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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Preview
Unable to display preview. Download preview PDF.
Abbreviations
- ACTH:
-
corticotrophin
- APAD:
-
3’acetylpyridine adenine dinucleotide
- CaM:
-
calmodulin
- DTT:
-
dithiothreitol
- Gpp(NH)p:
-
5’ guanylylimidodiphosphate
- HPLC:
-
high performance liquid chromatography
- NAD+ :
-
nicotinamide adenine 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
References
Zwiers H, Veldhuis HD, Schotman P, Gispen WH: ACTH, cyclic nucleotides, and brain protein phosphorylation in vitro. Neurochem Res 1: 669–677, 1976
Kristjansson GI, Zwiers H, Oestreicher AB, Gispen WH: Evidence that synaptic phosphoprotein B-50 is located exclusively in nerve tissue. J Neurochem 39: 371–378, 1982
Benowitz LI, Perrone-Bizzozero N, Finklestein S: Molecular properties of the growth-associated protein GAP-43 (B-50). J Neurochem 48: 1640–1647, 1987
Skene JHP, Virag I: Post-translational membrane attachment and dynamic fatty acylation of a neuronal growth cone protein, GAP-43. J Cell Biol 108: 613–625, 1989
Liu Y, Fisher DA, Storm DR: Analysis of the palmitoylation and membrane argetting domain of neuromodulin (GAP-43) by site-specific mutagenesis. Biochem 32: 10714–10719, 1993
Coggins PJ, Zwiers H: Evidence for a single PKC-mediated phosphorylation ite in rat brain protein B-50. J Neurochem 53: 1895–1901, 1989
Aloyo VJ, Zwiers H, De Graan PNE, Gispen WH: Phosphorylation of the neuronal PKC substrate B-50: in vitro assay conditions alter sensitivity to ACTH. Neurochem Res 13: 343–348, 1988
Paudel HK, Zwiers H, Wang JH: Phosphorylase kinase phosphorylates the calmodulin-binding regulatory regions of neuronal tissue-specific proteins B-50 (GAP-43) and neurogranin. J Biol Chem 268: 6207–6213, 1993
Apel ED, Litchfield DW, Clark RH, Krebs EG, Storm DR: Phosphorylation of neuromodulin (GAP-43) by casein kinase II. J Biol Chem 266: 10544–10551, 1991
Andreason TJ, Leutie CW, Heideman W, Storm DR: Purification of a novel calmodulin binding protein from bovine cerebral cortex membranes. Biochem 22: 4615–4618, 1983
Chapman ER, Au D, Nicolson TA, Storm DR: Mutagenesis of the calmodulin binding domain of neuromodulin, In: W.H. Gispen and A. Routtenberg (eds). Progress in Brain Research Elsevier, Amsterdam, 1991, pp 37–44
Slemmon JR Martzen M: Neuromodulin (GAP-43) can regulate a calmodulin-dependent target in vitro. Biochem 33: 5653–5660, 1994
Dekker LV, De Graan PNE, Versteeg DHG, Oestreicher AB, Gispen WH: Phosphorylation of B-50 (GAPt-43) is correlated with neurotransmitter release in rat hippocampal slices. J Neurochem 52: 24–30, 1989
Nelson RB, Linden DJ, Routtenberg A: Phosphoproteins localized to presynaptic terminal linked to persistence of long-term potentiation (LTFt): quantitative analysis of two-dimensional gels. Brain Res 497: 30–42, 1989
Skene JHP: Axonal growth-associated proteins. Ann Rev Neurosci 12: 127–156, 1989
Strittmatter SM, Valenzuela D, Kennedy TE, Neer EJ, Fishman MC: Go is a major growth cone protein subject to regulation by GAP-43. Nature 344: 836–841, 1990
Strittmatter SM, Valenzuela D, Sudo Y, Linder ME, Fishman MC: An intracellular guanine nucleotide release protein for Go. J Biol Chem 266: 22465–22471, 1991
Strittmatter SM, Valenzuela D, Fishman MC: An amino-terminal domain of the growth-associated protein GAP-43 mediates its effects on filopodial formation and cell spreading. J Cell Science 107: 195–204, 1994
Sudo Y, Valenzuela D, Bech-Sickinger AG, Fishman MC, Strittmatter SM: Palmitoylation alters protein activity: blockade of Go stimulation by GAP-43. EMBO J 11: 2095–2102, 1992
Coggins PJ, McLean K, Nagy A, Zwiers H: ADP-Ribosylation of the neuronal phosphoprotein B-50 (GAP-43). J Neurochem 60: 368–371, 1993
Brune B. Lapetina EG: Activation of a cytosolic ADP-ribosyl-mtransferase by nitric oxide-generating agents. J Biol Chem 264: 8455–8458, 1989
Scaife RM, Wilson L, Purich DL: Microtubule protein ADP-ribosylation in vitro leads to assembly inhibition and rapid depolymerization. Biochem 31: 310–316, 1992
Coggins PJ, McLean K, Zwiers H: Neurogranin, a B-50/GAP-43-immunoreactive C-kinase substrate (BICKS), is ADP-ribosylated. FEBS letters 335: 109–113, 1993
Bradford MM: A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of proteindye binding. Anal Biochem 72: 284–254, 1976
McMaster D, Zwiers H, Lederis K: The growth-associated neuronal phosphoprotein B-50: Improved purification, partial primary structure, and characterization and localization of proteolysis products. Brain Res Bull 21: 265–276, 1988
Bazzet-Jones DP: Electron spectroscopic imaging of chromatin and other nucleoprotein complexes. Electron Microsc Rev 5: 37–58, 1992
Aloyo VJ, Zwiers H, Gispen WH: Phosphorylation of B-50 by calcium-activated phospholipid dependent protein kinase and B-50 protein kinase. J Neurochem 41: 649–653, 1983
Williams MB, Li X, Gu X, Jope RS: Modulation of endogenous ADP-ribosylation in rat brain. Brain Res 592: 49–56, 1992
McDonald LJ, Moss J: Stimulation by nitric oxide of an NAD linkage to glyceraldehyde-3-phosphate dehydrogenase. Proc Natl Acad Sci USA 90: 6238–6241, 1993
Coggins PJ, Zwiers H: Detergents and peptides alter proteolysis and calmodulin binding of B-50/GAP-43 in vitro. J Neurochem 63: 1491–1498, 1994
Zhang M, Vogel HJ, Zwiers H: Nuclear magnetic resonance studies of the structure of B-50/Neuromodulin and its interaction with calmodulin. Can J Biochem Cell Biol 72: 109–116, 1994
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1995 Springer Science+Business Media Dordrecht
About this chapter
Cite this chapter
Philibert, K., Zwiers, H. (1995). Evidence for multisite ADP-ribosylation of neuronal phosphoprotein B-50/GAP-43. In: Barnes, J.A., Coore, H.G., Mohammed, A.H., Sharma, R.K. (eds) Signal Transduction Mechanisms. Developments in Molecular and Cellular Biochemistry, vol 15. Springer, Boston, MA. https://doi.org/10.1007/978-1-4615-2015-3_19
Download citation
DOI: https://doi.org/10.1007/978-1-4615-2015-3_19
Publisher Name: Springer, Boston, MA
Print ISBN: 978-1-4613-5833-6
Online ISBN: 978-1-4615-2015-3
eBook Packages: Springer Book Archive