Tyrosine kinases are important regulators of growth, differentiation, and apoptosis in eukaryotic cells. Constitutive or 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 regulation and substrate recognition. These studies could provide a basis for the design of molecules to interfere with the phosphorylation of specific substrates by NRTKs.
Aim 1 focuses on the roles of noncatalytic domains in NRTK signaling.
In Aim 1 A, we will study Src- catalyzed 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. We will also use a novel single molecule fluorescence assay to study the mechanism of processive phosphorylation.
In Aim 1 B, we will study the evolution of phosphotyrosine signaling. The pTyr-based signaling system was originally thought to be unique to multicellular animals. Surprisingly, recent genomic analyses have demonstrated that choanoflagellates and other unicellular organisms possess numerous tyrosine kinases. Many of these ancestral kinases contain domain combinations that are not seen in metazoans. We hypothesize that these domains are involved in substrate targeting, and that kinase autoinhibition arose more recently in metazoan evolution. We will clone representatives of NRTK families from premetazoans and screen their cellular activity. We will also express and purify the kinases and compare their enzymatic properties with their mammalian counterparts. We will study the importance of non-catalytic domains in NRTK substrate recognition, and carry out a proteomic screen for potential substrates from choanoflagellates. The proposed work has the potential to uncover new pathway components, connections, and regulatory mechanisms that function in NRTK signaling.
Aim 2 uses the approaches developed in Aim 1 to study Brk (breast tumor kinase), a NRTK that is overexpressed in human breast cancer. We previously showed that Brk is regulated by intramolecular interactions involving the SH3 and SH2 domains. In this project, we investigate the hypothesis that these interactions are disrupted in cancer cells. We will combine biochemical and cell biological approaches to investigate the effects of cancer-associated mutations that activate Brk. Brk is negatively regulated by C- terminal phosphorylation, but the kinase responsible has not been identified. We present evidence that a previously uncharacterized NRTK, Srms, phosphorylates the C-terminus of Brk. We will characterize the enzymatic activity of Srms, and study its interaction with Brk in vitro. We will also examine the ability of Srms to regulate Brk in breast epithelial cells. The information provided by these studies will be important in determining how Brk activity is deregulated in breast cancer, and may provide a framework for strategies to block the activity of Brk.

Public Health Relevance

This project examines how normal cellular growth control mechanisms can become inappropriately activated in cancer cells. The first aim studies basic science questions: how signaling enzymes recognize their substrates, and the evolutionary origins of cell signaling pathways. The second aim studies how the activity of the signaling protein Brk (breast tumor kinase) is regulated. The proposed work has the potential to uncover new regulatory mechanisms, and may provide new strategies to block the activity of Brk in breast cancer.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
2R01CA058530-19A1
Application #
8691276
Study Section
Macromolecular Structure and Function E Study Section (MSFE)
Program Officer
Knowlton, John R
Project Start
1993-09-01
Project End
2019-04-30
Budget Start
2014-05-14
Budget End
2015-04-30
Support Year
19
Fiscal Year
2014
Total Cost
Indirect Cost
Name
State University New York Stony Brook
Department
Physiology
Type
Schools of Medicine
DUNS #
City
Stony Brook
State
NY
Country
United States
Zip Code
11794
Aleem, Saadat U; Georghiou, George; Kleiner, Ralph E et al. (2016) Structural and Biochemical Basis for Intracellular Kinase Inhibition by Src-specific Peptidic Macrocycles. Cell Chem Biol 23:1103-12
Cabail, M Zulema; Chen, Emily I; Koller, Antonius et al. (2016) Auto-thiophosphorylation activity of Src tyrosine kinase. BMC Biochem 17:13
Fan, Gaofeng; Aleem, Saadat; Yang, Ming et al. (2015) Protein-tyrosine Phosphatase and Kinase Specificity in Regulation of SRC and Breast Tumor Kinase. J Biol Chem 290:15934-47
Touchette, Megan H; Bommineni, Gopal R; Delle Bovi, Richard J et al. (2015) Diacyltransferase Activity and Chain Length Specificity of Mycobacterium tuberculosis PapA5 in the Synthesis of Alkyl β-Diol Lipids. Biochemistry 54:5457-68
Tsui, Tiffany; Miller, W Todd (2015) Cancer-Associated Mutations in Breast Tumor Kinase/PTK6 Differentially Affect Enzyme Activity and Substrate Recognition. Biochemistry 54:3173-82
Cabail, M Zulema; Li, Shiqing; Lemmon, Eric et al. (2015) The insulin and IGF1 receptor kinase domains are functional dimers in the activated state. Nat Commun 6:6406
Aleem, Saadat U; Craddock, Barbara P; Miller, W Todd (2015) Constitutive Activity in an Ancestral Form of Abl Tyrosine Kinase. PLoS One 10:e0131062
Krishnan, Harini; Retzbach, Edward P; Ramirez, Maria I et al. (2015) PKA and CDK5 can phosphorylate specific serines on the intracellular domain of podoplanin (PDPN) to inhibit cell motility. Exp Cell Res 335:115-22
Yokoyama, Noriko; Miller, W Todd (2015) Molecular characterization of WDCP, a novel fusion partner for the anaplastic lymphoma tyrosine kinase ALK. Biomed Rep 3:9-13
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

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