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

Biochem J. 1996 Jul 1;317 ( Pt 1)(Pt 1):219-24. doi: 10.1042/bj3170219.

Abstract

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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • GAP-43 Protein
  • Isoenzymes / antagonists & inhibitors
  • Isoenzymes / genetics
  • Isoenzymes / metabolism*
  • Kinetics
  • Membrane Glycoproteins / metabolism*
  • Molecular Sequence Data
  • Multigene Family
  • Nerve Tissue Proteins / metabolism*
  • Oligopeptides / metabolism*
  • Peptide Mapping
  • Peptides / metabolism*
  • Phosphopeptides / analysis
  • Phosphorylation
  • Protein Kinase C / antagonists & inhibitors
  • Protein Kinase C / genetics
  • Protein Kinase C / metabolism*
  • Recombinant Proteins / metabolism
  • Substrate Specificity

Substances

  • GAP-43 Protein
  • Isoenzymes
  • Membrane Glycoproteins
  • Nerve Tissue Proteins
  • Oligopeptides
  • Peptides
  • Phosphopeptides
  • Recombinant Proteins
  • Protein Kinase C