How activating mutations affect MEK1 regulation and function

Abstract

The MEK1 kinase directly phosphorylates ERK2, after the activation loop of MEK1 is itself phosphorylated by Raf. Studies over the past decade have revealed a large number of disease-related mutations in the MEK1 gene that lead to tumorigenesis and abnormal development. Several of these mutations result in MEK1 constitutive activity, but how they affect MEK1 regulation and function remains largely unknown. Here, we address these questions focusing on two pathogenic variants of the Phe-53 residue, which maps to the well-characterized negative regulatory region of MEK1. We found that these variants are phosphorylated by Raf faster than the wild-type enzyme, and this phosphorylation further increases their enzymatic activity. However, the maximal activities of fully phosphorylated wild-type and mutant enzymes are indistinguishable. On the basis of available structural information, we propose that the activating substitutions destabilize the inactive conformation of MEK1, resulting in its constitutive activity and making it more prone to Raf-mediated phosphorylation. Experiments in zebrafish revealed that the effects of activating variants on embryonic development reflect the joint control of the negative regulatory region and activating phosphorylation. Our results underscore the complexity of the effects of activating mutations on signaling systems, even at the level of a single protein.

Keywords: MEK1; Raf kinase; cancer biology; mitogen-activated protein kinase (MAPK); protein phosphorylation; zebrafish.

Figure 1.

Activating mutations affect thermal stability of MEK1 and its activity toward ERK2. A, location of position 53 in helix A from the structure of MEK1 in PDB code 5bx0 is indicated by a red dot. Shown are helix A (magenta), helix C (green), and helix AL (red), an inhibitory helix in the activation loop. B, top, close connection between helix A, helix C, and helix AL is shown in inactive MEK1. MEK1 is rotated 180° from A. Side chains involved in ionic interactions along helix C are shown. Phe-53 stabilizes these interactions through Lys-57. Glu-114, a catalytic residue, interacts with helix AL. Bottom, model of the three helices in the active configuration of MEK1 based on the structure of active PKA (PDB code 1ATP). Helix AL is refolded, building the active site. Glu-114 points back, adopting interactions in the active site (red dot, Glu-114). C, model for active MEK1, the whole protein based on PKA (PDB code 1ATP). Helix A adopts different interactions with helix C. D, thermal stability of wild-type MEK1 (black), F53L (red), and F53S (green) and ΔΝ60 (eliminating helix A) (gray) as visualized with SYPRO Orange dye. E and F, MEK1/F53L and MEK1/F53S are intrinsically active and, upon phosphorylation, become as active as phosphorylated wild-type MEK1. The normalized dpERK level is an average of four experimental replicates in E and F. Error bars, S.E.

Figure 2.

Activating mutations enhance the rate of MEK1 phosphorylation by Raf. A, complex between B-Raf and MEK1 is based on a structure (PDB code 4mne), with helix A modeled from PDB code 5bx0 (a more complete structure of MEK1). B and C, phosphorylation of MEK1/F53L and MEK1/F53S occurs faster than phosphorylation of wild-type MEK1. The normalized dpMEK is an average of three experimental replicates. The reaction was run at a Raf/MEK ratio of 1:∼217. Error bars, S.E.

Figure 3.

Joint control of MEK1 by mutations and phosphorylation. A, MEK1/F53L and MEK1/F53L/S218A/S222A are indistinguishable in their ability to phosphorylate ERK2 in vitro. The normalized dpERK is an average of three experimental replicates. Error bars, S.E. B, MEK1/F53L variant leads to a severe oval embryo phenotype, whereas the aspect ratio of embryos injected with the MEK1/F53L/S218A/S222A variant is indistinguishable from the uninjected embryos. N_UI = 103, N_F53L = 100, N_{F53L SSAA} = 101 (three experimental replicates). C, MEK1/F53S variant leads to a milder oval embryo phenotype, whereas the aspect ratio of embryos injected with the MEK1/F53S/S218A/S222A variant is indistinguishable from the uninjected embryos. N_UI = 61, N_F53S = 83, N_{F53S SSAA} = 80 (three experimental replicates). D, inhibition of FGFR signaling dramatically reduces elongation of the embryo. N_UI = 37 (two experimental replicates), N_{F53L, DMSO} = 56 (three experimental replicates), N_{F53L, 5 μm SU5402} = 56 (three experimental replicates). B–D, S218A/S222A is abbreviated as SSAA in the figure. B–D, pairwise Student's t tests (two-sided, homoscedastic) were performed to compare groups: **, p < 0.0005; *, p < 0.05, n.s., not significant. B–D, Error bars, S.D.