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In vivo phosphorylation with TKB1 competent cells
David L. Dankort William J. Muller
Institute for Molecular Biology and Biotechnology, McMaster University,
Ontario, Canada
GST-fusion proteins expressed in TKB1 bacteria are used to affinity purify cellular proteins, which interact with a specific autophosphorylation site on the ErbB2/Neu receptor tyrosine kinase (RTK). This approach is aptly suited to rapidly identify phosphotyrosine-protein interactions.
When receptor tyrosine kinases (RTKs) are activated, a number of proteins within the cell are rapidly phosphorylated, with the predominate tyrosine-phosphorylated proteins being the RTKs themselves. RTK tyrosine phosphorylation primarily functions to generate binding sites for cytoplasmic- or plasma membrane-associated proteins involved in transducing proliferative or differentiating signals to the nucleus. These signaling proteins each contain modular Src homology 2 (SH2) or protein tyrosine binding/interacting domains (PTB/PID), which enable them to directly interact with specific receptors in a phosphotyrosine-dependent and sequence-specific manner and, therefore, bind to short linear peptide sequences.1,2,3
Extensive mutagenesis of known autophosphorylation sites in the RTK Neu/ErbB-2 has revealed that four (Y1144, Y1201, Y1227, Y1253) of the five autophosphorylation sites are capable of mediating transformation from the receptor.4 Subsequent coimmunoprecipitation analyses from stable cell lines disclose that SHC and GRB2 proteins bind directly to the receptor at tyrosines 1227 and 1144, respectively. 4
Signaling Molecules Identified
Figure 1
To more quickly identify candidate signaling molecules, we developed
an in vitro association assay based on the work of Pawson, et al.,5'"/>
Source:
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