Molecular mechanisms underlying cellular effects of human MEK1 mutations

INTRODUCTION

Somatic mutations in genes encoding components of the RAS pathway have been recognized as drivers of tumorigenesis since the 1990s (Hanahan and Weinberg, 2000; Avruch, 2007; Roberts and Der, 2007; Hanahan and Weinberg, 2011; Poulikakos and Solit, 2011; Chapman et al., 2014). More recently, germline mutations in the same genes have been associated with developmental diseases, collectively known as RASopathies (Rauen, 2013; Edwards and Gelb, 2016). These mutations, estimated to affect ∼1/1000 human births, produce a broad spectrum of phenotypes, including heart defects, stunted growth, and neurocognitive impairments (Tartaglia and Gelb, 2010; Maher et al., 2016, 2018b). Hundreds of such mutations have already been identified and many more are likely to be discovered by personalized sequencing projects. What is sorely lacking, however, is a rigorous strategy for investigating the functional consequences of these mutations in vivo. To address this challenge, we have recently started to use quantitative experiments in Drosophila, capitalizing on the reproducible nature of endogenous signaling events and on the large suite of techniques for targeted genetic perturbations at essentially all levels of the RAS pathway (Ashton-Beaucage and Therrien, 2017).

The present study is motivated by a feedback model that has emerged from our work on mutations affecting MEK1 kinase (Goyal et al., 2017a), a core component of pathway (Bromberg-White et al., 2012; Futran et al., 2013). Kinase activity of MEK requires its phosphorylation by RAF, an upstream kinase of the RAS cascade (Futran et al., 2013). We discovered that although pathogenic variants are clearly activating at the molecular level, they can cause both ectopic pathway activation and reduction of its sensitivity to extracellular signals (Jindal et al., 2015; Goyal et al., 2017a, b). Both effects could be explained using a mathematical model whereby the RAF-independent activity of a mutant kinase triggers the expression of inhibitors that desensitize the pathway to future activation by extracellular cues. Here we provide direct proof of this model by identifying the molecular nature of the negative feedback. Furthermore, we extend our mechanistic analysis of activating mutations and show that they make MEK1 independent of KSR, an adaptor protein which is essential for ligand-dependent signal transduction under normal conditions (Patel and Shvartsman, 2018).

Our approach to quantitative studies of disease mutations relies on imaging pathway activation in the early Drosophila embryo, a time when all intracellular pathway components are uniformly expressed but remain inactive until signal transduction is initiated by locally secreted extracellular ligands of the cell surface receptor tyrosine kinases (RTKs) (Figure 1A). The first of these RTKs is Torso, which is provided with the active form of ligand (Trunk) at the poles of the embryo (Goyal et al., 2018). Torso signaling is responsible for the RAS-dependent induction of genes involved in the formation of the nonsegmented terminal structures of the future larva. RAS signaling induced by Torso can be visualized in several ways, including immunohistochemistry using antibodies recognizing the dually phosphorylated form of ERK (dpERK), the terminal kinase of the RAS pathway, and a direct substrate of active MEK (Gabay et al., 1997). We established a robust approach for pairwise comparison of ERK activation between normal (wild type [WT]) and genetically perturbed embryos (Coppey et al., 2008). In particular, one can study the effect of targeted expression of disease variants of MEK.

We used the Gal4-UAS system to overexpress MEK variants on top of normal levels of WT MEK (Duffy, 2002). We used GFP-labeled WT embryos as internal control for all experimental quantification Additionally, we have previously demonstrated that mutant MEK has no difference in protein stability or localization (Goyal et al., 2017a). Importantly, we additionally showed that increased levels of WT MEK do not affect the pattern of ERK activation, which means that MEK is not a limiting pathway component and provides a reference point for quantifying the effects caused by targeted expression of disease variants (Goyal et al., 2017a). We use this feature of RAS signaling in the early embryo to experimentally test the main assumptions of our model: ligand-independent MEK activity and transcriptional induction of a negative regulator. Most of our experiments use the MEK^F53S variant, which was identified in individuals with cardiofaciocutaneous syndrome (CFC), a form of RASopathy that is characterized by mild craniofacial dysmorphia and pulmonary valve stenosis (Figure 1, B and C) (Rauen, 1993). Since the MEK^F53S substitution impacts the negative regulatory region (NRR) which keeps MEK inactive in the absence of phosphorylation by RAF, MEK^F53S can be partially active even in cells not exposed to RTK signaling (Fischmann et al., 2009). Here we provide direct proof of RAF-independent activity in vivo and show that it causes the expression of an ERK phosphatase. We used genetic experiments to show that it is indeed the critical component of the feedback loop. We also show that MEK^F53S is more sensitive to activation by ligands and suggest that this is a common property of disease variants.

RESULTS

RAF-independent kinase activity

In contrast to what is observed with WT MEK, overexpression of MEK^F53S and other disease variants leads to significant changes in the spatial pattern of active ERK: it is significantly elevated in the middle of the embryo and strongly down-regulated at the poles (Goyal et al., 2017a). Ectopic activation in the region maximally removed from the poles, where the diffusible ligand of Torso is produced, can be explained in two, nonmutually exclusive ways. Mutant MEKs can be enzymatically active even in the absence of ligand-dependent phosphorylation by RAF, which can be caused by disrupted intramolecular interactions with the NRR of MEK (Fischmann et al., 2009; Jindal et al., 2017a). Mutant MEKs can also be more sensitive to extracellular ligands, which can induce RAF-dependent phosphorylation and kinase activation even at residual levels of Torso ligand that are insufficient for causing activation of WT MEK (Yeung et al., 2020).

The differential contributions of two effects can be probed using a protein that combines the MEK^F53S substitution with two alanine substitutions of the two serines that are phosphorylated by RAF and are essential for activation of WT MEK (Mansour et al., 1996). If ectopic activation in the middle of the embryo is caused solely by RAF-independent activity, it should be indistinguishable from the effect caused by overexpression of the unphosphorylatable MEK^F53S variant (MEK^{F53S SSAA}) (Figure 1D). To test this prediction, we generated transgenic flies in which the UAS construct controlling such a variant is inserted in the same region of the genome as the UAS-MEK constructs used in our earlier studies. We found that the midembryo ERK activation by the double mutant was significantly increased relative to the WT (p = 0.000131), just like the variant with an intact activation loop (p = 0.000471) (Figure 1, E–G; Supplemental Figure S1). We also detected significant down-regulation of ERK activation at the poles, since strongly overexpressed mutant protein competes with endogenous MEK and interferes with its phosphorylation and activation by RAF. Caution should be utilized when comparing the poles of these two mutants, since this dominant-negative effect caused by the SSAA mutation is different from the induced negative feedback that we have previously described with GOF mutations (Goyal et al., 2017a). We will address this in the final section of the paper. What is more important for the current work is the effect in the middle of the embryo, which unambiguously establishes small but measurable catalytic activity of unphosphorylated MEK^F53S and is consistent with previous kinetic data from studies with purified MEK and ERK proteins (Jindal et al., 2017a; Yeung et al., 2020).

Increased sensitivity to activation by RAF

Several MEK disease variants, including MEK^F53S, display an increased rate of phosphorylation by RAF in vitro, suggesting that they are more sensitive to activation by extracellular signals in vivo (Yeung et al., 2020). However, in vitro studies that led to this conclusion relied on a binary system containing MEK1 and preactivated RAF, whereas MEK activation in vivo requires kinase suppressor of Ras (KSR), a protein scaffold essential for the RTK-dependent ERK signaling (Therrien et al., 1995; Udell et al., 2011). KSR forms heterotrimeric complexes with both MEK and RAF, which is critical both for the allosteric activation of RAF and for the RAF-dependent activation of WT MEK (Brennan et al., 2011; Dhawan et al., 2016; Lavoie et al., 2018). Given the critical role of KSR in the activation of WT MEK, we examined the effects on disease variants.

Consistent with its essential role in the RTK-dependent signaling, we found that ERK activation by Torso is abolished when ksr expression is depleted by RNA interference (RNAi) (Figure 2, A and D). Remarkably, this effect could be significantly rescued by expressing the MEK^F53S variant (Figure 2, B and E). Importantly, expression of the WT MEK could not rescue the effect of ksr knockdown (Figure 2, C and F). The rescuing effect is also seen by examining the transcriptional effects of ERK activation by Torso (Casanova and Struhl, 1989; Smits and Shvartsman, 2020). Specifically, ERK signaling at the termini induces the expression of zygotic genes needed for the formation of the terminal structures of the future larva. One of these genes, tailless (tll), is expressed in both anterior and posterior domains (Strecker et al., 1986). The anterior domain of tll reflects the joint action of several patterning cues, but the posterior domain depends on Torso alone and serves as a sensitive readout of pathway activation (Figure 2G) (Kim et al., 2013). Consistent with the effects of ksr depletion on Torso-dependent ERK activation, we found that complete loss of posterior tll expression in the ksr knockdown embryos is substantially rescued by the concurrent overexpression of MEK^F53S but not WT MEK (Figure 2, H and I). Thus, in contrast to WT MEK, the disease MEK variant can be activated by RAF independently of KSR. The middle of the embryo, which normally is not subject to ERK signaling, provides a readout of the quantity of constitutive activity by mutant MEK. The slight increase that we see in the middle of the MEK^F53S mutant is not enough to explain the substantial increase of signaling that we measure in the termini, which strongly suggests that rescue is indeed ligand dependent.

This conclusion is further supported by analyzing the morphogenetic effects of terminal signaling, such as the formation of the larval filzkörper, tubular structures needed for respiration (Figure 3, A and A’, green arrowhead) (Jurgens et al., 1984; Dalton et al., 1989). Consistent with previous studies, embryos with RNAi-depleted ksr or raf fail to form filzkörper (Figure 3, B–C’, B’ and C’, gray arrowhead). However, filzkörper are restored in a significant fraction of embryos where ksr depletion is combined with expression of MEK^F53S (Figure 3, D and D’). Importantly, overexpression of WT MEK does not rescue filzkörper (Figure 3, E and E’). Even though the rescuing capacity of the MEK^F53S variant does not require KSR, we found that it still depends on RAF and is lost in embryos where expression of MEK^F53S was combined with the knockdown raf (Figure 3, F and F’). This means that the rescuing effect of MEK^F53S is strictly dependent on upstream signaling. Importantly, the KSR-independent rescuing ability was not limited to a single MEK variant and was also observed for the E203K and Y130C variants that were identified in human cancer and CFC syndrome, respectively (Figure 3, G and I) (Rodriguez-Viciana et al., 2006; Bentivegna et al., 2008).

Transcriptional induction of Mkp3 by constitutively active MEK

How can MEK variants that have signal-independent activity and are also more prone to activation by extracellular signals cause a strong reduction of ERK activation by Torso in the terminal regions of the embryo? We proposed that this paradoxical behavior is caused by a hypothetical inhibitor of ERK activation that is induced by constitutive activity of MEK variants and desensitizes the ERK cascade to activation by RTK ligands (Goyal et al., 2018). Genetic studies in Drosophila and other model systems have identified several inhibitors, such as Sprouty, that are transcriptionally induced by ERK and can inhibit ERK activation at multiple points within the ERK cascade (Musacchio and Perrimon, 1996; Sawamoto et al., 1996; Casci et al., 1999; Neben et al., 2019). Negative regulators may be expressed in a tissue-specific manner, rendering certain tissues particularly sensitive to increased RAS signaling as was suggested for germline mutations of HRAS in mice (Chen et al., 2009). Functional effects of these feedbacks on pathway activation and function in vivo are underexplored.

As an unbiased way of identifying the candidate regulators that could potentially explain the observed desensitization of the ERK cascade in our system, we used RNA sequencing (RNA-seq) to compare maternally deposited transcripts in embryos from mothers expressing either MEK^F53S or WT MEK (Figure 4A). We found that signal-independent constitutive activity of MEK^F53S leads to pronounced ERK activation in the nurse cells, which are connected to the oocyte by stable cytoplasmic bridges and supply the future oocyte with multiple cytoplasmic components, including all maternally deposited transcripts encoding the RAS pathway components (Supplemental Figure S2). This observation is crucial, since it highlights that previous stages of development contribute to subsequent developmental events. We hypothesized that ectopic ERK signaling changes the sensitivity to subsequent ERK signaling through transcriptional feedbacks in the maternal contribution in the unfertilized embryo. This is important, since we have not observed the negative feedback in optogenetic stimulation of the ERK pathway, where pathway stimulation was limited to the early embryo (Johnson et al., 2017).

Our analysis revealed significant changes in the expression of known transcriptional targets of ERK signaling, such as pnt, aos, and mkp3, demonstrating that maternal expression of constitutively active MEK indeed changes the composition of maternally deposited transcriptome. We focused on Mkp3, a dual specificity phosphatase that dephosphorylates dually phosphorylated ERK and is transcriptionally induced by the ERK pathway in developing and adult tissues (Figure 4B) (Keyse, 2008; Haagenson and Wu, 2010; Seternes et al., 2019). We note that these signaling changes occur before the maternal-zygotic transition, which leaves no time for a patterned transcriptional response. The up-regulation of Mkp3 that we measure in our RNA seq experiments must therefore be occurring already during oogenesis in the nurse cells and this RNA is then imported into the oocyte during oogenesis. Therefore, if the additional maternal Mkp3 remains uniformly distributed in the early embryo as it is in WT (Gomez et al., 2005), we expect a global effect on ERK signaling, both at the poles and the middle of the embryo. Indeed, that is what we see. Furthermore, if the MEK-induced induction of Mkp3 is responsible for the reduction of ERK signaling at the poles (Figure 4, C, E, F, and I), knockdown of mkp3 should restore normal levels of ERK signaling. We could directly test this prediction by using the same maternal driver to express both constitutively active MEK and RNAi targeting Mkp3. Remarkably, embryos from these mothers had near-normal levels of active ERK at the poles and even increased active ERK in the middle of the embryo, confirming a global effect of Mkp3 (Figure 4, D, E, G, and J). Importantly, maternal RNAi knockdown of Mkp3 had no effect without constitutively active MEK (Figure 4, H and K). These results strongly support the model whereby the Mkp3-dependent feedback is activated by the constitutively active MEK and causes reduction of the ERK pathway activation by Torso (Goyal et al., 2017a).

DISCUSSION

Understanding how coding mutations in human MEK1 affect ERK signaling in vivo is important both for clinical applications and for fundamental research. First, since multiple components of the ERK cascade, including MEK, are drug targets in oncology and other therapeutic areas, it is critical to determine how the effects of candidate drugs are impacted by mutations (Ohren et al., 2004; Caunt et al., 2015). Second, it is important to investigate how disease mutations perturb the elaborate mechanisms that have been elucidated by decades of work on MEK regulation and function. In theory, one could start from structural information for WT MEK and try to predict the effects of mutations using molecular dynamics simulations (Nikolaev et al., 2011; Ordan et al., 2018). In practice, however, computational tools are yet to access the timescales relevant for protein complexes involved in MEK regulation and function. Another approach is based on reconstitution of isolated processes in vitro, such as MEK phosphorylation by RAF (Jindal et al., 2017a; Yeung et al., 2020). While this approach is commonly used as a useful step in functional characterization of disease mutations, extrapolating the results of in vitro studies to signaling in crowded and compartmentalized intracellular systems is nontrivial.

Given the challenges faced by computational and reconstitution strategies to mechanistic studies of disease mutations, it appears more promising to analyze how they influence ERK signaling in cells and tissues. The terminal patterning system is well suited for this purpose because it is readily accessible to quantitative analysis of ERK signaling and its transcriptional and morphogenetic defects and can be genetically perturbed at multiple levels of the ERK cascade (Goyal et al., 2018). We used these advantages to investigate the signaling properties of MEK variants that have been predicted to cause signal-independent activity of MEK and/or accelerate signal-dependent MEK activation (Fischmann et al., 2009; Jindal et al., 2017a; Yeung et al., 2020). Using a variant that combines alanine substitutions with the activation loop with mutations within the negative regulation of MEK, we established that disease variants indeed have enzymatic activity even in the absence of upstream phosphorylation by RAF. We also found that signal-dependent activation of disease variants is independent of KSR, a critical signaling adaptor and a recent target of potent allosteric inhibitors (Dhawan et al., 2016). Our results suggest that these inhibitors may be ineffective for individuals with activating mutations in MEK.

Measurements of ERK activation offer a striking example of a context-dependent response to mutations in a ubiquitously expressed pathway component. Depending on their location in the embryo, cells respond to gain-of-function MEK variants with either up-regulating or down-regulating their ERK activation levels. We proposed that this divergent response reflects a feedback loop whereby mutation-imparted constitutive activation of the pathway triggers expression of negative pathway regulators, making it less sensitive to activation by endogenous patterning cues. We provided clear support for this mechanism by identifying the dual specificity ERK phosphatase Mkp3 as a critical component of the hypothesized feedback and showing that RNAi knockdown of Mkp3 eliminates the divergent response to expression of activating MEK variants. Mkp3 was also found to be up-regulated in cardiomyocytes from an individual with a developmental abnormality with a germline mutation in RAF, suggesting that this effect is not limited to Drosophila (Jaffre et al., 2019). Importantly, we have previously detected CIC binding to the Mkp3 locus (Keenan et al., 2020). Furthermore, while this study was in review, the mammalian homologue to Mkp3, DUSP6, was demonstrated to be transcriptionally regulated by CIC promoter binding (Ren et al., 2020). Thus, this regulatory loop through Mkp3 is highly conserved. Studies in systems amenable to quantitative imaging, such as the terminal system, offer an opportunity to explore these effects in the most controlled and comprehensive way. We have done this for MEK, but the presented approach can be readily extended to the rest of the RAS pathway (Rauen, 2013; Maher et al., 2018a; Tajan et al., 2018; Taylor et al., 2019). In particular, all of our approaches are directly useful for quantitative investigation of newly discovered human mutations affecting ERK, the only known enzymatic substrate of MEK and an emerging drug target in cancer (Futran et al., 2013; Taylor et al., 2019; Motta et al., 2020; Smorodinsky-Atias et al., 2020).

Methods AND MATERIALS

Request a protocol through Bio-protocol.

Fly stocks

Fly stocks were maintained under standard conditions and crosses were performed at 25°C unless otherwise specified. OregonR, Histone-GFP, MTD-GAL4, UAS-MEK^WT, UAS-MEK^F53S, UAS-MEK^Y130C, and UAS-MEK^E203K flies were described in Jindal et al. (2017b). S218A/S222A were introduced into UAS-MEK^F53S construct to generate UAS-MEK^{F53S SSAA} with site-directed mutagenesis using Q5 hot start DNA Polymerase (NEB M0493) with the primers mek_218_228S2A_F: CCAACgCCTTTGTGGGCACCCGTA and mek_218_228S2A_R: CCATCGcGTCGATCAGTTGACCGGAG and verified by sequencing. The UAS-MEK^{F53S SSAA} construct was integrated into attP40 site using the ΦC31-based integration system. UAS-ksr RNAi (41598) and UAS-RAF RNAi (41589) were from the Bloomington Drosophila Stock Center. The UAS-Mkp3 RNAi line was generated by integrating a previously established RNAi construct HMS04475 (a gift from Norbert Perrimon) onto the third chromosome by ΦC31 integration into attP2 (Sopko et al., 2014). The newly developed fly lines are available on request. Egg lays and embryonic development were conducted at 22°C.

Cuticle phenotyping

Embryos were dechorionated after being aged for more than 24 h as previously reported (Goyal et al., 2017a; Johnson et al., 2017). Dechorionated embryos were shaken in methanol and heptane (1:1) and incubated overnight in a media containing lactic acid and Hoyer’s media (1:1) at 65°C. Embryos were imaged on a Nikon Eclipse Ni in darkfield.

Immunostaining and FISH

dpERK antibody staining and tailless FISH protocols were performed as described previously (Goyal et al., 2017a). Rabbit anti-dpERK (1:100; Cell Signaling Technology #4370S), sheep anti-GFP (1:1000, Bio-Rad #4745-1051), and sheep anti-digoxigenin (DIG) (1:125; Roche #11333089001) were used as primary antibodies. DAPI (1:10,000; Molecular probes #D1306) was used to stain for nuclei, and Alexa Fluor conjugates (1:500; Invitrogen) were used as secondary antibodies.

Microscopy and image processing

Fluorescent imaging of fixed embryos for immunostaining and FISH experiments were performed on a Nikon A1-RS scanning confocal microscope with a 20× objective, similar to previously reported procedures (Goyal et al., 2017a). For pairwise comparisons of WT and mutant backgrounds, embryos were collected, stained, and imaged together under the same experimental conditions. Our software allows the MEK measurements to be quantified while the experimenter is blinded to the genotype. Broken embryos, embryos with intact vitelline membrane, or embryos undergoing mitosis were excluded from the analysis.

RNA-seq analysis

MTD>MEK^WT and MTD>MEK^F53S overexpressing females were mated to sterile males of the βTub85D^D genotype (Bloomington Stock #2149). One half-hour collections of ∼20 embryos were stored at 4°C in RNAlater solution, transferred to TRIzol, and prepped with a TRIzol RNA column from Zymo Research. Directional RNA-seq was conducted in biological triplicates for each genotype by the Princeton Sequencing Core. The raw data and gene expression measurements will be available on NCBI GEO with the accession identifier: GSE162684.

Each RNA-seq library was inspected for quality and subsequently mapped to the fly transcriptome (r6.19) using Salmon (v0.8.2) in quasi-mapping mode with GC content and sequence-specific bias corrections to produce a gene-level read count matrix. Differential expression analysis was performed between the MEK^WT and the MEK^F53S replicate per-gene read counts using the edgeR software package’s exact test option after removing lowly expressed genes. Specifically, we removed genes with fewer than 1 count per million (corresponding to approximately 37 mapped reads in the smallest library) in at least half of the samples. False discovery rate control was applied to the per-gene p values using the Benjamini–Hochberg procedure, and genes with FDR < = 0.01 were determined to be differentially expressed. The limits set on the axes of the volcano plot does limit the display of several extreme examples for visual clarity.

FOOTNOTES

ACKNOWLEDGMENTS

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