Evolutionary history of MEK1 illuminates the nature of deleterious mutations

Abstract

Mutations in signal transduction pathways lead to various diseases including cancers. MEK1 kinase, encoded by the human MAP2K1 gene, is one of the central components of the MAPK pathway and more than a hundred somatic mutations in the MAP2K1 gene were identified in various tumors. Germline mutations deregulating MEK1 also lead to congenital abnormalities, such as the cardiofaciocutaneous syndrome and arteriovenous malformation. Evaluating variants associated with a disease is a challenge, and computational genomic approaches aid in this process. Establishing evolutionary history of a gene improves computational prediction of disease-causing mutations; however, the evolutionary history of MEK1 is not well understood. Here, by revealing a precise evolutionary history of MEK1, we construct a well-defined dataset of MEK1 metazoan orthologs, which provides sufficient depth to distinguish between conserved and variable amino acid positions. We matched known and predicted disease-causing and benign mutations to evolutionary changes observed in corresponding amino acid positions and found that all known and many suspected disease-causing mutations are evolutionarily intolerable. We selected several variants that cannot be unambiguously assessed by automated prediction tools but that are confidently identified as "damaging" by our approach, for experimental validation in Drosophila. In all cases, evolutionary intolerant variants caused increased mortality and severe defects in fruit fly embryos confirming their damaging nature. We anticipate that our analysis will serve as a blueprint to help evaluate known and novel missense variants in MEK1 and that our approach will contribute to improving automated tools for disease-associated variant interpretation.

Keywords: Drosophila; damaging mutations; serine–threonine kinase; variants of unknown significance.

Fig. 1.

Central role of MAP2K kinases in the MAPK/ERK pathway. Arrows indicate known interactions. Adapted from ref. .

Fig. 2.

Maximum likelihood phylogenetic tree of the MEK protein family and their closest homologs from the representative set of eukaryotic species. Bootstrap support values >50% are indicated as circles.

Fig. 3.

Evolutionary history of the MEK family across the major eukaryotic lineages. Schematic eukaryotic tree of life was extracted from NCBI Taxonomy Browser. Each member of MEK family (MEK1 through MEK7) is indicated by a different color. Filled squares represent confidently assigned orthologs. Empty squares represent paralogs (placed next to the filled square of corresponding orthologs) or proteins that could not be confidently assigned to a specific MEK class.

Fig. 4.

Conserved and variable positions in MEK1 proteins from various species. Small subset of more than 300 MAP2K1 orthologs is shown. Positions highlighted in black are invariable in the entire dataset of MEK1 orthologs. Variable positions are shown in teal.

Fig. 5.

Prediction of functional consequences for selected MEK1 variants of unknown significance by different automated tools (, , –81). The green circle represents “benign” and red circle represents “pathogenic.”

Fig. 6.

Activating mutations F53Y, G128D, and Q56P in MEK1 cause developmental defects. (A) Lethality of all three mutations is increased when expressed under control of maternal driver (MTD-gal4). The control is overexpression of mek^WT (n = 1,476). mek^F53Y (n = 2,596); mek^G128D (n = 1,742); mek^Q56P (n = 727). (B) Defects in embryo patterning are absent without the Gal4 (Top). Defects in embryo patterning are frequent in all three mutants, when under control of a maternal germline driver (MTD-Gal4) (Bottom, red). (C) Normal embryonic cuticle with the 8 denticle belts shown as well as a normal head (white arrow). mek^WT is phenotypically normal (n = 46). (D–F) Representative embryo cuticles show defects in the maternal patterning system. Head pattering is affected by all three mutants (green arrow). (D) mek^F53Y cuticles have disrupted heads as well as fused or missing denticle belts (n = 83). (E) mek^G128D cuticles have missing heads as well as fused or missing denticle belts (n = 24). (F) mek^Q56P cuticles have missing heads as well as fused or missing denticle belts as well as a failure to retract the germband (n = 61). Additionally, 61% of embryos fail to form a cuticle (Inset). (G) Lethality of all three mutations is increased when expressed under control of the somatic follicle cell driver (TJ-gal4). The control is overexpression of mek^WT (n = 2,606). mek^F53Y (n = 1,167); mek^G128D (n = 1,351); mek^Q56P (n = 223). (H) A graded effect is seen in the embryo cuticles of the three mutants, when under control of a maternal somatic driver (TJ-Gal4) (red). (I) Normally, embryos have 8 segments, visualized by the presence of 8 denticle belts on the ventral side of the embryo. mek^WT is phenotypically normal (n = 13). (J–L) Severity increases between the three mutants, which are dorsalized in a graded manner. Dorsalized eggshells result in dorsalized embryos with malformed heads (green arrows), as evidenced in the mutants mek^G128D and mek^Q56P. (J) mek^F53Y is phenotypically normal (n = 48). (K) mek^G128D is weakly dorsalized (n = 43). (L) mek^Q56P is very strongly dorsalized and has no detectable denticle belts (n = 30). (M) Normal eggshells show normal spacing of the respiratory filaments (n = 18). (N–P) Dorsalized embryos are caused by dorsalized eggshells. (N) F53Y eggshells are phenotypically normal (n = 14). (O) G128D eggshells are weakly dorsalized, as evidenced by the increase in the spacing between the two respiratory appendages (n = 12). (P) Q56P eggshells are more strongly dorsalized (n = 16).