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Sci. STKE, 23 July 2002
Vol. 2002, Issue 142, p. pl11
[DOI: 10.1126/stke.2002.142.pl11]

PROTOCOLS

Analysis of Phosphorylation-Dependent Protein-Protein Interactions Using a Bacterial Two-Hybrid System

Adam J. Shaywitz1, Simon L. Dove2*, Michael E. Greenberg3, and Ann Hochschild4

1Program in Biological and Biomedical Sciences, Division of Medical Sciences, Harvard Medical School, and Division of Neuroscience, Children's Hospital, Boston, MA 02115, USA.
2Division of Infectious Diseases, Children's Hospital, Harvard Medical School, Boston, MA 02115, USA.
3Department of Neurobiology, Harvard Medical School, and Division of Neuroscience, Children's Hospital, Boston, MA 02115, USA.
4Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115, USA.

Abstract: Phosphorylation-dependent protein-protein interactions provide the foundation for a multitude of intracellular signal transduction pathways. One of the goals of signal transduction research is to more precisely understand the nature of these phosphorylation-dependent interactions. Here, we describe a bacterial two-hybrid assay that allows for the rapid, efficient analysis of phosphorylation-dependent protein-protein interactions. In this system, the interacting protein domains are provided as fusion proteins in Escherichia coli. cells that contain a eukaryotic kinase. Specific phosphorylation of one of the fused protein domains results in a protein-protein interaction that can be detected as a change in the expression of a reporter gene. We also describe how this system can be modified to permit the use of cDNA libraries to identify either novel binding partners for a phosphorylated substrate or novel kinases that can induce a specific protein-protein interaction.

*Corresponding author. E-mail, simon.dove{at}tch.harvard.edu

Citation: A. J. Shaywitz, S. L. Dove, M. E. Greenberg, A. Hochschild, Analysis of Phosphorylation-Dependent Protein-Protein Interactions Using a Bacterial Two-Hybrid System. Sci. STKE 2002, pl11 (2002).

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