Defining protein interaction networks involving the atypical MAP kinases ERK4 and ERK7

摘要 丝裂原活化蛋白激酶（MAPK）是响应于细胞凋亡而活化的信号传导网络的核心组分。 各种细胞刺激。ERK 1/2、p38、JNK和ERK 5家族中的经典MAPK位于三个- 分层激酶级联，并已深入研究。相反，MAPK的几个“非典型”成员 家族不参与典型的MAPK级联反应，我们对它们的功能知之甚少。 特别是，虽然已经鉴定了数百种经典MAPK的底物，但只有少数下游底物是必需的。 已知靶点是研究最少的非典型MAPK，ERK 4（MAPK 4）和ERK 7（MAPK 15）。这里我们 我建议利用MAPK底物靶向的基本机制来鉴定新的ERK 4和ERK 7相互作用物 和印刷受体.经典的MAPKs被建立为与其底物中的短线性序列基序相互作用 和调节剂通过激酶结构域中的“对接槽”与催化裂缝分离。当ERK 4 和ERK 7似乎有一个类似的对接槽，我们不知道靶向的序列基序， 这些激酶。在未发表的研究中，我们开发了一种基于酵母的筛选平台， 发现与ERK 4的对接槽相互作用的人蛋白质组衍生肽序列， ERK7。我们将通过确定一组命中的MAPK结合亲和力来验证这些筛选的结果。 肽及其变体。我们将随后研究是否全长蛋白对应的命中 从我们的筛选中筛选出的蛋白质组成了真正的ERK 4或ERK 7相互作用伴侣和/或底物。我们将在 体外激酶测定以确定MAPK是否以某种方式磷酸化这些候选底物 依赖于它们的对接槽，并检查hit蛋白是否与培养细胞中的MAPK缔合。 最后，我们将检查CRISPR/Cas9介导的激酶敲除或突变是否与底物对接。 序列破坏候选底物的磷酸化。通过这些研究，我们希望发现新的 ERK 4和ERK 7的底物和调节剂，从而提供对它们如何发挥作用的机制的了解， 潜在地识别在其控制下的新生物过程。

ABSTRACT Mitogen-activated protein kinases (MAPKs) are core components of signaling networks activated in response to a variety of cellular stimuli. Classical MAPKs in the ERK1/2, p38, JNK and ERK5 families are situated in three- tiered kinase cascades and have been intensely studied. In contrast, several “atypical” members of the MAPK family do not participate in canonical MAPK cascades, and we know comparatively little about how they function. In particular, while hundreds of substrates have been identified for classical MAPKs, only a few downstream targets are known for the most understudied atypical MAPKs, ERK4 (MAPK4) and ERK7 (MAPK15). Here we propose to exploit basic mechanisms of MAPK substrate targeting to identify new ERK4 and ERK7 interactors and substrates. Classical MAPKs are established to interact with short linear sequence motifs in their substrates and regulators through a “docking groove” within the kinase domain separate from the catalytic cleft. While ERK4 and ERK7 appear to have an analogous docking groove, we have no knowledge of sequence motifs targeted by these kinases. In unpublished studies, we have developed a yeast-based screening platform that we will apply to discover human proteome-derived peptide sequences that interact with the docking grooves of ERK4 and ERK7. We will validate results from these screens by determining MAPK binding affinities for a panel of hit peptides and their variants. We will subsequently investigate whether full length proteins corresponding to hits from our screens constitute authentic ERK4 or ERK7 interaction partners and/or substrates. We will perform in vitro kinase assays to determine whether the MAPKs phosphorylate these candidate substrates in a manner dependent on their docking groove and examine whether hit proteins associate with the MAPKs in cultured cells. Finally, we will examine whether CRISPR/Cas9-mediated kinase knockout or mutation of the substrate docking sequence disrupts phosphorylation of candidate substrates. Through these studies, we aim to discover new substrates and regulators of ERK4 and ERK7, thereby providing mechanistic insight into how they function and potentially identifying new biological processes under their control.