Mechanisms of selective signaling in MAP kinase phosphorylation networks

摘要 丝裂原活化蛋白激酶(MAPK)级联信号通路是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。尽管有很高的序列相似性，但主要的 MAPK家族在不同的刺激下被激活，并磷酸化很大程度上独特的底物。 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，结合到与催化中心分离的激活域区域。但是，对接 与所有MAPK家族结合的位点符合共同的序列基序，因此尚不清楚如何 对于单个MAPK选择性地实现。我们提议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 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信令网络中。在初步研究中，我们开发了一个基于酵母的筛选平台来鉴定 与MAPKs相互作用的人类蛋白质组衍生序列。ERK2、p38α和JNK1的屏幕点击量 已知相互作用伙伴的对接序列高度丰富，并符合 似乎赋予了MAPK选择性的相互作用。我们将研究这些序列基序的中介能力 体外和培养细胞中的选择性相互作用。我们进一步建议研究可能的新的MAPK 在我们的筛选中确定了底物，特别是参与调节Rho GTPase信号的JNK底物。 为了了解MAPK对接槽如何编码特定的相互作用，我们将解决X射线晶体结构 不同类肽与ERK2和p38α的复合体。在这些结构的指导下，以前的其他结构 报道的和饱和突变筛选，我们将确定区分对接的关键决定因素 不同MAPK的凹槽。我们还将调查已报道的ERK MAPK功能增益突变是如何 改变其结合特异性，扰乱细胞内ERK信号的网络特性。最后，我们会 研究为什么MAPK激酶对它们的同源MAPK具有如此精致的特异性，尽管它们没有 选择性对接位置和催化位置的相互作用。总体而言，这些研究将在 MAPK通路，并阐明这些联系是如何建立的。这项研究将提供更完整的 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.