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.
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.
|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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