeptide (AKPKES*ARQDEGKEDPEADQE) that corresponds to the C-terminus sequence of protein F1 (from position 204-224) was synthesized and used to produce anti-protein F1 antibodies. Immunoblot analysis has demonstrated that the prepared antibodies recognized intact protein F1. Protein F1 and the synthesized F1 peptide were phosphorylated in vitro by PKC. Furthermore, phosphorylated protein F1 was immunoprecipitated by anti-F1 peptide antibodies demonstrating that these antibodies recognized both native, non-phosphorylated and phosphorylated protein. The anti-protein F1 antibodies also stained the plasma membranes of cell bodies and neuritis of mouse neuronal cultures obtained from 14-day old spinal 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 raised against F1 peptide revealed protein F1 immunoreactivity that outlined all neurites of cultured mouse spinal neurons.
Azzazy
H M
HM
Department of Biochemistry and Molecular Biology, University of North Texas Health Science Center at Fort Worth 76107.
Gross
G W
GW
Wu
M C
MC
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001921:Brain / Q000201:enzymology / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D019922:GAP-43 Protein; D015151:Immunoblotting; D007150:Immunohistochemistry; D007700:Kinetics; D008297:Male; D008562:Membrane Glycoproteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis* / Q000276:immunology / Q000378:metabolism*; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology / Q000378:metabolism; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D011493:Protein Kinase C / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits / Q000276:immunology; D051381:Rats; D017207:Rats, Sprague-Dawley; D012694:Serine; D013116:Spinal Cord / Q000166:cytology* / Q000378:metabolism
D000906:Antibodies; D019922:GAP-43 Protein; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D010455:Peptides; D010748:Phosphopeptides; D010768:Phosphoserine; D012694:Serine; D011493:Protein Kinase C