Mapping ERK2-MKP3 Binding Interfaces by Hydrogen/Deuterium Exchange Mass Spectrometry

Bo Zhou,Fang Wang,Jialin Zhang,Sharanya Reddy,Zhong-Yin Zhang,Sijiu Liu

doi:10.1074/jbc.m608916200

Abstract

ERK2, a prototypic member of the MAPK family, plays a central role in regulating cell growth and differentiation. MKP3, an ERK2-specific phosphatase, terminates ERK2 signaling. To understand the molecular basis of ERK2 recognition by MKP3, we carried out hydrogen/deuterium exchange mass spectrometry experiments to map the interaction surfaces between the two proteins. The results show that the exquisite specificity of MKP3 for ERK2 is governed by two distinctive protein-protein interactions. To increase the "effective concentration" of the interacting molecules, the kinase interaction motif in MKP3 ((64)RRLQKGNLPVR(74)) and an MKP3-specific segment ((101)NSSDWNE(107)) bind the common docking site in ERK2 defined by residues in L(16), L(5), beta(7)-beta(8), and alpha(d)-L(8)-alpha(e), located opposite the kinase active site. In addition to this "tethering" effect, additional interactions between the (364)FTAP(367) sequence in MKP3 and the ERK2 substrate-binding site, formed by residues in the activation lip and the P+1 site (beta(9)-alpha(f) loop), L(13) (alpha(f)-alpha(g) loop), and the MAPK insert (L(14)-alpha(1L14)-alpha(2L14)), are essential for allosteric activation of MKP3 and formation of a productive complex whereby the MKP3 catalytic site is correctly juxtaposed to carry out the dephosphorylation of phospho-Thr(183)/phospho-Tyr(185) in ERK2. This bipartite protein-protein interaction model may be applicable to the recognition of other MAPKs by their cognate regulators and substrates.

Highlights

Using a combination of hydrogen/deuterium exchange mass spectrometry (H/DX-MS), mutagenesis, and modeling, we have further established the importance of the KIM sequence and the FXFP peptide for high affinity Extracellular signal-regulated protein kinase 2 (ERK2) binding and ERK2-induced Mitogen-activated protein kinase (MAPK) phosphatase 3 (MKP3) activation
This study has uncovered a striking reduction in the conformational/dynamic flexibility of the MKP3 catalytic domain upon association with ERK2, which likely correlates with ERK2-induced MKP3 activation
In addition to providing solution evidence that the common docking (CD) site is important for binding the KIM and N-␤4-␣4 peptides in MKP3, this study has defined the ERK2 substrate binding (SB) site, which directly interacts with the 364FTAP367 peptide, triggering MKP3 activation

Summary

Introduction

The importance of MAPKs in cellular signaling is well established, there is limited understanding of the molecular basis for MAPK recognition by its activators, inactivators, and substrates. MKP3 substrate specificity is linked to the ability of the substrate to induce productive orientation in the active site This provides a powerful mechanism to ensure high fidelity in MKP3-mediated ERK2 inactivation. Stand the molecular basis of specific ERK2 recognition by MKP3, we have employed hydrogen/deuterium exchange mass spectrometry (H/DX-MS) to map the interaction surfaces between the two proteins concomitant with complex formation. The results yield a structural model for understanding how efficient and precise docking interactions between ERK2 and its cognate substrates and regulators can be achieved

Methods

Results

Conclusion