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A Crosslinking Analysis of GAP-43 Interactions with Other Proteins in Differentiated N1E-115 Cells
Department of Ophthalmology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229, USA
* Author to whom correspondence should be addressed.
Received: 13 August 2008; in revised form: 3 September 2008 / Accepted: 13 September 2008 / Published: 16 September 2008
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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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.
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.
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.