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Published March 2007 | Published + Supplemental Material
Journal Article Open

The Alliance for Cellular Signaling Plasmid Collection: A Flexible Resource for Protein Localization Studies and Signaling Pathway Analysis


Cellular responses to inputs that vary both temporally and spatially are determined by complex relationships between the components of cell signaling networks. Analysis of these relationships requires access to a wide range of experimental reagents and techniques, including the ability to express the protein components of the model cells in a variety of contexts. As part of the Alliance for Cellular Signaling, we developed a robust method for cloning large numbers of signaling ORFs into Gateway® entry vectors, and we created a wide range of compatible expression platforms for proteomics applications. To date, we have generated over 3000 plasmids that are available to the scientific community via the American Type Culture Collection. We have established a website at www.signaling-gateway.org/data/plasmid/ that allows users to browse, search, and blast Alliance for Cellular Signaling plasmids. The collection primarily contains murine signaling ORFs with an emphasis on kinases and G protein signaling genes. Here we describe the cloning, databasing, and application of this proteomics resource for large scale subcellular localization screens in mammalian cell lines.

Additional Information

┬ę 2007 The American Society for Biochemistry and Molecular Biology, Inc. Received, November 15, 2006, and in revised form, December 26, 2006. Published, MCP Papers in Press, December 27, 2006, DOI 10.1074/mcp.M600437-MCP200 We thank Linda Holloway, Judy Kantor, Barry Westfall, and Jennifer Zinck at the ATCC for assistance in facilitating the distribution of AfCS clones and Mike Brasch, Joel Jessee, Ray Harris, Virginia Heatwole, and Graziella Piras at Invitrogen for technical advice on the Gateway system and provision of plasmids prior to commercial release. We thank Gerard Manning for early access to the human and mouse kinome sequences; these sequence data were invaluable in helping to curate kinase ORFs prior to cloning. We are grateful to colleagues in the AfCS for insightful discussions on a wide range of plasmid vector development projects. This work was supported by contributions from public and private sources, including the NIGMS, National Institutes of Health, Glue Grant Initiative U54 GM062114. A complete listing of the AfCS sponsors can be found at www.signaling-gateway.org/aboutus/sponsors.html. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The on-line version of this article (available at http://www.mcponline.org) contains supplemental material.

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Published - ZAVmcp07.pdf

Supplemental Material - ZAVmcp07supp.pdf


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