A Bipartite Mechanism for ERK2 Recognition by Its Cognate Regulators and Substrates

Jialin Zhang,Bo Zhou,Chao-Feng Zheng,Zhong-Yin Zhang

doi:10.1074/jbc.m303909200

Jialin Zhang, Bo Zhou + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m303909200

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Abstract

Mitogen-activated protein (MAP) kinases control gene expression in response to extracellular stimuli and exhibit exquisite specificity for their cognate regulators and substrates. We performed a structure-based mutational analysis of ERK2 to identify surface areas that are important for recognition of its interacting proteins. We show that binding and activation of MKP3 by ERK2 involve two distinct protein-protein interaction sites in ERK2. Thus, the common docking (CD) site composed of Glu-79, Tyr-126, Arg-133, Asp-160, Tyr-314, Asp-316, and Asp-319 are important for high affinity MKP3 binding but not essential for ERK2-induced MKP3 activation. MKP3 activation requires residues Tyr-111, Thr-116, Leu-119, Lys-149, Arg-189, Trp-190, Glu-218, Arg-223, Lys-229, and His-230 in the ERK2 substrate-binding region, located distal to the common docking site. Interestingly, many of the residues important for MKP3 recognition are also used for Elk1 binding and phosphorylation. In addition to the shared residues, there are also residues that are unique to each target recognition. There is evidence indicating that the CD site and the substrate-binding region defined here are also utilized for MEK1 recognition, and indeed, we demonstrate that the binding of MKP3, Elk1, and MEK1 to ERK2 is mutually exclusive. Taken together, our data suggest that the efficiency and fidelity of ERK2 signaling is achieved by a bipartite recognition process. In this model, one part of the ERK2-binding proteins (e.g. the kinase interaction motif sequence) docks to the CD site located on the back side of the ERK2 catalytic pocket for high affinity association, whereas the interaction of the substrate-binding region with another structural element (e.g. the FXFP motif in MKP3 and Elk1) may not only stabilize binding but also provide contacts crucial for modulating the activity and/or specificity of ERK2 target molecules.

Highlights

Mitogen-activated protein (MAP) kinase deactivation occurs through the action of multiple protein phosphaidence indicating that the common docking (CD) site and the substratebinding region defined here are utilized for MEK1 recognition, and we demonstrate that the binding of MAP kinase phosphatase 3 (MKP3), Elk1, and MEK1 to ERK2 is mutually exclusive
Definition of ERK2 Common Docking Site for MKP3—Many ERK2-interacting proteins such as ERK2 activators (e.g. MEK1/2), inactivators (e.g. MKP3 and HePTP), and substrates (e.g. RSK1 and Elk1) contain a kinase interaction motif (KIM) characterized by a cluster of 2–3 positively charged Arg or Lys residues that are important for ERK2 binding (6, 11, 18 –23)
Deletion of the KIM sequence from MKP3 resulted in a 135-fold reduction in ERK2 binding affinity but did not affect the propensity of MKP3 to be activated by ERK2 [11]

Summary

EXPERIMENTAL PROCEDURES

DNA Constructs and Site-directed Mutagenesis—The coding sequence of ERK2 was subcloned into pET15b to yield the N-terminal His6-tagged ERK2. Determination of Dissociation Constants—The dissociation constants of ERK2 and its mutants for MKP3 were determined by the activation assay at 30 °C and pH 7.0, in 50 mM 3,3-dimethylglutarate buffer, containing 1 mM EDTA with an ionic strength of 0.15 M adjusted by addition of NaCl [11]. To determine the affinity of Elk or MEK1 for ERK2, the competitive binding assay was used [11] In this assay, the reaction was initiated by the addition of 0.1 ␮M MKP3 in a mixture (0.2 ml) containing 40 mM pNPP, 1.2 ␮M ERK2, and various concentrations of the Elk peptide, Elk1-(307– 428), or MEK1 at 30 °C and pH 7.0, in 50 mM 3,3-dimethylglutarate buffer, containing 1 mM EDTA with an ionic strength of 0.15.

RESULTS

67 Ϯ 7 89 Ϯ 7 87 Ϯ 5 58 Ϯ 4 97 Ϯ 10 95 Ϯ 6 20 Ϯ 2 25 Ϯ 2 42 Ϯ 5 117 Ϯ 8

DISCUSSION