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

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.

[1]  T. Hunter,et al.  The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. , 1988, Science.

[2]  S. Fields,et al.  A novel genetic system to detect protein–protein interactions , 1989, Nature.

[3]  H. Kawasaki,et al.  Two yeast genes encoding calmodulin-dependent protein kinases. Isolation, sequencing and bacterial expressions of CMK1 and CMK2. , 1991, The Journal of biological chemistry.

[4]  S. Inouye,et al.  A gene encoding a protein serine/threonine kinase is required for normal development of M. xanthus, a gram-negative bacterium , 1991, Cell.

[5]  B. Errede,et al.  A conserved kinase cascade for MAP kinase activation in yeast. , 1993, Current opinion in cell biology.

[6]  J. S. Parkinson.  Signal transduction schemes of bacteria , 1993, Cell.

[7]  Masatoshi Hagiwara,et al.  Phosphorylated CREB binds specifically to the nuclear protein CBP , 1993, Nature.

[8]  A. Mendelsohn,et al.  Applications of interaction traps/two-hybrid systems to biotechnology research. , 1994, Current opinion in biotechnology.

[9]  S. Inouye,et al.  Myxococcus xanthus, a gram-negative bacterium, contains a transmembrane protein serine/threonine kinase that blocks the secretion of beta-lactamase by phosphorylation. , 1995, Genes & development.

[10]  R. Evans,et al.  Phosphorylation of CREB at Ser-133 induces complex formation with CREB-binding protein via a direct mechanism , 1996, Molecular and cellular biology.

[11]  Cheng‐Cai Zhang,et al.  Bacterial signalling involving eukaryotic‐type protein kinases , 1996, Molecular microbiology.

[12]  M. Cobb,et al.  Reconstitution of Mitogen-activated Protein Kinase Phosphorylation Cascades in Bacteria , 1997, The Journal of Biological Chemistry.

[13]  J. Keith Joung,et al.  Activation of prokaryotic transcription through arbitrary protein–protein contacts , 1997, Nature.

[14]  Peter E Wright,et al.  Solution Structure of the KIX Domain of CBP Bound to the Transactivation Domain of CREB: A Model for Activator:Coactivator Interactions , 1997, Cell.

[15]  A. Hochschild,et al.  Conversion of the omega subunit of Escherichia coli RNA polymerase into a transcriptional activator or an activation target. , 1998, Genes & development.

[16]  Jon M. Kornhauser,et al.  Magnitude of the CREB-Dependent Transcriptional Response Is Determined by the Strength of the Interaction between the Kinase-Inducible Domain of CREB and the KIX Domain of CREB-Binding Protein , 2000, Molecular and Cellular Biology.