Int. J. Mol. Sci. 2008, 9(9), 1753-1771; doi:10.3390/ijms9091753
Article

A Crosslinking Analysis of GAP-43 Interactions with Other Proteins in Differentiated N1E-115 Cells

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Received: 13 August 2008; in revised form: 3 September 2008 / Accepted: 13 September 2008 / Published: 16 September 2008
(This article belongs to the Special Issue Protein Folding)
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract: It has been suggested that GAP-43 (growth-associated protein) binds to various proteins in growing neurons as part of its mechanism of action. To test this hypothesis in vivo, differentiated N1E-115 neuroblastoma cells were labeled with [35S]-amino acids and were treated with a cleavable crosslinking reagent. The cells were lysed in detergent and the lysates were centrifuged at 100,000 x g to isolate crosslinked complexes. Following cleavage of the crosslinks and analysis by two-dimensional gel electrophoresis, it was found that the crosslinker increased the level of various proteins, and particularly actin, in this pellet fraction. However, GAP-43 was not present, suggesting that GAP-43 was not extensively crosslinked to proteins of the cytoskeleton and membrane skeleton and did not sediment with them. GAP-43 also did not sediment with the membrane skeleton following nonionic detergent lysis. Calmodulin, but not actin or other proposed interaction partners, co-immunoprecipitated with GAP-43 from the 100,000 x g supernatant following crosslinker addition to cells or cell lysates. Faint spots at 34 kDa and 60 kDa were also present. Additional GAP-43 was recovered from GAP-43 immunoprecipitation supernatants with anti-calmodulin but not with anti-actin. The results suggest that GAP-43 is not present in complexes with actin or other membrane skeletal or cytoskeletal proteins in these cells, but it is nevertheless possible that a small fraction of the total GAP-43 may interact with other proteins.
Keywords: Neuromodulin; cytoskeleton; filopodia; lipid rafts; palmitoylation
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MDPI and ACS Style

Ollom, C.M.; Denny, J.B. A Crosslinking Analysis of GAP-43 Interactions with Other Proteins in Differentiated N1E-115 Cells. Int. J. Mol. Sci. 2008, 9, 1753-1771.

AMA Style

Ollom CM, Denny JB. A Crosslinking Analysis of GAP-43 Interactions with Other Proteins in Differentiated N1E-115 Cells. International Journal of Molecular Sciences. 2008; 9(9):1753-1771.

Chicago/Turabian Style

Ollom, Callise M.; Denny, John B. 2008. "A Crosslinking Analysis of GAP-43 Interactions with Other Proteins in Differentiated N1E-115 Cells." Int. J. Mol. Sci. 9, no. 9: 1753-1771.

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