Substrate specificity of nonreceptor tyrosine kinases

描述（申请人提供）：酪氨酸激酶是真核细胞生长、分化和凋亡的重要调节因子。酪氨酸激酶的不适当激活经常发生在人类癌症中。本项目的目标是了解非受体酪氨酸激酶（NRTKs）的催化和非催化结构域在底物识别和调控中的作用。这些研究可以为设计干扰NRTKs对特定底物识别的分子提供依据。在目的1中，我们将研究非催化结构域的身份、排列和位置控制Src激酶底物特异性的假设。首先，我们将对肽和蛋白质底物以及结构域重排的Src突变形式进行实验。其次，我们已经证明，Src底物特异性可以通过用外源PDZ结构域取代天然的SH3和SH2结构域来重新设计。我们将研究这些嵌合PDZ激酶对底物的识别，并通过PDZ靶向构建人工信号通路。第三，我们将从单细胞生物单链单胞菌中描述具有不寻常结构域组合的酪氨酸激酶。目的2侧重于Cas接头蛋白的过程磷酸化；我们假设过程磷酸化对下游信号传导至关重要。我们将测试Src SH2和SH3结构域在过程磷酸化、细胞转化和迁移中的重要性。为了深入了解过程磷酸化的生物学目的，我们将研究底物结构域被聚合磷酸化基序阵列取代的Cas突变体。我们将使用异核磁共振来确定结合在Src活性位点的Cas肽的结构。Aim 3关注Brk （breast tumor kinase，乳腺肿瘤激酶），这是一种与Src相关的NRTK。在与Senthil Muthuswamy博士的合作中，我们最近发现Brk和ErbB2在人类乳腺癌中共同扩增和共同过表达。我们将研究ErbB2和Brk相互作用的生化基础。特别是，我们将验证ErbB2上的自磷酸化位点与Brk的SH2结构域结合，释放自身抑制的假设。为了测试Brk的c端与酶的SH2结构域相互作用的模型，我们将使用氢交换质谱（HXMS）。我们将进行蛋白质组学实验，以鉴定在共表达Brk和ErbB2的细胞中与Brk相互作用的蛋白。这些研究可能为阻断erbb2阳性乳腺癌中Brk活性的策略提供一个框架。公共卫生相关性：酪氨酸激酶是一种酶，使正常哺乳动物细胞能够对来自环境的信号作出反应，从而导致细胞生长和分裂。然而，在许多形式的癌症中，酪氨酸激酶如Src或Brk（乳腺肿瘤激酶）是不适当的活跃。该项目研究Src和Brk识别细胞蛋白的机制；该实验将提供新的信息，可用于开发特定的激酶抑制剂作为抗癌剂。

DESCRIPTION (provided by applicant): 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.