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Evidence for multisite ADP-ribosylation of neuronal phosphoprotein B-50/GAP-43

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Abstract

The neuronal phosphoprotein B-50/GAP-43 is associated with neuronal growth and regeneration and is involved in the calcium/CaM and Go signal transduction systems. In particular, B-50 interacts uniquely with CaM by binding in the absence of Ca2+. Previously identified as a major neuronal substrate for protein kinase C, which releases CaM via phosphorylation, B-50 has more recently been shown to be a substrate for endogenous ADP-ribosyltransferases. In the present study, we utilized amino acid modification with iodoacetamide and chemical stability to mercury and neutral hydroxylamine to demonstrate that the predominant site of ADP-ribosylation is Cys 3 and/or Cys 4. Chymotryptic peptide mapping further revealed a second, less labelled site of ribosylation in the C-terminal region. The results also demonstrate that, in contrast to PKC phosphorylation, ADP-ribosylation of B-50 does not mediate CaM binding. Since Cys 3 and Cys 4, by palmitoylation, are important for membrane anchoring, our findings suggest that ADP-ribosylation of B-50 may have a role in directing the intracellular localization of the protein. Hence, ribosylation of B-50 may mediate where B-50 interacts with signal transduction pathways.

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Abbreviations

ACTH:

corticotrophin

APAD:

3′ acetylpyridine adenine dinucleotide

CaM:

calmodulin

DTT:

dithiothreitol

Gpp(NH)p:

5′ guanylylimidodiphosphate

HPLC:

high performance liquid chromatography

NAD+ :

nicotinamide ademine dinucleotide

NMR:

nuclear magnetic resonance

PKC:

protein kinase C

dp:

dephosphorylated

p:

protein kinase C phosphorylated

r:

ADP-ribosylated

SDS-PAGE:

sodium dodecyl sulphate-polyacrylamide gel electrophoresis

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Authors and Affiliations

  1. Departments of Medical Physiology and Medical Biochemistry, University of Calgary, Health Sciences Centre, 3330 Hospital Drive N.W., T2N 4N1, Calgary, Alberta, Canada

    Keith Philibert & Henk Zwiers

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  1. Keith Philibert
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  2. Henk Zwiers
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Philibert, K., Zwiers, H. Evidence for multisite ADP-ribosylation of neuronal phosphoprotein B-50/GAP-43. Mol Cell Biochem 149, 183–190 (1995). https://doi.org/10.1007/BF01076576

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