Evidence for a single protein kinase C-mediated phosphorylation site in rat brain protein B-50.
The neuronal protein B-50 may be involved in diverse functions including neural development, axonal regeneration, neural plasticity, and synaptic transmission. The rat B-50 sequence contains 226 amino acids which include 14 Ser and 14 Thr residues, all putative sites for phosphorylation by calcium/phospholipid-dependent protein kinase C (PKC). Phosphorylation of the protein appears to be a major factor in its biochemical and possibly its physiological activity. Therefore, we investigated rat B-50 phosphorylation and identified a single phosphorylated site at Ser41. Phosphoamino acid analysis eliminated the 14 Thr residues because only [32P]Ser was detected in an acid hydrolysate of [32P]B-50. Staphylococcus aureus protease peptide mapping produced a variety of radiolabelled [32P]B-50 products, none of which had the same molecular weights or HPLC retention times as several previously characterized fragments. Indirect confirmation of the results was provided by differential phosphorylation of major and minor forms of B-60 that have their N-termini at, or C-terminal to, the Ser41 residue and are the major products of specific B-50 proteolysis. Only those forms of B-60 that contained the Ser41 residue incorporated phosphate label. The results are discussed with reference to the substrate requirements for B-50 phosphorylation by PKC and the proposed structure of the B-50 calmodulin binding domain.
Coggins
P J
PJ
Department of Medical Physiology, University of Calgary, Alberta, Canada.
Zwiers
H
H
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D019922:GAP-43 Protein; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000302:isolation & purification / Q000378:metabolism*; D010449:Peptide Mapping; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats
D019922:GAP-43 Protein; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D011493:Protein Kinase C