Cell surface receptors linked to tyrosine kinases control a host of important cellular activities, including proliferation, differentiation, and motility. Disregulated tyrosine kinase signaling is a common feature of many human cancers, thus tyrosine kinases and their downstream effectors are targets for the development of new drugs for the treatment of cancer. In order to take full advantage of such promising new therapies, however, we need an understanding of how tyrosine kinase signaling networks process information on a systems level. While considerable progress has been made in developing quantitative models describing tyrosine kinase signaling networks, these efforts are severely hampered by a lack of quantitative information on how changes in tyrosine phosphorylation are coupled to their downstream effectors containing modular phosphotyrosine binding domains. The goals of this collaborative project are to take advantage of new experimental approaches to address this gap in knowledge directly. Specifically, we will use SH2 profiling, a phosphoproteomic approach that is highly complementary to mass spectrometry-based methods, to quantify dynamic changes in binding sites for specific effector proteins upon receptor tyrosine kinase activation. Responses to different receptors and in different cell types will be compared, allowing systems-level behavior to be correlated with biological outputs. We will also use single-molecule imaging methods to monitor the coupling of specific effectors to receptors in the intracellular environment. These studies will afford unprecedented insight into the interaction dynamics of receptor signaling complexes that will enable much more powerful and accurate models of tyrosine kinase signaling.

Public Health Relevance

Signaling from receptors with tyrosine kinase activity plays an important role in a number of human diseases, in particular cancer. Quantitative computer-based models that accurately describe the signaling mechanism used by these receptors will be very useful in designing new therapies for cancer and in deciding which patients will benefit most from those therapies (individualized medicine). The proposed studies use innovative experimental approaches to reveal new mechanistic insights necessary for building more powerful and accurate models.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project--Cooperative Agreements (U01)
Project #
5U01CA154966-04
Application #
8688931
Study Section
Special Emphasis Panel (ZCA1-SRLB-1 (M1))
Program Officer
Li, Jerry
Project Start
2011-07-01
Project End
2016-06-30
Budget Start
2014-07-01
Budget End
2015-06-30
Support Year
4
Fiscal Year
2014
Total Cost
$489,333
Indirect Cost
$121,248
Name
University of Connecticut
Department
Genetics
Type
Schools of Medicine
DUNS #
022254226
City
Farmington
State
CT
Country
United States
Zip Code
06030
Levy, Aaron D; Xiao, Xiao; Shaw, Juliana E et al. (2018) Noonan Syndrome-Associated SHP2 Dephosphorylates GluN2B to Regulate NMDA Receptor Function. Cell Rep 24:1523-1535
Jadwin, Joshua A; Curran, Timothy G; Lafontaine, Adam T et al. (2018) Src homology 2 domains enhance tyrosine phosphorylation in vivo by protecting binding sites in their target proteins from dephosphorylation. J Biol Chem 293:623-637
Machida, Kazuya (2017) In-Solution SH2 Domain Binding Assay Based on Proximity Ligation. Methods Mol Biol 1555:331-347
Locke, Clifford; Machida, Kazuya; Wu, Yi et al. (2017) Optogenetic activation of EphB2 receptor in dendrites induced actin polymerization by activating Arg kinase. Biol Open 6:1820-1830
Ng, Khong Y; Machida, Kazuya (2017) Rosette Assay: Highly Customizable Dot-Blot for SH2 Domain Screening. Methods Mol Biol 1555:437-451
Ogiue-Ikeda, Mari; Machida, Kazuya (2017) Functionally Altered SH2 Domains for Biochemical Studies: Loss-of-Function Mutant and Domain Concatenation. Methods Mol Biol 1555:199-223
Ogiue-Ikeda, Mari; Machida, Kazuya (2017) Expression and Purification of SH2 Domains Using Baculovirus Expression System. Methods Mol Biol 1555:183-198
Miller, Megan B; Yan, Yan; Machida, Kazuya et al. (2017) Brain Region and Isoform-Specific Phosphorylation Alters Kalirin SH2 Domain Interaction Sites and Calpain Sensitivity. ACS Chem Neurosci 8:1554-1569
Machida, Kazuya; Liu, Bernard (2017) Binding Assays Using Recombinant SH2 Domains: Far-Western, Pull-Down, and Fluorescence Polarization. Methods Mol Biol 1555:307-330
Liu, Bernard A; Machida, Kazuya (2017) Introduction: History of SH2 Domains and Their Applications. Methods Mol Biol 1555:3-35

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