Title: The development of protein microarrays to investigate receptor tyrosine kinase signaling
Description: In order for a multicellular organism to develop and function properly, its cells must communicate to one-another to coordinate the correct spatio-temporal ordering of processes such as cell growth, migration, adhesion, and apoptosis. In metazoans, many of these processes are coordinated by the receptor tyrosine kinases (RTKs) which activate a variety of kinase-dependent signaling cascades in the cell when stimulated by extracellular growth factors. Despite influencing cells differently, many RTKs appear to recruit and activate the same signaling pathways. To determine whether differences in RTK signaling could be explained by quantitative differences in their recruitment of shared signaling proteins, we sought to characterize the global recruitment profile of RTKs at a genomic level. We first developed a protein microarray assay that permitted the high-throughput testing of thousands of protein-peptide interactions in a systematic format. This in vitro assay provides control of variables such as protein post-modification and cross-reactivity that affect in vivo assays. Besides the identification of protein-peptide interactions, this assay also allows the quantification of these interactions. Thus, the texture , as well as the structure of networks produced by this assay can be analyzed.

We then used this assay to characterize the recruitment profile of the ErbB family of RTKs for all possible phospho-dependent interactions in the human genome. We found that even though these receptors recruit many of the same proteins, their affinities are not the same. As weaker interactions are added to the network, EGFR and ErbB2 increased the diversity of proteins they recruit, while ErbB3 does not. This observation appears to correlate with their oncogenicity, and reveals that quantitative differences in recruitment exist, even within a family of homologous receptors. We extended this work to more diverse RTKs, and used their recruitment profiles to predict the in vivo phosphorylation of signaling proteins using a linear model. We observed that phosphorylation that occurs prior to membrane modification can be predicted, while those that occur afterward cannot. Thus, linear approximations of in vivo phosphorylation can be predicted by the genomic recruitment profile of RTKs measured with an in vitro assay.