Intracellular signaling networks that involve protein tyrosine kinases are critical in the control of most cellular processes, including growth, adhesion, migration, differentiation, and apoptosis. Misregulation of these networks results in a variety of human diseases, including cancer, diabetes, and immune deficiency. Many of the proteins in these networks contain Src homology 2 (SH2) or phosphotyrosine binding (PTB) domains, which recognize tyrosine-phosphorylated proteins in a sequence-specific fashion. In this proposal, the molecular recognition properties of virtually every SH2 and PTB domain encoded in the human genome will be investigated with respect to physiologically-relevant ligands using protein microarray technology. Recombinant SH2/PTB domains will be arrayed in the wells of microtiter plates and subsequently probed with 258 fluorescently-labeled phosphopeptides representing experimentally-verified sites of tyrosine phosphorylation on human receptor tyrosine kinases, as well as with 604 peptides representing sites of tyrosine phosphorylation on downstream proteins (nonreceptor tyrosine kinases and SH2/PTB-containing proteins). By probing the arrays with eight different concentrations of each peptide, equilibrium dissociation constants will be determined for the binding of each peptide to each protein (~140 active SH2/PTB constructs). This effort will produce high quality, quantitative protein interaction networks which will reveal individual connections between signaling proteins, as well as how network connectivity changes with protein concentration. We have previously proposed that the extent to which a protein becomes more promiscuous when overexpressed contributes to its oncogenicity, and the study described here will generate the quantitative data needed to investigate this hypothesis further. In addition, the information revealed by our systematic efforts should prove invaluable to cell and cancer biologists who study tyrosine kinase-mediated signaling, to computational biologists who study molecular recognition, and to systems biologists who seek to model signal transduction networks. As such, we intend to make our data easily accessible though an interactive web site in formats suitable both for broad computational studies and for more focused hypothesis-driven inquiries. It is our hope that the studies described here will shed light on how signaling proteins are integrated into complex networks and how we can intervene most effectively when these networks go awry. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Exploratory/Developmental Grants Phase II (R33)
Project #
5R33CA128726-02
Application #
7484307
Study Section
Special Emphasis Panel (ZCA1-SRRB-C (M1))
Program Officer
Li, Jerry
Project Start
2007-08-13
Project End
2010-07-31
Budget Start
2008-08-01
Budget End
2009-07-31
Support Year
2
Fiscal Year
2008
Total Cost
$343,463
Indirect Cost
Name
Harvard University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
082359691
City
Cambridge
State
MA
Country
United States
Zip Code
02138
Gujral, T S; Karp, R L; Finski, A et al. (2013) Profiling phospho-signaling networks in breast cancer using reverse-phase protein arrays. Oncogene 32:3470-6
Koytiger, Grigoriy; Kaushansky, Alexis; Gordus, Andrew et al. (2013) Phosphotyrosine signaling proteins that drive oncogenesis tend to be highly interconnected. Mol Cell Proteomics 12:1204-13
Gujral, Taranjit S; Karp, Ethan S; Chan, Marina et al. (2013) Family-wide investigation of PDZ domain-mediated protein-protein interactions implicates ?-catenin in maintaining the integrity of tight junctions. Chem Biol 20:816-27
Krall, Jordan A; Beyer, Elsa M; MacBeath, Gavin (2011) High- and low-affinity epidermal growth factor receptor-ligand interactions activate distinct signaling pathways. PLoS One 6:e15945
Gujral, Taranjit S; MacBeath, Gavin (2010) A system-wide investigation of the dynamics of Wnt signaling reveals novel phases of transcriptional regulation. PLoS One 5:e10024
Mehlitz, Adrian; Banhart, Sebastian; Mäurer, André P et al. (2010) Tarp regulates early Chlamydia-induced host cell survival through interactions with the human adaptor protein SHC1. J Cell Biol 190:143-57
Boettcher, Jan Peter; Kirchner, Marieluise; Churin, Yuri et al. (2010) Tyrosine-phosphorylated caveolin-1 blocks bacterial uptake by inducing Vav2-RhoA-mediated cytoskeletal rearrangements. PLoS Biol 8:
Kaushansky, Alexis; Allen, John E; Gordus, Andrew et al. (2010) Quantifying protein-protein interactions in high throughput using protein domain microarrays. Nat Protoc 5:773-90
Gordus, Andrew; Krall, Jordan A; Beyer, Elsa M et al. (2009) Linear combinations of docking affinities explain quantitative differences in RTK signaling. Mol Syst Biol 5:235
Wolf-Yadlin, Alejandro; Sevecka, Mark; MacBeath, Gavin (2009) Dissecting protein function and signaling using protein microarrays. Curr Opin Chem Biol 13:398-405

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