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.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Macromolecular Structure and Function E Study Section (MSFE)
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Knowlton, John R
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State University New York Stony Brook
Schools of Medicine
Stony Brook
United States
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Suga, Hiroshi; Miller, W Todd (2018) Src signaling in a low-complexity unicellular kinome. Sci Rep 8:5362
Delle Bovi, Richard J; Miller, W Todd (2017) Expression and purification of functional insulin and insulin-like growth factor 1 holoreceptors from mammalian cells. Anal Biochem 536:69-77
Cabail, M Zulema; Chen, Emily I; Koller, Antonius et al. (2016) Auto-thiophosphorylation activity of Src tyrosine kinase. BMC Biochem 17:13
Aleem, Saadat; Georghiou, George; Kleiner, Ralph E et al. (2016) Structural and Biochemical Basis for Intracellular Kinase Inhibition by Src-specific Peptidic Macrocycles. Cell Chem Biol 23:1103-1112
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
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
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
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 ?-Diol Lipids. Biochemistry 54:5457-68
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
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

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