Mechanisms of selective signaling in MAP kinase phosphorylation networks

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。我们进一步建议研究推定的新型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。

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.