Identification of a Novel Mitogen-Activated Protein Kinase Kinase Activation Domain Recognized by the Inhibitor PD 184352

Abstract

Utilizing a genetic screen in the yeast Saccharomyces cerevisiae, we identified a novel autoactivation region in mammalian MEK1 that is involved in binding the specific MEK inhibitor, PD 184352. The genetic screen is possible due to the homology between components of the yeast pheromone response pathway and the eukaryotic Raf-MEK-ERK signaling cascade. Using the FUS1::HIS3 reporter as a functional readout for activation of a reconstituted Raf-MEK-ERK signaling cascade, randomly mutagenized MEK variants that were insensitive to PD 184352 were obtained. Seven single-base-change mutations were identified, five of which mapped to kinase subdomains III and IV of MEK. Of the seven variants, only one, a leucine-to-proline substitution at amino acid 115 (Leu115Pro), was completely insensitive to PD 184352 in vitro (50% inhibitory concentration >10 M). However, all seven mutants displayed strikingly high basal activity compared to wild-type MEK. Overexpression of the MEK variants in HEK293T cells resulted in an increase in mitogen-activated protein (MAP) kinase phosphorylation, a finding consistent with the elevated basal activity of these constructs. Further, treatment with PD 184352 failed to inhibit Leu115Pro-stimulated MAP kinase activation in HEK293T cells, whereas all other variants had some reduction in phospho-MAP kinase levels. By using cyclic AMP-dependent protein kinase (1CDK) as a template, an MEK homology model was generated, with five of the seven identified residues clustered together, forming a potential hydrophobic binding pocket for PD 184352. Additionally, the model allowed identification of other potential residues that would interact with the inhibitor. Directed mutation of these residues supported this region's involvement with inhibitor binding.The mitogen-activated protein (MAP) kinase cascade, comprised of MAP kinase (ERK), MAP kinase kinase (MEK), and MAP kinase kinase kinase (Raf), is an evolutionarily conserved signaling module that regulates growth, differentiation, and movement in eukaryotic cells in response to extracellular stimulation (reviewed in reference 23). A key regulatory component of this pathway is MAP kinase kinase, or MEK, a dualspecificity kinase that phosphorylates MAP kinase (ERK) on specific threonine and tyrosine residues. This phosphorylation activates MAP kinase and induces a host of downstream cellular responses (reviewed in reference 10).MEK itself, is subject to regulation and activation by Raf phosphorylation on two serine residues, Ser218 and Ser222 (2, 33), which lie in a regulatory loop between conserved kinase subdomains VII and VIII (16). Substitution of these serine residues with negatively charged amino acids, such as aspartate or glutamate, partially mimics the phosphorylation modification and results in a constitutively active kinase, presumably through stabilization of the regulatory loop, allowing the enzyme to retain an active conformation (2).Another regulatory feature of MEK is a proline-rich region, located carboxy terminal to the regulatory loop, whic...

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