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Mutation and overexpression of receptor tyrosine kinases or the proteins they regulate serve as oncogenic drivers in diverse cancers. To better understand receptor tyrosine kinase signaling and its link to oncogenesis, we used protein microarrays to systematically and quantitatively measure interactions between virtually every SH2 or PTB domain encoded in the human genome and all known sites of tyrosine phosphorylation on 40 receptor tyrosine kinases and on most of the SH2 and PTB domain-containing adaptor proteins. We found that adaptor proteins, like RTKs, have many high affinity bindings sites for other adaptor proteins. In addition, proteins that drive cancer, including both receptors and adaptor proteins, tend to be much more highly interconnected via networks of SH2 and PTB domain-mediated interactions than nononcogenic proteins. Our results suggest that network topological properties such as connectivity can be used to prioritize new drug targets in this well-studied family of signaling proteins.

Although many of the signaling networks activated by receptor tyrosine kinases (RTKs) and cytokine receptors are well understood, how these networks interconnect is much less clear. We set out to determine how cells respond to simultaneous exposure to opposing signals and how their downstream networks process this information. Using six isogenic cell lines, each stably transfected with a different RTK, we found that, in each case, the cognate growth factor induced proliferation, whereas TNFα induced apoptosis. Surprisingly, when the cells were treated simultaneously with growth factor and TNFα, the growth factor enhanced, rather than antagonized, TNFα-induced cell death. In contrast, TNFα had no effect on growth factor-induced proliferation, suggesting that cross-talk between these networks is unidirectional. A quantitative, system-wide study of signaling at early and late time points corroborated this observation: proteins in the RTK networks were not affected by TNFα treatment, but proteins in the TNFα network were affected by growth factors. These studies also highlighted the stress mitogen-activated protein kinase proteins p38 and c-Jun N-terminal kinase as the key nodes of signal integration, and their activation states at an early time point correlated well with subsequent measurements of apoptosis. Knocking down cRaf reduced the growth factor enhancement of TNFα-induced apoptosis, highlighting its role as a regulator of network cross-talk upstream of p38 and c-Jun N-terminal kinase. Overall, we found that when cells encounter conflicting stimuli, their phenotypic response is determined not by the sum of isolated processes, but by how their signaling networks interconnect. This underscores the need to build mechanistic models of network integration as a first step in predicting cellular behavior in complex settings and in rationally designing combination therapies.

Lysate microarrays (reverse-phase protein arrays) hold great promise as a tool for systems-level investigations of signaling and multiplexed analyses of disease biomarkers. To date, however, widespread use of this technology has been limited by questions concerning data quality and the specificity of detection reagents. To address these concerns, we developed a strategy to identify high-quality reagents for use with lysate microarrays. In total, we tested 383 antibodies for their ability to quantify changes in protein abundance or modification in 20 biological contexts across 17 cell lines. Antibodies yielding significant differences in signal were further evaluated by immunoblotting and 82 passed our rigorous criteria. The large-scale data set from our screen revealed that cell fate decisions are encoded not just by the identities of proteins that are activated, but by differences in their signaling dynamics as well. Overall, our list of validated antibodies and associated protocols establish lysate microarrays as a robust tool for systems biology.