Tyrosine kinases are important regulators of growth, differentiation, and apoptosis in eukaryotic cells. Inappropriate activation of tyrosine kinases often occurs in human cancers. The goal of this project is to understand the contributions of the catalytic and noncatalytic domains of nonreceptor tyrosine kinases (NRTKs) in substrate recognition and regulation. These studies could provide a basis for the design of molecules to interfere with the recognition of specific substrates by NRTKs.
In Aim 1, we will investigate the hypothesis that the identity, arrangement, and placement of the noncatalytic domains govern Src kinase substrate specificity. First, we will carry out experiments with peptide and protein substrates, and mutant forms of Src in which the domains have been rearranged. Second, we have shown that Src substrate specificity can be reengineered by replacing the natural SH3 and SH2 domains with heterologous PDZ domains. We will study substrate recognition by these chimeric PDZ-kinases, and construct artificial signaling pathways by PDZ targeting. Third, we will characterize tyrosine kinases with unusual domain combinations from the unicellular organism Monosiga brevicollis.
Aim 2 focuses on the processive phosphorylation of the Cas adaptor protein;we hypothesize that processive phosphorylation is critical for downstream signaling. We will test the importance of the Src SH2 and SH3 domains on processive phosphorylation, cell transformation, and migration. To gain insight into the biological purpose of processive phosphorylation, we will study Cas mutants in which the substrate domain has been replaced by arrays of polymerized phosphorylation motifs. We will use heteronuclear NMR to determine the structure of a Cas peptide bound in the active site of Src.
Aim 3 focuses on Brk (breast tumor kinase), a NRTK related to Src. In collaboration with Dr. Senthil Muthuswamy, we recently showed that Brk and ErbB2 are co-amplified and co-overexpressed in human breast cancers. We will study the biochemical basis for the interaction between ErbB2 and Brk. In particular, we will test the hypothesis that autophosphorylation sites on ErbB2 bind to the SH2 domain of Brk, releasing auto inhibition. To test the model that the C-terminus of Brk interacts with the enzyme's SH2 domain, we will use hydrogen-exchange mass spectrometry (HXMS). We will carry out proteomic experiments to identify proteins that interact with Brk in cells co-expressing Brk and ErbB2. These studies may provide a framework for strategies to block the activity of Brk in ErbB2-positive breast cancers.

Public Health Relevance

Tyrosine kinases are enzymes that enable normal mammalian cells to respond to signals from their environment that cause the cells to grow and divide. In many forms of cancer, however, tyrosine kinases such as Src or Brk (breast tumor kinase) are inappropriately active. This project investigates the mechanisms by which Src and Brk recognize cellular proteins;the experiments will provide new information that could be used to develop specific kinase inhibitors as anticancer agents.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA058530-18
Application #
8259482
Study Section
Macromolecular Structure and Function E Study Section (MSFE)
Program Officer
Knowlton, John R
Project Start
1993-09-01
Project End
2014-05-31
Budget Start
2012-06-01
Budget End
2014-05-31
Support Year
18
Fiscal Year
2012
Total Cost
$201,990
Indirect Cost
$72,274
Name
State University New York Stony Brook
Department
Physiology
Type
Schools of Medicine
DUNS #
804878247
City
Stony Brook
State
NY
Country
United States
Zip Code
11794
Schultheiss, Kira P; Craddock, Barbara P; Suga, Hiroshi et al. (2014) Regulation of Src and Csk nonreceptor tyrosine kinases in the filasterean Ministeria vibrans. Biochemistry 53:1320-9
Li, Wanqing; Scarlata, Suzanne; Miller, W Todd (2009) Evidence for convergent evolution in the signaling properties of a choanoflagellate tyrosine kinase. Biochemistry 48:5180-6
Yadav, Shalini S; Yeh, Brian J; Craddock, Barbara P et al. (2009) Reengineering the signaling properties of a Src family kinase. Biochemistry 48:10956-62
Patwardhan, Parag; Shiba, Kiyotaka; Gordon, Chris et al. (2009) Synthesis of functional signaling domains by combinatorial polymerization of phosphorylation motifs. ACS Chem Biol 4:751-8
Yadav, Shalini S; Miller, W Todd (2008) The evolutionarily conserved arrangement of domains in SRC family kinases is important for substrate recognition. Biochemistry 47:10871-80
King, Nicole; Westbrook, M Jody; Young, Susan L et al. (2008) The genome of the choanoflagellate Monosiga brevicollis and the origin of metazoans. Nature 451:783-8
Wu, Jinhua; Li, Wanqing; Craddock, Barbara P et al. (2008) Small-molecule inhibition and activation-loop trans-phosphorylation of the IGF1 receptor. EMBO J 27:1985-94
Manning, Gerard; Young, Susan L; Miller, W Todd et al. (2008) The protist, Monosiga brevicollis, has a tyrosine kinase signaling network more elaborate and diverse than found in any known metazoan. Proc Natl Acad Sci U S A 105:9674-9
Li, Wanqing; Young, Susan L; King, Nicole et al. (2008) Signaling properties of a non-metazoan Src kinase and the evolutionary history of Src negative regulation. J Biol Chem 283:15491-501
Yadav, Shalini S; Miller, W Todd (2007) Cooperative activation of Src family kinases by SH3 and SH2 ligands. Cancer Lett 257:116-23

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