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, p38? 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 p38?. 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.
Enzymes called protein kinases are important regulators of cell responses to environmental cues, and their deregulation is a hallmark of multiple human diseases. We aim to understand how closely related protein kinases called MAP kinases, which are targeted by drugs in current clinical use, can effect completely different changes to cell physiology. This research will help explain both beneficial and undesired effects of protein kinase inhibitor drugs, and may lead to improved strategies to treat disease.
