Mechanisms of selective signaling in MAP kinase phosphorylation networks

摘要 丝裂原激活蛋白激酶 (MAPK) 级联是介导信号网络的核心组成部分 真核生物对多种细胞刺激的反应。尽管具有很高的序列相似性，但主要 MAPK 家族响应不同的刺激而被激活，并磷酸化大部分独特的底物组。 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赋予的 在底物和调节器中，激酶结构域的区域与催化中心分开。然而，对接 与 MAPK 所有家族结合的位点符合常见的序列基序，因此尚不清楚如何 选择性地针对单个 MAPK 实现。我们提出的研究的目标是确定新的底物 和 MAPK 信号网络中的调节器，以了解信号特异性是如何在初级中编码的 MAPK 及其相互作用因子的序列，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来识别 与 MAPK 相互作用的人类蛋白质组衍生序列。 ERK2、p38α 和 JNK1 屏幕点击数 对接序列高度富集已知的相互作用伙伴并符合序列基序 似乎赋予 MAPK 选择性相互作用。我们将检查这些序列基序介导的能力 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假定的小说 MAPK 我们的筛选中鉴定出的底物，特别是参与 Rho GTPase 信号传导调节的 JNK 底物。 为了了解 MAPK 对接槽如何编码特定的相互作用，我们将求解 X 射线晶体结构 不同类别的肽与 ERK2 和 p38α 形成复合物。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。我们还将研究如何报道 ERK MAPK 的功能获得突变 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 激酶对其同源 MAPK 具有如此精细的特异性，尽管具有非 选择性对接位点和催化位点相互作用。总的来说，这些研究将在以下领域建立新的联系： MAPK 通路并阐明这些连接是如何建立的。这项研究将提供更完整的 了解对正常生理和疾病中基本细胞过程至关重要的信号通路。

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.