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Mechanisms of selective signaling in MAP kinase phosphorylation networks

MAP 激酶磷酸化网络中的选择性信号传导机制

基本信息

批准号：
10624431
负责人：
BENJAMIN E TURK
金额：
$ 35.18万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-08-15 至 2024-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10624431
关键词：
Affinity Animals Binding Binding Sites Biological Process Catalytic Domain Cell physiology Cells Clinical Complex Critical Pathways Cues Cultured Cells Disease Docking Drug Targeting Enzymes Eukaryota Family Feedback Goals Growth Factor Human Impairment In Vitro Individual KRP protein Libraries MAP Kinase Kinase Kinase MAPK1 gene MAPK8 gene Malignant Neoplasms Maps Mediating Mitogen-Activated Protein Kinase Kinases Mitogen-Activated Protein Kinases Mutagenesis Mutation Nature Osmotic Shocks Output Oxidative Stress Pathway interactions Peptide Conformation Peptides Phosphoric Monoester Hydrolases Phosphorylation Phosphotransferases Physiology Property Protein Kinase Protein Kinase Interaction Proteins Proteome Recurrence Regulation Reporter Reporting Research Roentgen Rays Role Screening Result Signal Pathway Signal Transduction Site Specificity Stimulus Structure Variant Yeasts cytokine gain of function mutation human disease improved inhibitor therapy mutant novel p38 Mitogen Activated Protein Kinase peptide structure protein kinase inhibitor recruit response rho GTP-Binding Proteins screening

项目摘要

ABSTRACT Mitogen-activated protein kinase (MAPK) cascades are core components of signaling networks mediating responses to a diverse array of cellular stimuli in eukaryotes. Despite having high sequence similarity, the major MAPK families are activated in response to different stimuli and phosphorylate largely unique sets of substrates. Specificity in MAPK signaling pathways is thought to be conferred by interaction of short linear sequence motifs in substrates and regulators to regions of the kinase domain separate from the catalytic center. However, docking sites binding to all families of MAPK conform to common sequence motifs, and it is therefore unclear how selectively is achieved for an individual MAPK. The goals of our proposed studies are to identify new substrates and regulators in MAPK signaling networks, to understand how signaling specificity is encoded in the primary sequence of MAPKs and their interactors, and to reveal how disease-associated mutations change connections in MAPK signaling networks. In preliminary studies, we developed a yeast-based screening platform to identify human proteome-derived sequences that interact with MAPKs. Hits from screens of ERK2, p38α and JNK1 docking sequences were highly enriched for known interaction partners and conformed to sequence motifs that appear to confer MAPK-selective interactions. We will examine the capacity of these sequence motifs to mediate selective interactions in vitro and in cultured cells. We further propose to investigate putative novel MAPK substrates identified in our screens, in particular JNK substrates involved in regulation of Rho GTPase signaling. To understand how the MAPK docking groove encodes specific interactions, we will solve X-ray crystal structures of different classes of peptides in complex with ERK2 and p38α. Guided by these structures, others previously reported, and saturation mutagenesis screens, we will identify key determinants that distinguish the docking grooves of different MAPKs. We will also investigate how reported gain-of-function mutations in ERK MAPKs change its binding specificity and perturb the network properties of ERK signaling in cells. Finally, we will investigate why MAPK kinases have such exquisite specificity for their cognate MAPKs despite having non- selective docking site and catalytic site interactions. Overall these studies will establish new connections in MAPK pathways and elucidate how those connections are made. This research will provide a more complete understanding of signaling pathways critical for basic cellular process in normal physiology and in disease.

摘要丝裂原活化蛋白激酶（MAPK）级联是介导细胞凋亡的信号网络的核心组成部分。真核生物对各种细胞刺激的反应。尽管具有很高的序列相似性， MAPK家族响应于不同的刺激而被激活，并且磷酸化主要独特的底物组。 MAPK信号通路的特异性被认为是由短线性序列基序的相互作用所赋予的在底物和调节剂中，激酶结构域的区域与催化中心分离。然而，对接与所有MAPK家族结合的位点符合共同的序列基序，因此不清楚如何结合。选择性地针对单个MAPK实现。我们提出的研究目标是确定新的底物以及MAPK信号网络中的调节因子，以了解信号特异性如何在初级细胞中编码。 MAPKs及其相互作用物的序列，并揭示疾病相关突变如何改变连接在MAPK信号网络中。在初步研究中，我们开发了一个基于酵母的筛选平台，与MAPK相互作用的人类蛋白质组衍生序列。来自ERK 2、p38α和JNK 1筛选的命中对接序列高度富集了已知的相互作用配偶体，并符合似乎赋予MAPK选择性相互作用。我们将研究这些序列基序介导体外和培养细胞中的选择性相互作用。我们进一步建议调查推定的新型MAPK 在我们的筛选中鉴定的JNK底物，特别是参与Rho GT3信号传导调节的JNK底物。为了理解MAPK对接沟如何编码特定的相互作用，我们将解析X射线晶体结构，不同种类的肽与ERK 2和p38α的复合物。在这些结构的指导下，其他人以前报告，和饱和诱变筛选，我们将确定区分对接的关键决定因素，不同MAPK的凹槽。我们还将研究已报道的ERK MAPKs功能获得性突变改变其结合特异性并扰乱细胞中ERK信号传导的网络特性。最后我们将研究为什么MAPK激酶对它们的同源MAPK具有如此精确的特异性，选择性对接位点和催化位点相互作用。总的来说，这些研究将建立新的联系， MAPK通路，并阐明这些连接是如何建立的。这项研究将提供一个更完整的了解在正常生理和疾病中对基本细胞过程至关重要的信号通路。