Intrinsically active MEK variants are differentially regulated by proteinases and phosphatases

Abstract

MAPK/ERK kinase (MEK) 1/2 are central signaling proteins that serve as specificity determinants of the MAPK/ERK cascade. More than twenty activating mutations have been reported for MEK1/2, and many of them are known to cause diseases such as cancers, arteriovenous malformation and RASopathies. Changes in their intrinsic activity do not seem to correlate with the severity of the diseases. Here we studied four MEK1/2 mutations using biochemical and molecular dynamic methods. Although the studied mutants elevated the activating phosphorylation of MEK they had no effect on the stimulated ERK1/2 phosphorylation. Studying the regulatory mechanism that may explain this lack of effect, we found that one type of mutation affects MEK stability and two types of mutations demonstrate a reduced sensitivity to PP2A. Together, our results indicate that some MEK mutations exert their function not only by their elevated intrinsic activity, but also by modulation of regulatory elements such as protein stability or dephosphorylation.

Drosophila RASopathy Models Identify Disease Subtype Differences and Biomarkers of Drug Efficacy

“…Two lines-PTPN11 Y279C , PTPN11 Q510E -displayed higher levels of pMEK, indicating more complex regulation of MAPK activity in which pMEK and pERK do not mirror one another. This phenomenon has been reported in mammalian studies: recruitment of additional cofactors differentially altered phosphorylation levels of pERK vs. pMEK (Kuang et al, 2009;Lito et al, 2014;Ordan et al, 2018) . PTPN11 Y279C , PTPN11 Q510P lines displayed initial activation of pERK followed by suppression of pERK within 24 hours, suggesting negative feedback regulation, a well-described feature of the MAPK pathway (Fritsche-Guenther et al, 2011;Lake et al, 2016;Shin et al, 2009) .…”

An ultrasensitive fiveplex activity assay for cellular kinases

“…2a–e). Undetectable MEK catalysis in quiescent cells was likely the result of cytoplasmic phosphatases, which deactivate MEK under resting conditions 54 . Stimulation with T + E + I for 15 min increased the assay RFUs by ~8–30-fold, and the background-corrected coefficient of variation for all measured kinases was less than 20% (Fig.…”

Catalytic domain plasticity of MKK7 reveals structural mechanisms of allosteric activation and new targeting opportunities

“…Since the introduction of two phosphorylation-mimetic mutations was insufficient to induce an active state in the crystal structure, we postulated a potential contribution of other regulatory elements that could trigger kinase activation. Previous studies have demonstrated that MEK1 harbors the N-terminal helix preceding the kinase domain, a regulatory element that controls kinase activity 41,42 . Consistent with previous prediction 43 , comparative sequence analyses suggested that despite slight variations for example in the C glutamate position, MAP2K members shared highly similar domain architecture, prompting us to postulate existence of the N-terminal helix in MKK7 with a similar regulatory role to that in the counterpart MEK1/2 (Figure 2A and Supplementary Figure S1 To investigate the presence of the N-terminal regulatory helix in MKK7 as well as its role in kinase activation, we attempted to crystallize three different MKK7 with extended N-terminal regions, full-length, ΔN20 and ΔN75, all of which had S287 and T291 mutated to aspartic acid, a mutation known to mimic phosphorylation 44,45 .…”