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

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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 In unpublished studies, we have developed a yeast-based screening platform that we will apply to discover human proteome-derived peptide sequences that interact with the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.