MKP5 allostery in MAPK regulation and signaling in the heart

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ABSTRACT The catalytic activity and specificity of the mitogen-activated protein kinase (MAPK) phosphatases (MKPs) is governed through their ability to interact with the MAPKs. The MKPs set the fidelity and kinetics of MAPK signaling by regulating, in a spatially distinct manner, both the magnitude and duration of MAPK activity. However, these regulatory mechanisms alone are insufficient to explain the complexity of MKP/MAPK- mediated signaling. This is exemplified by the observation that inactivation of MKPs deliver unique, and in some cases, non-obvious physiological and pathophysiological outcomes that has led to the realization that MKPs represent valuable therapeutic targets for the treatment of certain diseases. We have found that MKP5, which directly dephosphorylates p38 MAPK and JNK, is involved in the development of cardiac fibrosis, a serious sequalae that precedes heart failure. Mice lacking MKP5 are protected from the development of cardiac fibrosis and heart failure in a model of pressure overload. A high throughput small molecule screen identified a first-in-class inhibitor of MKP5. Resolution of the co-crystal structure of the inhibitor in complex with MKP5 revealed that the inhibitor bound to an allosteric site on MKP5. We hypothesize that the MKP5 allosteric site represents an additional mode of MKP5 regulation that governs signaling specificity and that this allosteric site represents the “Achilles heel” of MKP5 for small molecule targeting. The broad goal of this work is two-fold. First, we will define the molecular basis for how the allosteric site regulates MKP5 catalysis and MAPK signaling. Second, whether disruption of the allosteric site produces outcomes in a disease context that provide proof-of-concept that if successfully “drugged” this site represents a platform for development of first-in-class MKP inhibitors. We will execute the specifics of these two broad goals through three specific aims. Aim 1, will employ structural biology and biophysical approaches to elucidate the structural determinants and binding mechanisms conferred by the MKP5 allosteric site to determine how this site regulates MKP5 catalysis and MAPK binding. A co-complex between MKP5 and p38 MAPK will be obtained to define how p38 MAPK modulates MKP5 through the allosteric site. In Aim 2, the effects of mutants in the MKP5 allosteric site on MAPK signaling will be determined by generating knockin cells harboring MKP5 allosteric site mutants using CRISPR-Cas approaches. The specificity of the MKP5 allosteric site to “fine-tune” MAPK-mediated signaling will be determined using non-biased phosphoproteomic and transcriptomic approaches. In Aim 3, the effects of targeting the MKP5 allosteric site in cardiac fibrosis and heart failure will be determined through the generation of a novel MKP5 allosteric site mutant knock-in mouse. These studies will define a new mode of operation for MKP5 that will provide information on the actions of the allosteric site at the atomic level. How the MKP5 allosteric site impacts physiological signaling and the validity of the allosteric site as a “druggable” target in disease therapeutics will be defined.