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 β1 and the novel PKCs Δ and ϵ effectively phosphorylated recombinant GAP-43 in vitro; atypical PKC ζ did not. The Km values (between 0.6 and 2.3 μM) 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 Vmax values of PKC β1 and PKC ϵ 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 β1 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.
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July 1996
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Research Article|
July 01 1996
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
Silke A. OEHRLEIN;
Silke A. OEHRLEIN
*Institute of Physiological Chemistry, Johannes-Gutenberg University, Duesbergweg 6, 55 099 Mainz, Germany
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Peter J. PARKER;
Peter J. PARKER
†Imperial Cancer Research Fund, Protein Phosphorylation Laboratory, Lincoln's Inn Fields, London WC2A 3PX, U.K.
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Thomas HERGET
Thomas HERGET
‡
‡To whom correspondence should be addressed.
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Publisher: Portland Press Ltd
Received:
August 30 1995
Revision Received:
February 22 1996
Accepted:
March 01 1996
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1996
1996
Biochem J (1996) 317 (1): 219–224.
Article history
Received:
August 30 1995
Revision Received:
February 22 1996
Accepted:
March 01 1996
Citation
Silke A. OEHRLEIN, Peter J. PARKER, Thomas HERGET; 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 1 July 1996; 317 (1): 219–224. doi: https://doi.org/10.1042/bj3170219
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