Phosphorylation of GAP-43 (growth-associated protein of 43 kDa) by conventional, novel and atypical isotypes of the protein kinase C gene family: differences between oligopeptide and polypeptide phosphorylation.
GAP-43 (growth-associated protein of 43 kDa; also known as neuromodulin, P-57, B-50 and F-1) is a neuronal calmodulin binding protein and a major protein kinase C (PKC) substrate in mammalian brain. Here we describe the phosphorylation by and the site specificity of different PKC isotypes. The conventional PKC beta 1 and the novel PKCs delta and epsilon effectively phosphorylated recombinant GAP-43 in vitro; atypical PKC zeta did not. The K(m) values (between 0.6 and 2.3 microM) were very low, demonstrating a high-affinity interaction between kinase and substrate. All PKC isotypes were shown to phosphorylate serine-41 in GAP-43. When using a 19-amino-acid oligopeptide based on the GAP-43 phosphorylation site as substrate, there was a significant difference compared with polypeptide phosphorylation. The V(max) values of PKC beta 1 and PKC epsilon were much higher for this oligopeptide than for the complete protein (up to 10-fold); in contrast, their apparent affinities for the peptide were much lower (up to 100-fold) than for the intact GAP-43 polypeptide. Furthermore, phosphorylation of the GAP-43 oligopeptide by PKC beta 1 was more sensitive to a catalytic-site inhibitor than was phosphorylation of intact GAP-43. These results suggest that there are multiple sites of interaction between GAP-43 and PKC.
Oehrlein
S A
SA
Institute of Physiological Chemistry, Johannes-Gutenberg University, Mainz, Germany.
Parker
P J
PJ
Herget
T
T
D000595:Amino Acid Sequence; D019922:GAP-43 Protein; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D005810:Multigene Family; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism*; D010449:Peptide Mapping; D010455:Peptides / Q000378:metabolism*; D010748:Phosphopeptides / Q000032:analysis; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
D019922:GAP-43 Protein; D007527:Isoenzymes; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D009842:Oligopeptides; D010455:Peptides; D010748:Phosphopeptides; D011994:Recombinant Proteins; D011493:Protein Kinase C