Molecular basis of ERK2/MAPK regulation by its cognate regulators and substrates

Abstract

Mitogen activated protein kinases (MAPKs), including ERKs, JNKs, and p38s, compose an important protein kinase family and regulate various cellular processes, such as cell growth, proliferation, differentiation, survival, and apoptosis, in response to extracellular stimulations from hormones, growth factors, cytokines, and environmental stresses. ERK2 is a member of the MAP kinase family, which is involved in many cellular functions and responsive primarily to growth factors and hormones signals. ERK2 plays a key role in intracellular signaling by regulating ∼70 substrates spreading from plasma membrane receptors to nuclear transcription factors. The MAPK/ERK pathway has been well studied. ERK2 can only be activated by MAPK kinase, MEK1/2. However, it is not clear how ERK2 is down regulated and how the specificity and efficiency of ERK2 regulation through protein-protein interactions are achieved.;In my studies, I found that ERK2 interacts with its cognate proteins, including its activator MEK1, inactivator MKP3, and substrate Elk1 via a bipartite interface. Through the mutagenesis analysis and MKP3 activation assay, we found that two surface areas on ERK2, common docking (CD) site \and substrate binding (SB) site, are involved in MKP3 binding and ERK2-induced MKP3 activation, respectively. In addition, we also found those two sites are required for ERK2 substrate, Elk1, phosphorylation. Furthermore we tested the ERK2-MEK1 interaction by both in vitro and in vivo experiments. The results confirmed our hypothesis that the fidelity and efficiency of the regulation of ERK2 signaling is achieved via a bipartite model. Finally we carried out hydrogen-deuterium exchange mass spectrometry to map the ERK2-MKP3 binding surface. The results are consistent with our biochemical data that the interaction between ERK2 and MKP3 requires both the CD and SB sites.;In this bipartite interaction model, the CD site, located opposite to the ERK2 activation site, is responsible for high affinity binding with ERK2 cognate proteins. Originally, the CD site was defined as a linear stretch of amino acid in L16 (residues 311-324). I showed the requirement of Tyr314, Asp316, and Asp319 in this sequence for MKP3 and MEK1 binding. More importantly, my results revealed additional residues, which are topographically close to the CD domain and are involved in MKP3 or MEK1 binding and Elk1 recognition.;The SB site includes residues in alphaD, L12/P+1, L13, alpha G, and MAPK insert, which is considered a putative ERK2 substrate binding site. Mutations in this region not only disrupt MKP3 or MEK1 binding, but also hamper the ability of ERK2 to either activate MKP3 and Elk1, or to be activated by MEK1. Thus, the SB site not only stabilizes the binding but also provides contacts important for modulating the activity and/or specificity of ERK2 target molecules.;Finally, given the structural similarity among the MAPKs, this bipartite protein-protein interaction model may be applicable to the recognition of other MAPKs by their cognate regulators and substrates.