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A compendium of ERK targets
FEBS Letters
Abstract
The RAF-MEK-ERK cascade is one of the most studied signaling pathways as it controls many vital cellular programs. There has been an immense amount of effort to determine ERK target proteins that are involved in regulating these programs. Classical biochemical and genetic approaches have identified hundreds of direct ERK substrates, and with the advent of phospho-proteomic technologies, numerous studies have expanded the number of ERK target proteins. Here, we compile a comprehensive ERK target phospho-site archive in which we gathered information from various research studies in a database to form a searchable compendium of ERK targets. This article is protected by copyright. All rights reserved.
... Three of the hyperactivated sites are activatory phosphosites of ERK1/2 (red) demonstrating a direct ERK hyperactivation. We then checked other phosphosites for known ERK-targets, using a previously published compendium of ERK targets [55] Seven of the p38 inhibitor-dependent phosphosites are described as ERK targets, of which six follow the same hyperactivation pattern as ERK phosphorylation itself. This further corroborates that next to ERK also downstream partners are hyperactivated by p38 inhibition. ...
... Left panel denotes which site was found to be significantly regulated (blue-down; red-up) by the indicated comparison. Sites are annotated as follows: 'HGNC symbol'_'amino acid'_'position'_'number. of phosphosites'; ERK activation sites and known target sites of ERK [55] are indicated by green and orange circles, respectively. ...
B cell receptor (BCR) signaling is required for the survival and maturation of B cells and is deregulated in B cell lymphomas. While proximal BCR signaling is well studied, little is known about the crosstalk of downstream effector pathways, and a comprehensive quantitative network analysis of BCR signaling is missing. Here, we semi-quantitatively modelled BCR signaling in Burkitt lymphoma (BL) cells using systematically perturbed phosphorylation data of BL-2 and BL-41 cells. The models unveiled feedback and crosstalk structures in the BCR signaling network, including a negative crosstalk from p38 to MEK/ERK. The relevance of the crosstalk was verified for BCR and CD40 signaling in different BL cells and confirmed by global phosphoproteomics on ERK itself and known ERK target sites. Compared to the starting network, the trained network for BL-2 cells was better transferable to BL-41 cells. Moreover, the BL-2 network was also suited to model BCR signaling in Diffuse large B cell lymphoma cells lines with aberrant BCR signaling (HBL-1, OCI-LY3), indicating that BCR aberration does not cause a major downstream rewiring.
... However, MS analysis revealed a small number of ERK substrates that were differentially phosphorylated ( Figure 5C,D; Table S3). Of 399 proteins specially immunoprecipitated (i.e., enriched >2-fold over a control immunoprecipitation with an isotype-matched IgG) in KSR1 KO1-3 cells, 85 were known ERK substrates [20]. Analysing differences between parental and KSR1 −/− cells using a fold change of >2 and p-value of <0.05 as the cut-off for differential phosphorylation, we identified 29-34 substrates showing enhanced and 28-33 substrates showing decreased phosphorylation in the KSR1 KO1-3 clones versus parental cells. ...
... Interestingly, only six up-and four downregulated substrate phosphorylations were shared between all three KSR1 knockout clones, suggesting that cells can adapt to KSR1 loss via different mechanisms that share common core processes ( Figure 5C,D). Of these 10 proteins, only two are listed in the "Compendium of ERK targets" [20], specifically, BAG3 and NEDD4L. In both cases, ERK phosphorylation inhibits their function. ...
Citation: Liu, Z.; Krstic, A.; Neve, A.; Casalou, C.; Rauch, N.; Wynne, K.; Cassidy, H.; McCann, A.; Kavanagh, E.; McCann, B.; et al. Kinase Suppressor of RAS 1 (KSR1) Maintains the Transformed Phenotype of BRAFV600E Mutant Human Melanoma Cells. Int. J. Mol. Sci. 2023, 24, 11821. https:// Abstract: Kinase Suppressor of RAS 1 (KSR1) is a scaffolding protein for the RAS-RAF-MEK-ERK pathway, which is one of the most frequently altered pathways in human cancers. Previous results have shown that KSR1 has a critical role in mutant RAS-mediated transformation. Here, we examined the role of KSR1 in mutant BRAF transformation. We used CRISPR/Cas9 to knock out KSR1 in a BRAFV600E-transformed melanoma cell line. KSR1 loss produced a complex phenotype charac-terised by impaired proliferation, cell cycle defects, decreased transformation, decreased invasive migration, increased cellular senescence, and increased apoptosis. To decipher this phenotype, we used a combination of proteomic ERK substrate profiling, global protein expression profiling, and biochemical validation assays. The results suggest that KSR1 directs ERK to phosphorylate substrates that have a critical role in ensuring cell survival. The results further indicate that KSR1 loss induces the activation of p38 Mitogen-Activated Protein Kinase (MAPK) and subsequent cell cycle aberrations and senescence. In summary, KSR1 function plays a key role in oncogenic BRAF transformation.
... However, MS analysis revealed a small number of ERK substrates that were differentially phosphorylated ( Figure 5C,D; Table S3). Of 399 proteins specially immunoprecipitated (i.e., enriched >2-fold over a control immunoprecipitation with an isotype-matched IgG) in KSR1 KO1-3 cells, 85 were known ERK substrates [20]. Analysing differences between parental and KSR1 −/− cells using a fold change of >2 and p-value of <0.05 as the cut-off for differential phosphorylation, we identified 29-34 substrates showing enhanced and 28-33 substrates showing decreased phosphorylation in the KSR1 KO1-3 clones versus parental cells. ...
... Interestingly, only six up-and four downregulated substrate phosphorylations were shared between all three KSR1 knockout clones, suggesting that cells can adapt to KSR1 loss via different mechanisms that share common core processes ( Figure 5C,D). Of these 10 proteins, only two are listed in the "Compendium of ERK targets" [20], specifically, BAG3 and NEDD4L. In both cases, ERK phosphorylation inhibits their function. ...
Citation: Liu, Z.; Krstic, A.; Neve, A.; Casalou, C.; Rauch, N.; Wynne, K.; Cassidy, H.; McCann, A.; Kavanagh, E.; McCann, B.; et al. Kinase Suppressor of RAS 1 (KSR1) Maintains the Transformed Phenotype of BRAFV600E Mutant Human Melanoma Cells. Int. J. Mol. Sci. 2023, 24, 11821. https:// Abstract: Kinase Suppressor of RAS 1 (KSR1) is a scaffolding protein for the RAS-RAF-MEK-ERK pathway, which is one of the most frequently altered pathways in human cancers. Previous results have shown that KSR1 has a critical role in mutant RAS-mediated transformation. Here, we examined the role of KSR1 in mutant BRAF transformation. We used CRISPR/Cas9 to knock out KSR1 in a BRAFV600E-transformed melanoma cell line. KSR1 loss produced a complex phenotype charac-terised by impaired proliferation, cell cycle defects, decreased transformation, decreased invasive migration, increased cellular senescence, and increased apoptosis. To decipher this phenotype, we used a combination of proteomic ERK substrate profiling, global protein expression profiling, and biochemical validation assays. The results suggest that KSR1 directs ERK to phosphorylate substrates that have a critical role in ensuring cell survival. The results further indicate that KSR1 loss induces the activation of p38 Mitogen-Activated Protein Kinase (MAPK) and subsequent cell cycle aberrations and senescence. In summary, KSR1 function plays a key role in oncogenic BRAF transformation.
... Our work highlights the fact that the activity, rather than the level, of ERK is critical for Pbody dissolution. However, since ERKs can phosphorylate more than 200 intracellular targets [36], we investigated whether one of these targets was part of a protein essential for P-body composition [14]. While LSM14A appears to be a putative indirect target of ERK in rats, the ERK phosphorylation motif is not conserved in human. ...
... While LSM14A appears to be a putative indirect target of ERK in rats, the ERK phosphorylation motif is not conserved in human. For EIF4ENF1 (4E-T), a direct site is present in mouse, while an indirect site is present in humans [36]. In addition, our experimental data combined with bioinformatics analysis of many cell lines showed an inverse correlation between P-body protein components and RAS translation. ...
Simple Summary
Cancer therapies directly targeting the mitogen-activated protein kinase (MAPK) pathway lead to cancer drug resistance. Resistance has been linked to compensatory RAS overexpression, but the mechanisms underlying this overexpression remain unclear. Here, we find that MEK inhibitors (MEKi) increases translation of the KRAS and NRAS oncogenes through a mechanism involving liquid–liquid phase separation (LLPS), and more particularly processing body (P-body) dissolution. Overall, we describe a new feedback loop mechanism involving P-bodies and phase separation that regulates RAS translation. Identification of key components that regulate LLPS will be important for future targeted therapeutic strategies.
Abstract
Overactivation of the mitogen-activated protein kinase (MAPK) pathway is a critical driver of many human cancers. However, therapies directly targeting this pathway lead to cancer drug resistance. Resistance has been linked to compensatory RAS overexpression, but the mechanisms underlying this response remain unclear. Here, we find that MEK inhibitors (MEKi) are associated with an increased translation of the KRAS and NRAS oncogenes through a mechanism involving dissolution of processing body (P-body) biocondensates. This effect is seen across different cell types and is extremely dynamic since removal of MEKi and ERK reactivation result in reappearance of P-bodies and reduced RAS-dependent signaling. Moreover, we find that P-body scaffold protein levels negatively impact RAS expression. Overall, we describe a new feedback loop mechanism involving biocondensates such as P-bodies in the translational regulation of RAS proteins and MAPK signaling.
... Next, we used an unbiased approach to identify specific cell populations in which ERK was activated during regeneration. We leveraged four independent databases, three of which contained effectors of the known ERK targets Myc, E2F, and AP-1 (41, 53-55) and a phospho-ERK target database (56). To identify Myc, E2F, and AP-1 targets in our datasets, we first interrogated the genes that were significantly up-regulated during regeneration and down-regulated upon ERK, FGFR, and ErbB-2 inhibition against the Myc, E2F, and AP-1 databases (41,53,55). ...
... S14F). These data supported genes and gene sets previously identified as ERK targets (56) and reinforced genes we identified from our bulk RNA-seq experiments as involved in regeneration (Figs. 3, B to D, and 5A and fig. S9B). ...
Although most mammals heal injured tissues and organs with scarring, spiny mice (Acomys) naturally regenerate skin and complex musculoskeletal tissues. Now, the core signaling pathways driving mammalian tissue regeneration are poorly characterized. Here, we show that, while immediate extracellular signal-regulated kinase (ERK) activation is a shared feature of scarring (Mus) and regenerating (Acomys) injuries, ERK activity is only sustained at high levels during complex tissue regeneration. Following ERK inhibition, ear punch regeneration in Acomys shifted toward fibrotic repair. Using single-cell RNA sequencing, we identified ERK-responsive cell types. Loss- and gain-of-function experiments prompted us to uncover fibroblast growth factor and ErbB signaling as upstream ERK regulators of regeneration. The ectopic activation of ERK in scar-prone injuries induced a pro-regenerative response, including cell proliferation, extracellular matrix remodeling, and hair follicle neogenesis. Our data detail an important distinction in ERK activity between regenerating and poorly regenerating adult mammals and open avenues to redirect fibrotic repair toward regenerative healing.
... An examination of the amino-acid sequence around the pS225 showed that it contains a consensus P-X-S/T-P motif for phosphorylation by ERK. 24 As expected, ERK1/2, but not cyclindependent kinase 1 (CDK1; in which the consensus phosphorylation motif is S/T-P-X-K/R), physically interacted with SIX1 ( Fig. 1c, d, Supplementary Fig. S1c). SIX1 (1-183) encompassing both the highly conserved SIX1 domain (SD) and the DNA-binding homeobox domain (HD) interacted with ERK2 ( Supplementary Fig. S1d). ...
Aerobic glycolysis is a hallmark of cancer and is regulated by growth factors, protein kinases and transcription factors. However, it remains poorly understood how these components interact to regulate aerobic glycolysis coordinately. Here, we show that sine oculis homeobox 1 (SIX1) phosphorylation integrates growth factors (e.g. TGFβ, EGF) to control aerobic glycolysis and determines its tumor-promoting activity. SIX1 is phosphorylated at serine 225 (S225) by growth factors-activated protein kinases ERK1/2 and its phosphorylation is responsible for glycolysis stimulated by some growth factors. SIX1 is dephosphorylated by the atypical protein phosphatase eyes absent 4 (EYA4). Phosphorylation blocks non-canonical ubiquitination and degradation of SIX1 through the E3 ubiquitin ligase FZR1. Unexpectedly, the non-canonical phosphorylation mimic SIX1 (S225K), but not the canonical phosphorylation mimic SIX1 (S225D/E), phenocopies the effects of SIX1 phosphorylation on glycolysis and cancer cell growth and metastasis in vitro and in mice. Compared to normal liver tissues, SIX1 phosphorylation at S225 (pS225) is upregulated in human liver cancer tissues. ERK1/2 expression is positively correlated with pS225 and EYA4 expression is negatively associated with pS225 in liver cancer specimens. Moreover, low expression of pS225 had longer disease-free survival and overall survival in patients with liver cancer. Thus, we identify a common mechanism underlying growth factors-mediated glycolysis, and provide a previously unidentified mode for non-classical phosphorylation mimics of a protein. Targeting growth factors/SIX1 signaling pathway may be beneficial to cancer treatment.
... We treated the A375 and MelJuso cell lines with 100 nM RocA for 20 h, followed by cell lysis and immunoprecipitation of the lysates using PTMScan® Phospho-MAPK/CDK Substrate Motif (PXS*P and S*PXK/R) Kit and LC-MS/MS analysis of enriched peptides (Fig. 1B). Then we took advantage of the online database Compendium of ERK targets (18) to assign the previously identified ERK kinase targets. Results presented in Fig. 1C and Datasets S1 and S2 indicate that a proportion of ERK kinase targets might be downregulated in melanoma cells upon eIF4F inhibition. ...
The eIF4F translation initiation complex plays a critical role in melanoma resistance to clinical BRAF and MEK inhibitors. In this study, we uncover a function of eIF4F in the negative regulation of the rat sarcoma (RAS)/rapidly accelerated fibrosarcoma (RAF)/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) signaling pathway. We demonstrate that eIF4F is essential for controlling ERK signaling intensity in treatment-naïve melanoma cells harboring BRAF or NRAS mutations. Specifically, the dual-specificity phosphatase DUSP6/MKP3, which acts as a negative feedback regulator of ERK activity, requires continuous production in an eIF4F-dependent manner to limit excessive ERK signaling driven by oncogenic RAF/RAS mutations. Treatment with small-molecule eIF4F inhibitors disrupts the negative feedback control of MAPK signaling, leading to ERK hyperactivation and EGR1 overexpression in melanoma cells in vitro and in vivo. Furthermore, our quantitative analyses reveal a high spare signaling capacity in the ERK pathway, suggesting that eIF4F-dependent feedback keeps the majority of ERK molecules inactive under normal conditions. Overall, our findings highlight the crucial role of eIF4F in regulating ERK signaling flux and suggest that pharmacological eIF4F inhibitors can disrupt the negative feedback control of MAPK activity in melanomas with BRAF and NRAS activating mutations.
... ERK is known to directly phosphorylate hundreds of substrates that are localized in either the cytoplasm, various organelles, or nucleus [22,23]. We used the PROMO website (https:// alggen. ...
Background and Aims
Tumor growth and progression are affected by interactions between tumor cells and stromal cells within the tumor microenvironment. We previously showed that the expression of an integral membrane protein, called stomatin, was increased in cancer cells following their association with stromal cells. Additionally, stomatin impaired the Akt signaling pathway to suppress tumor growth. However, it remains unclear how stomatin expression is regulated. To explore this, we examined the cell surface molecules that can transduce the intercellular communication signals between cancer cells and stromal cells.
Results
Among these molecules, EphA3 and EphA7 receptors and their ligand ephrin‐A5 were found to be expressed in prostate cancer cells, but not in prostate stromal cells. Cell‐to‐cell contact of prostate cancer cells through the EphA–ephrin‐A interaction suppressed stomatin expression, while knockdown of EphA3/7 or ephrin‐A5 increased stomatin expression. This increase contributed to an inhibition of prostate cancer cell proliferation. Intracellularly, the binding of ephrin‐A to EphA attenuated extracellular signaling‐regulated kinase (ERK) activation that promoted stomatin expression. Furthermore, ELK1 and ELK4, which are Ets family transcription factors phosphorylated by ERK, were involved in the induction of stomatin expression. We also found that higher Gleason score prostate cancer tissue samples had increased activation of EphA, while the stomatin expression and activated ERK and ELK levels were all low. In the mouse xenograft tumor samples generated by implantation of prostate cancer cells, EphA3 phosphorylation was attenuated and the ERK–ELK signaling and stomatin expression were enhanced in the area where stromal cells infiltrated the tumor.
Conclusion
The EphA‐mediated signaling suppresses the ERK–ELK pathway, leading to the reduction of stomatin expression that affects prostate cancer malignancy.
... [75] ERK was activated by MEK and has both self-activation and self-inhibition effect. [77] PPAR and C/EBP form a positive feedback loop with each other. [78] The three drugs corresponding to TGF-, MEKi and Rosiglitazone were also modeled by introducing three input nodes in the CAC network ( Figure 1A, see Supporting Information for detailed model). ...
Cancer is a systemic heterogeneous disease involving complex molecular networks. Tumor formation involves an epithelial‐mesenchymal transition (EMT), which promotes both metastasis and plasticity of cancer cells. Recent experiments have proposed that cancer cells can be transformed into adipocytes via a combination of drugs. However, the underlying mechanisms for how these drugs work, from a molecular network perspective, remain elusive. To reveal the mechanism of cancer‐adipose conversion (CAC), this study adopts a systems biology approach by combing mathematical modeling and molecular experiments, based on underlying molecular regulatory networks. Four types of attractors are identified, corresponding to epithelial (E), mesenchymal (M), adipose (A) and partial/intermediate EMT (P) cell states on the CAC landscape. Landscape and transition path results illustrate that intermediate states play critical roles in the cancer to adipose transition. Through a landscape control approach, two new therapeutic strategies for drug combinations are identified, that promote CAC. These predictions are verified by molecular experiments in different cell lines. The combined computational and experimental approach provides a powerful tool to explore molecular mechanisms for cell fate transitions in cancer networks. The results reveal underlying mechanisms of intermediate cell states that govern the CAC, and identified new potential drug combinations to induce cancer adipogenesis.
... Christen et al. Cell Communication and Signaling (2024) 22:428 Background Upon RAS-dependent recruitment to the plasma membrane, BRAF activates the MEK/ERK pathway [1]. Activated ERK phosphorylates thousands of proteins in the cytoplasm and nucleus, thereby controlling numerous processes in normal and malignant cells [2]. BRAF alterations are frequently observed in cancer, either as point mutants like BRAF V600E or as fusion proteins resulting from chromosomal recombination [3,4]. ...
BRAF serves as a gatekeeper of the RAS/RAF/MEK/ERK pathway, which plays a crucial role in homeostasis. Since aberrant signalling of this axis contributes to cancer and other diseases, it is tightly regulated by crosstalk with the PI3K/AKT/mTOR pathway and ERK mediated feedback loops. For example, ERK limits BRAF signalling through phosphorylation of multiple residues. One of these, T401, is widely considered as an ERK substrate following acute pathway activation by growth factors. Here, we demonstrate that prominent T401 phosphorylation (pT401) of endogenous BRAF is already observed in the absence of acute stimulation in various cell lines of murine and human origin. Importantly, the BRAF/RAF1 inhibitor naporafenib, the MEK inhibitor trametinib and the ERK inhibitor ulixertinib failed to reduce pT401 levels in these settings, supporting an alternative ERK-independent pathway to T401 phosphorylation. In contrast, the mTOR inhibitor torin1 and the dual-specific PI3K/mTOR inhibitor dactolisib significantly suppressed pT401 levels in all investigated cell types, in both a time and concentration dependent manner. Conversely, genetic mTOR pathway activation by oncogenic RHEB (Q64L) and mTOR (S2215Y and R2505P) mutants substantially increased pT401, an effect that was reverted by dactolisib and torin1 but not by trametinib. We also show that shRNAmir mediated depletion of the mTORC1 complex subunit Raptor significantly enhanced the suppression of T401 phosphorylation by a low torin1 dose, while knockdown of the mTORC2 complex subunit Rictor was less effective. Using mass spectrometry, we provide further evidence that torin1 suppresses the phosphorylation of T401, S405 and S409 but not of other important regulatory phosphorylation sites such as S446, S729 and S750. In summary, our data identify the mTOR axis and its inhibitors of (pre)clinical relevance as novel modulators of BRAF phosphorylation at T401.
Supplementary Information
The online version contains supplementary material available at 10.1186/s12964-024-01808-2.
... MYC) (Kapeli and Hurlin, 2011), INTS11 (Yue et al., 2017), Y-box binding protein 1 , KRAB zinc finger proteins (Reggiardo et al., 2022) and Kruppel-like factor 4 (KLF4) (Yang et al., 2013). The number of known nuclear phospho-ERK substrates is constantly increasing along with progress in phosphoproteome analysis (Unal et al., 2017). The functions of MYC (Cole, 1986;Ingvarsson, 1990) and AP1 (FOS/JUN) (Deschamps et al., 1985;Jochum et al., 2001) and other proteins (Ghaleb and Yang, 2017;Lupo et al., 2013) suggest that MAPK-responsive transcription factors not only mediate malignancy-inducing signals, but can also regulate normal growth control and differentiation. ...
The expression of mutated RAS genes drives extensive transcriptome alterations. Perturbation experiments have shown that the transcriptional responses to downstream effector pathways are partially unique and non-overlapping, suggesting a modular organization of the RAS-driven expression program. However, the relationship between individual deregulated transcription factors and the entire cancer cell-specific genetic program is poorly understood. To identify potential regulators of the RAS/MAPK-dependent fraction of the genetic program, we monitored transcriptome and proteome changes following conditional, time-resolved expression of mutant HRASG12V in human epithelial cells during neoplastic conversion. High mobility group AT hook2 (HMGA2), an architectural chromatin modulating protein and oncofetal tumour marker, was recovered as the earliest upregulated transcription factor. Knock-down of HMGA2 reverted anchorage-independent growth and epithelial-mesenchymal transition not only in HRAS-transformed cells but also in an independent, KRASG12V-driven rat epithelial model. Moreover, HMGA2 silencing reverted the deregulated expression of 60% of RAS-responsive target genes. These features qualify HMGA2 as a master regulator of mutant RAS-driven expression patterns. The delayed deregulation of FOSL1, ZEB1 and other transcription factors with known oncogenic activity suggests that HMGA2 acts in concert with a network of regulatory factors to trigger full neoplastic conversion. Although transcription factors are considered difficult to drug, the central role of HMGA2 in the transcription factor network as well as its relevance for cancer prognosis has motivated attempts to block its function using small molecular weight compounds. The further development of direct HMGA2 antagonists may prove useful in cancer cells that have developed resistance to signalling chain inhibition.
... [29] These cytokines interact with respective receptors on the plasma membrane to activate the ERK axis. [30] ERK is one of the serine/ threonine protein kinases, which are crucial components of the PI3K-AKT signaling pathway that is tightly connected to inflammation, apoptosis, and differentiation. [31,32] Activation of the ERK signal is closely related to phosphorylation of Tyr/Thr sites in the protein. ...
In this study, a series of novel compounds were synthesized by introducing the 3,4,5-trimethoxyphenyl and isatin groups into the monocarbonyl skeleton of curcumin. The possible biological activities and potential targets for these compounds were explored through network pharmacology. The results revealed that these compounds could significantly inhibit production of the inflammatory factors IL-6 and TNF-α, and suppress phosphorylation of the extracellular signal-regulated kinase (ERK) protein. Moreover, molecular docking experiments showed that the ERK protein was the potential target for these compounds. In summary, this study, through network pharmacology, presents a novel series of methoxy curcumin analogs as potent anti-inflammatory drugs.
... However, this requires far more complex methods than peripheral blood smears and an investment that would several-fold surpass the resources needed for brightfield microscopy of peripheral blood smears. Furthermore, these methods do not report on cellular morphologies, which are often linked to important signaling networks [25,26] of biological processes, such as ERK and PI3K/AKT/mTOR pathways [17,[27][28][29][30][31][32][33], which have been shown to be associated with pathological conditions and morphological changes. Furthermore, it has been published that the mechanical qualities of immune cells are closely related to their activity state [34][35][36][37], erythrocytes' membrane flexibility reflects their ability to take up oxygen [38][39][40][41][42], and the size distribution of platelets can be used to trace back abnormal physiological conditions [43]. ...
Citation: Campos-Medina, M.; Blumer, A.; Kraus-Füreder, P.; Mayrhofer-Reinhartshuber, M.; Kainz, P.; Schmid, J.A. AI-Enhanced Blood Cell Recognition and Analysis: Advancing Traditional Microscopy with the Web-Based Platform IKOSA. Abstract: Microscopy of stained blood smears is still a ubiquitous technique in pathology. It is often used in addition to automated electronic counters or flow cytometers to evaluate leukocytes and their morphologies in a rather simple manner and has low requirements for resources and equipment. However, despite the constant advances in microscopy, computer science, and pathology, it still usually follows the traditional approach of manual assessment by humans. We aimed to extend this technique using AI-based automated cell recognition methods while maintaining its technical simplicity. Using the web platform IKOSA, we developed an AI-based workflow to segment and identify all blood cells in DAPI-Giemsa co-stained blood smears. Thereby, we could automatically detect and classify neutrophils (young and segmented), lymphocytes, eosinophils, and monocytes, in addition to erythrocytes and platelets, in contrast to previously published algorithms, which usually focus on only one type of blood cell. Furthermore, our method delivers quantitative measurements, unattainable by the classical method or formerly published AI techniques, and it provides more sophisticated analyses based on entropy or gray-level co-occurrence matrices (GLCMs), which have the potential to monitor changes in internal cellular structures associated with disease states or responses to treatment. We conclude that AI-based automated blood cell evaluation has the potential to facilitate and improve routine diagnostics by adding quantitative shape and structure parameters to simple leukocyte counts of classical analysis.
... This results in the activation of Ras small nucleotide guanosine triphosphate hydrolases (Ras GTPases) and the downstream phosphorylation cascade composed of Raf, MEK, and ERK kinases (9)(10)(11). Once phosphorylated, ERK can modify a wide range of cytoplasmic and cytoskeletal protein substrates, as well as several nuclear transcription factors, and hence activate cell division, differentiation, survival, and growth (12). ...
Pharmacological therapies are promising interventions to slow down aging and reduce multimorbidity in the elderly. Studies in animal models are the first step toward translation of candidate molecules into human therapies, as they aim to elucidate the molecular pathways, cellular mechanisms, and tissue pathologies involved in the anti-aging effects. Trametinib, an allosteric inhibitor of MEK within the Ras/MAPK (Ras/Mitogen-Activated Protein Kinase) pathway and currently used as an anti-cancer treatment, emerged as a geroprotector candidate because it extended lifespan in the fruit fly Drosophila melanogaster . Here, we confirm that trametinib consistently and robustly extends female lifespan, and reduces intestinal stem cell (ISC) proliferation, tumor formation, tissue dysplasia, and barrier disruption in guts in aged flies. In contrast, pro-longevity effects of trametinib are weak and inconsistent in males, and it does not influence gut homeostasis. Inhibition of the Ras/MAPK pathway specifically in ISCs is sufficient to partially recapitulate the effects of trametinib. Moreover, in ISCs, trametinib decreases the activity of the RNA polymerase III (Pol III), a conserved enzyme synthesizing transfer RNAs and other short, non-coding RNAs, and whose inhibition also extends lifespan and reduces gut pathology. Finally, we show that the pro-longevity effect of trametinib in ISCs is partially mediated by Maf1, a repressor of Pol III, suggesting a life-limiting Ras/MAPK-Maf1-Pol III axis in these cells. The mechanism of action described in this work paves the way for further studies on the anti-aging effects of trametinib in mammals and shows its potential for clinical application in humans.
... PI3K regulates the phosphorylation of AKT, and the subsequent PI3K/AKT downstream signaling pathway has been suggested to play a regulatory role in numerous cell proliferation and activation processes [12,13]. In contrast, the ERK signaling pathway has been reported to play a key role in transducing signals from cell-surface receptors to the nucleus, thereby regulating the activation and proliferation of cells [53,54]. Therefore, the phosphorylation of PI3K, AKT and ERK1/2 was also examined in the present study. ...
Background
Ultrasound-targeted microbubble destruction (UTMD) has emerged as a promising strategy for the targeted delivery of bone marrow mesenchymal stem cells (MSCs) to the ischemic myocardium. However, the limited migration capacity and poor survival of MSCs remains a major therapeutic barrier. The present study was performed to investigate the synergistic effect of UTMD with platelet-derived growth factor BB (PDGF-BB) on the homing of MSCs for acute myocardial infarction (AMI).
Methods
MSCs from male donor rats were treated with PDGF-BB, and a novel microbubble formulation was prepared using a thin-film hydration method. In vivo, MSCs with or without PDGF-BB pretreatment were transplanted by UTMD after inducing AMI in experimental rats. The therapeutic efficacy of PDGF-BB-primed MSCs on myocardial apoptosis, angiogenesis, cardiac function and scar repair was estimated. The effects and molecular mechanisms of PDGF-BB on MSC migration and survival were explored in vitro.
Results
The results showed that the biological effects of UTMD increased the local levels of stromal-derived factor-1 (SDF-1), which promoted the migration of transplanted MSCs to the ischemic region. Compared with UTMD alone, UTMD combined with PDGF-BB pretreatment significantly increased the cardiac homing of MSCs, which subsequently reduced myocardial apoptosis, promoted neovascularization and tissue repair, and increased cardiac function 30 days after MI. The vitro results demonstrated that PDGF-BB enhanced MSC migration and protected these cells from H2O2-induced apoptosis. Mechanistically, PDGF-BB pretreatment promoted MSC migration and inhibited H2O2-induced MSC apoptosis via activation of the phosphatidylinositol 3-kinase/serine-threonine kinase (PI3K/Akt) pathway. Furthermore, crosstalk between PDGF-BB and stromal-derived factor-1/chemokine receptor 4 (SDF-1/CXCR4) is involved in the PI3K/AKT signaling pathway.
Conclusion
The present study demonstrated that UTMD combined with PDGF-BB treatment could enhance the homing ability of MSCs, thus alleviating AMI in rats. Therefore, UTMD combined with PDGF-BB pretreatment may offer exciting therapeutic opportunities for strengthening MSC therapy in ischemic diseases.
... The ERK signaling cascade is triggered by the small GTPase Ras, which is activated in response to mitogen stimulations of receptor tyrosine kinases at the plasma membrane. Once phosphorylated, ERK enters the nucleus and alters gene expression by phosphorylating transcription factors such as Elk1, Ets-2 and c-Myc in addition to cytoplasmic substrates (Unal et al., 2017). ERK has been suggested to be mechanoresponsive downstream of stiffness-induced receptor tyrosine kinase activation (Hwang et al., 2015;Zhang et al., 2017;Yang et al., 2018;Farahani et al., 2021). ...
Breast cancer is characterized by physical changes that occur in the tumor microenvironment throughout growth and metastasis of tumors. Extracellular matrix stiffness increases as tumors develop and spread, with stiffer environments thought to correlate with poorer disease prognosis. Changes in extracellular stiffness and other physical characteristics are sensed by integrins which integrate these extracellular cues to intracellular signaling, resulting in modulation of proliferation and invasion. However, the co-ordination of mechano-sensitive signaling with functional changes to groups of tumor cells within 3-dimensional environments remains poorly understood. Here we provide evidence that increasing the stiffness of collagen scaffolds results in increased activation of ERK1/2 and YAP in human breast cancer cell spheroids. We also show that ERK1/2 acts upstream of YAP activation in this context. We further demonstrate that YAP, matrix metalloproteinases and actomyosin contractility are required for collagen remodeling, proliferation and invasion in lower stiffness scaffolds. However, the increased activation of these proteins in higher stiffness 3-dimensional collagen gels is correlated with reduced proliferation and reduced invasion of cancer cell spheroids. Our data collectively provide evidence that higher stiffness 3-dimensional environments induce mechano-signaling but contrary to evidence from 2-dimensional studies, this is not sufficient to promote pro-tumorigenic effects in breast cancer cell spheroids.
... The cellular effects of ERK dynamics are exerted largely through the regulation of gene expression. ERK has over 1000 identified target genes [25], many of which are themselves involved in transcriptional regulation, allowing ERK to exert widespread influence on the expressed genome. ERK activity can stimulate cell proliferation, differentiation, metabolism, and drug resistance, by modulating the expression of target genes including CCND1 (encoding Cyclin D1), FOS (FOS), MYC (MYC), and FOSL1 (FRA-1). ...
Signaling by the extracellular signal-regulated kinase (ERK) pathway controls many cellular processes, including cell division, death, and differentiation. In this second installment of a two-part review, we address the question of how the ERK pathway exerts distinct and context-specific effects on multiple processes. We discuss how the dynamics of ERK activity induce selective changes in gene expression programs, with insights from both experiments and computational models. With a focus on single-cell biosensor-based studies, we summarize four major functional modes for ERK signaling in tissues: adjusting the size of cell populations, gradient-based patterning, wave propagation of morphological changes, and diversification of cellular gene expression states. These modes of operation are disrupted in cancer and other related diseases and represent potential targets for therapeutic intervention. By understanding the dynamic mechanisms involved in ERK signaling, there is potential for pharmacological strategies that not only simply inhibit ERK, but also restore functional activity patterns and improve disease outcomes.
... Our studies identified D-sites corresponding to many known MAPK binding partners and substrates (12,(53)(54)(55) but also identified a large number of other docking sequences (Datasets S4 and S5), indicating that multiple complementary approaches will be important to completely define MAPK interactomes. For example, prior research using a structure-guided in silico screen based on the p38-MEF2A peptide complex (18) predicted only 20% of D-sites selected by p38α in this study (Dataset S5). ...
Mitogen-activated protein kinase (MAPK) cascades are essential for eukaryotic cells to integrate and respond to diverse stimuli. Maintaining specificity in signaling through MAPK networks is key to coupling distinct inputs to appropriate cellular responses. Docking sites—short linear motifs found in MAPK substrates, regulators, and scaffolds—can promote signaling specificity through selective interactions, but how they do so remains unresolved. Here, we screened a proteomic library for sequences interacting with the MAPKs extracellular signal-regulated kinase 2 (ERK2) and p38α, identifying selective and promiscuous docking motifs. Sequences specific for p38α had high net charge and lysine content, and selective binding depended on a pair of acidic residues unique to the p38α docking interface. Finally, we validated a set of full-length proteins harboring docking sites selected in our screens to be authentic MAPK interactors and substrates. This study identifies features that help define MAPK signaling networks and explains how specific docking motifs promote signaling integrity.
... This suggests that the activation of ERK1/2 may be closely related to the proliferation of tumor cells. The localization of ERK1/2 in cells differs and it can be distributed in the cytoplasm or nucleus [28]. Because ERK1/2 in the nucleus can bind with transcription factors, it can regulate cell proliferation. ...
Cell clusters are a prevalent form of cells in the body, and recent studies have found that they play a significant role in the malignant progression of tumors. Cell clusters can become malignant through cell–cell connectivity and the cell-interstitial microenvironment. However, the differences between anterior and posterior cells are still unclear. In this study, we identified that the proliferation rate of cells in the posterior cell zone was significantly higher than that in the anterior cell zone. This difference was positively related to the activation of Ras-ERK1/2-YAP axis. The leader cells in the anterior cell region had low expression of Ras-ERK1/2-YAP, which may be why they had inactive proliferation. This study also confirmed the importance of cytoskeleton in the process of cell proliferation. Loss of myosin inhibited both cell proliferation and the nuclear entry activation of ERK1/2-YAP. These findings are significant for understanding the proliferation of tumor cell clusters and identifying screening targets for anticancer drugs that weaken the mobility of tumor cells.
... PI3K regulates the phosphorylation of AKT, where the subsequent PI3K/AKT downstream signaling pathway has been suggested to serve a regulatory role in a number of cell proliferation and activation processes [12][13] . By contrast, the ERK signaling pathway has been reported to serve a key role in transducing signals from cell-surface receptors to the nucleus, thereby regulating the activation and proliferation of cells [53][54] . Therefore, the phosphorylation of PI3K, AKT and ERK1/2 was also examined in the present study. ...
Background
Ultrasound-targeted microbubble destruction (UTMD) has emerged as a promising strategy for the targeted delivery of bone marrow mesenchymal stem cells (MSCs) to the ischemic myocardium. However, the limited migration capacity and poor survival of MSCs remains a major therapeutic barrier. The present study was performed to investigate the synergistic effect of UTMD with PDGF-BB on the homing of MSCs for acute myocardial infarction (AMI).
Methods
MSCs from male donor rats were treated by PDGF-BB, and a novel microbubble formulation were prepared by a thin-film hydration method. In vivo, MSCs with or without PDGF-BB pretreatment were transplanted by UTMD after inducing AMI in experimental rats. The therapeutic efficacy of PDGF-BB-Primed MSCs on myocardial apoptosis, angiogenesis, cardiac function and scar repair was estimated. In vitro, the effect and molecular mechanism of PDGF-BB on MSCs migration and survival were explored.
Results
The results showed that the biological effects of UTMD increased local levels of SDF-1, which promoted the migration of transplanted MSCs to the ischemic region. Compared with UTMD alone, UTMD combined with PDGF-BB pretreatment significantly increased the cardiac homing of MSCs, which subsequently reduced myocardial apoptosis, promoted neovascularization and tissue repair, and increased cardiac function 30 days after MI. In vitro results showed that PDGF-BB enhanced MSC migration, and protected these cells from H2O2-induced apoptosis. Mechanistically, PDGF-BB pretreatment promoted MSCs migration and inhibited H2O2-induced MSC apoptosis via activation of the PI3K/Akt pathway. Further, crosstalk between PDGF-BB and SDF-1/CXCR4 is involved in the PI3K/AKT signaling pathway.
Conclusion
The present study demonstrated that UTMD combined with PDGF-BB treatment could enhance the homing ability of MSCs, thus alleviating AMI in rats. Therefore, UTMD combined with PDGF-BB pretreatment may offer exciting therapeutic opportunities for strengthening MSC therapy in ischemic diseases.
... Therefore, in the current study, we explored whether secreted EVs from the micrografts in culture speci cally affected urothelial cell migration and proliferation. The ERK pathway effects have mainly been linked to cell proliferation and migration in skin, however, to our knowledge, the role of pERK within urothelial micrografting has previously not been evaluated [26][27][28][29][30]. We hypothesized, that the conditioned medium (CM) of micrografted urothelial cells contained EVs with pro-regenerative content properties. ...
Autologous micrografting is a technique currently applied within skin wound healing, however, the potential use for surgical correction of other organs with epithelial lining, including the urinary bladder, remains largely unexplored. Currently, little is known about the micrograft expansion potential and the micromolecular events that occur in micrografted urothelial cells. In this study, we aimed to evaluate the regenerative potential of different porcine urothelial micrograft sizes in vitro , and, furthermore, to explore how urothelial micrografts communicate and which microcellular events are triggered. We demonstrated that increased tissue fragmentation subsequently potentiated the yield of proliferative cells and the cellular expansion potential, which confirms, that the micrografting principles of skin epithelium also apply to uroepithelium. Furthermore, we targeted the expression of the extracellular signal-regulated kinase (ERK) pathway and demonstrated that ERK activation occurred predominately at the micrograft borders and that ERK inhibition led to decreased urothelial migration and proliferation. Finally, we successfully isolated extracellular vesicles from the micrograft culture medium and evaluated their contents and relevance within various enriched biological processes. Our findings substantiate the potential of applying urothelial micrografting in future tissue-engineering models for reconstructive urological surgery, and, furthermore, highlights certain mechanisms as potential targets for future wound healing treatments.
... Next, Imp7 escorts ERK1/2 into the nucleus through the nuclear pores, detachment of these two proteins, which is mediated by Ran-GTP and export of Imp7 back to the cytoplasm. The released active ERK1/2 are now free to phosphorylate its nuclear targets including transcription factors and other nuclear proteins [2,38]. We also found that the nuclear translocation of the other MAPKs, p38 and JNK, has some similarities to ERK1/2, but the importins involved are either Imp 3/7 or Imp3/9 dimers [39]. ...
... This indicates that 6-G exerts a regulatory effect on cell survival or apoptosis by affecting the dynamic balance between the growth factor-activated ERK pathway and the stress-activated JNK-p38 pathway [38]. On the one hand, this regulation depends on ERK pathway-mediated direct targeting and regulation of the cell cycle and indirect regulation of RNA metabolism and transport [39]. On the other hand, it also depends on the regulation of the JNK pathway by various cellular stress and growth factors [40]. ...
6-Gingerol, the main active ingredient in ginger, exhibits a variety of biological activities, such as antioxidant, anti-inflammatory, and anticancer activities, and can affect cell development. However, the effects of 6-gingerol on mammalian reproductive processes, especially early embryonic development, are unclear. This study explored whether 6-gingerol can be used to improve the quality of in vitro-cultured porcine embryos. The results showed that 5 μM 6-gingerol significantly increased the blastocyst formation rates of porcine early embryos. 6-Gingerol attenuated intracellular reactive oxygen species accumulation and autophagy, increased intracellular glutathione levels, and increased mitochondrial activity. In addition, 6-gingerol upregulated NANOG, SRY-box transcription factor 2, cytochrome c oxidase subunit II, mechanistic target of rapamycin kinase, and RPTOR independent companion of MTOR complex 2 while downregulating Caspase 3, baculoviral IAP repeat containing 5, autophagy related 12, and Beclin 1. Most importantly, 6-gingerol significantly increased the levels of p-extracellular regulated protein kinase 1/2 while reducing the levels of p-c-Jun N-terminal kinase 1/2/3 and p-p38. These results indicate that 6-gingerol can promote the development of porcine early embryos in vitro.
RASopathies are caused by variants in more than 20 genes functionally converging toward the upregulation of intracellular signaling through the RAS/mitogen-associated protein kinase (MAPK) cascade. These disorders are largely transmitted as autosomal dominant traits, though recessive forms are emerging. More than 10 genes have been causally linked to Noonan syndrome, the most common and clinically variable RASopathy; genetic heterogeneity characterizes also cardio-facio-cutaneous syndrome and Noonan syndrome with loose anagen hair, also known as Mazzanti syndrome. On the other hand, allelic heterogeneity also occurs, as in the case of PTPN11, KRAS, and BRAF. A large proportion of cases result from de novo mutations; however, families transmitting the disorder are common in Noonan syndrome. In RASopathies, increased RAS/MAPK signaling can result from the upregulated activity of various GTPases of the RAS family, increased function of signal transducers positively controlling RAS activity or favoring RAS interactions with RAF kinases, functional upregulation of the three tiers belonging to the MAPK cascade, or inefficient signaling switch-off operating at different levels. While the MAPK cascade is virtually upregulated in almost all RASopathies, upregulation of the PI3K-AKT-mTOR pathway also represents an alternative driver or contributing mechanism. Genetic evidence also suggests the involvement of signaling dysregulation via other networks (e.g., small RHO GTPases signaling) to disease pathogenesis. Here, we summarize key concepts on the molecular genetics of these disorders, discussing the involved genes and the molecular circuits implicated in RAS/MAPK signaling dysregulation, including those that had previously remained uncharacterized due to their minor impact on oncogenesis.
The RAS small GTPases (HRAS, KRAS, and NRAS) comprise the most frequently mutated oncogene family in human cancer. RAS proteins reside on the cytoplasmic face of the plasma membrane where they act as binary on-off switches that relay signals from extracellular signal-activated cell surface receptors to diverse cytoplasmic signaling networks. RAS signaling is dysregulated by diverse mechanisms in cancer. These include hyperactivating point mutations in RAS itself, aberrant activation of upstream components (e.g., receptor tyrosine kinases), loss of negative regulators (e.g., neurofibromin), and mutational activation of downstream components (e.g., RAF, PI3Kα). Aberrant RAS activation drives persistent activation of downstream effector signaling networks, primarily through the RAF-MEK-ERK mitogen-activated protein kinase (MAPK) cascade. Mutational activation of RAS-ERK signaling is also the defining feature of rare germline developmental disorders termed RASopathies. As the terminal node of the ERK MAPK pathway, ERK regulates a complex phosphoproteome and transcriptome, with the MYC transcription factor and oncoprotein a major component of ERK-driven cancer growth. In this review, we summarize key proximal and distal features of the RAS signaling network and highlight approaches for targeting RAS and the ERK MAPK signaling network for cancer treatment.
VEGFA administration has been explored as a pro-angiogenic therapy for cardiovascular diseases including heart failure for several years, but with little success. Here, we investigate a different approach to augment VEGFA bioavailability: by deleting the VEGFA decoy receptor VEGFR1 (also known as FLT1), one can achieve more physiological VEGFA concentrations. We find that after cryoinjury, zebrafish flt1 mutant hearts display enhanced coronary revascularization and endocardial expansion, increased cardiomyocyte dedifferentiation and proliferation, and decreased scarring. Suppressing Vegfa signaling in flt1 mutants abrogates these beneficial effects of flt1 deletion. Transcriptomic analyses of cryoinjured flt1 mutant hearts reveal enhanced endothelial MAPK/ERK signaling and downregulation of the transcription factor gene egr3. Using newly generated genetic tools, we observe egr3 upregulation in the regenerating endocardium, and find that Egr3 promotes myofibroblast differentiation. These data indicate that with enhanced Vegfa bioavailability, the endocardium limits myofibroblast differentiation via egr3 downregulation, thereby providing a more permissive microenvironment for cardiomyocyte replenishment after injury.
Doxorubicin (Dox) is frequently employed as a chemotherapy agent for breast cancer. As the chemotherapy moves forward, breast cancer cells tend to develop resistance to Dox, besides that, Dox are also easy to cause cardiotoxicity related to cumulative dose. Therefore, how to potentiate the chemosensitivity of breast cancer cells to Dox while attenuating its cardiotoxicity has become a research hotspot. Tanshinone IIA (Tan IIA) is known for its anticancer activity as well as for its cardioprotective effects. In view of the aforementioned facts, we assessed whether Tan IIA possesses synergism and attenuation effect on Dox for breast cancer chemotherapy. Our studies in vitro indicated that, Tan IIA could potentiate the effect of Dox on breast cancer cells proliferation inhibition and apoptosis promotion by inhibiting ERK1/2 pathway, but interestingly, Tan IIA attenuated the cytotoxicity of Dox to myocardial cells by activating ERK1/2 pathway. Additionally, our studies in vivo also suggested that Tan IIA potentiated the chemotherapeutic effect of Dox against breast cancer while attenuating Dox‐induced myocardial injury. Given that Tan IIA had a synergism and attenuation effect on Dox, we believed that Tan IIA can be used as an ideal drug in combination with Dox for breast cancer therapy.
RAS and MYC rank amongst the most commonly altered oncogenes in cancer, with RAS being the most frequently mutated and MYC the most amplified. The cooperative interplay between RAS and MYC constitutes a complex and multifaceted phenomenon, profoundly influencing tumor development. Together and individually, these two oncogenes regulate most, if not all, hallmarks of cancer, including cell death escape, replicative immortality, tumor-associated angiogenesis, cell invasion and metastasis, metabolic adaptation, and immune evasion. Due to their frequent alteration and role in tumorigenesis, MYC and RAS emerge as highly appealing targets in cancer therapy. However, due to their complex nature, both oncogenes have been long considered “undruggable” and, until recently, no drugs directly targeting them had reached the clinic. This review aims to shed light on their complex partnership, with special attention to their active collaboration in fostering an immunosuppressive milieu and driving immunotherapeutic resistance in cancer. Within this review, we also present an update on the different inhibitors targeting RAS and MYC currently undergoing clinical trials, along with their clinical outcomes and the different combination strategies being explored to overcome drug resistance. This recent clinical development suggests a paradigm shift in the long-standing belief of RAS and MYC “undruggability”, hinting at a new era in their therapeutic targeting.
The MHC class I–related molecule MR1 is ubiquitously expressed, is highly conserved among mammals, and presents bacterial and endogenous antigens in tumor cells. These features indicate that tumor-specific T cells restricted to MR1 may represent ideal candidates for novel cancer-directed T-cell immunotherapy. The very low expression of the MR1 protein at the cell surface is a potential challenge limiting the possible use of MR1-directed immunotherapies. To overcome this challenge, it is important that understanding of the mechanisms regulating MR1 expression is increased, as little is known about this currently. This study identified ERK1/2 as negative regulators of the MR1 gene and protein expression. Inhibition of ERK1/2 in tumor cells or treatment of BRAF-mutant tumor cells with drugs specific for mutated BRAF increased MR1 protein expression and recognition by tumor-reactive and MR1-restricted T cells. The ERK1/2 inhibition of MR1 was mediated by the ELF1 transcription factor, which was required for MR1 gene expression. The effects of ERK1/2 inhibition also occurred in cancer cell lines of different tissue origins, cancer cell lines resistant to drugs that inhibit mutated BRAF, and primary cancer cells, making them potential targets of specific T cells. In contrast to tumor cells, the recognition of healthy cells was very poor or absent after ERK1/2 inhibition. These findings suggest a pharmaceutical approach to increase MR1 protein expression in tumor cells and the subsequent activation of MR1-restricted T cells, and they have potential therapeutic implications.
Human’s robust cognitive abilities, including creativity and language, are made possible, at least in large part, by evolutionary changes made to the cerebral cortex. This paper reviews the biology and evolution of mammalian cortical radial glial cells (primary neural stem cells) and introduces the concept that a genetically step wise process, based on a core molecular pathway already in use, is the evolutionary process that has molded cortical neurogenesis. The core mechanism, which has been identified in our recent studies, is the extracellular signal-regulated kinase (ERK)-bone morphogenic protein 7 (BMP7)-GLI3 repressor form (GLI3R)-sonic hedgehog (SHH) positive feedback loop. Additionally, I propose that the molecular basis for cortical evolutionary dwarfism, exemplified by the lissencephalic mouse which originated from a larger gyrencephalic ancestor, is an increase in SHH signaling in radial glia, that antagonizes ERK-BMP7 signaling. Finally, I propose that: (1) SHH signaling is not a key regulator of primate cortical expansion and folding; (2) human cortical radial glial cells do not generate neocortical interneurons; (3) human-specific genes may not be essential for most cortical expansion. I hope this review assists colleagues in the field, guiding research to address gaps in our understanding of cortical development and evolution.
RASopathies are rare developmental genetic syndromes caused by germline pathogenic variants in genes that encode components of the RAS/mitogen-activated protein kinase (MAPK) signal transduction pathway. Although the incidence of each RASopathy syndrome is rare, collectively, they represent one of the largest groups of multiple congenital anomaly syndromes and have severe developmental consequences. Here, we review our understanding of how RAS/MAPK dysregulation in RASopathies impacts skeletal muscle development and the importance of RAS/MAPK pathway regulation for embryonic myogenesis. We also discuss the complex interactions of this pathway with other intracellular signaling pathways in the regulation of skeletal muscle development and growth, and the opportunities that RASopathy animal models provide for exploring the use of pathway inhibitors, typically used for cancer treatment, to correct the unique skeletal myopathy caused by the dysregulation of this pathway.
To delineate the mechanisms by which the ERK1 and ERK2 mitogen-activated protein kinases support mutant KRAS–driven cancer growth, we determined the ERK-dependent phosphoproteome in KRAS-mutant pancreatic cancer. We determined that ERK1 and ERK2 share near-identical signaling and transforming outputs and that the KRAS-regulated phosphoproteome is driven nearly completely by ERK. We identified 4666 ERK-dependent phosphosites on 2123 proteins, of which 79 and 66%, respectively, were not previously associated with ERK, substantially expanding the depth and breadth of ERK-dependent phosphorylation events and revealing a considerably more complex function for ERK in cancer. We established that ERK controls a highly dynamic and complex phosphoproteome that converges on cyclin-dependent kinase regulation and RAS homolog guanosine triphosphatase function (RHO GTPase). Our findings establish the most comprehensive molecular portrait and mechanisms by which ERK drives KRAS-dependent pancreatic cancer growth.
Abnormal Extracellular Regulated Kinase 1/2 (ERK1/2) signaling is linked to multiple neurodevelopmental diseases, especially the RASopathies, which typically exhibit ERK1/2 hyperactivation in neurons and non-neuronal cells. To better understand how excitatory neuron-autonomous ERK1/2 activity regulates forebrain development, we conditionally expressed hyperactive MEK1S217/221E in cortical excitatory neurons. MEK1S217/221E expression led to persistent hyperactivation of ERK1/2 in cortical axons, but not in soma/nuclei. We noted reduced axonal arborization in multiple target domains in mutants and reduced expression of the activity dependent gene, ARC. These changes did not lead to deficits in voluntary locomotion or accelerating rotarod performance. However, skilled motor learning in a single-pellet retrieval task was significantly diminished in these MEK1S217/221E mutants. Restriction of MEK1S217/221E expression to layer V cortical neurons recapitulated axonal outgrowth deficits, but did not effect motor learning. These results suggest that cortical excitatory neuron-autonomous hyperactivation of MEK1 is sufficient to drive deficits in axon outgrowth, which coincide with reduced ARC expression, and deficits in skilled motor learning. Our data indicate that neuron-autonomous decreases in long-range axonal outgrowth may be a key aspect of neuropathogenesis in RASopathies.
Understanding the molecular and cellular mechanisms driving pediatric low-grade glioma (pLGG)—the most prevalent brain tumor in children—is essential for the identification and evaluation of novel effective treatments. This review explores the intricate relationship between the mitogen-activated protein kinase (MAPK) pathway, oncogene-induced senescence (OIS), the senescence-associated secretory phenotype (SASP), and the tumor microenvironment (TME), integrating these elements into a unified framework termed the MAPK/OIS/SASP/TME (MOST) axis. This integrated approach seeks to deepen our understanding of pLGG and improve therapeutic interventions by examining the MOST axis’ critical influence on tumor biology and response to treatment. In this review, we assess the axis’ capacity to integrate various biological processes, highlighting new targets for pLGG treatment, and the need for characterized in vitro and in vivo preclinical models recapitulating pLGG’s complexity to test targets. The review underscores the need for a comprehensive strategy in pLGG research, positioning the MOST axis as a pivotal approach in understanding pLGG. This comprehensive framework will open promising avenues for patient care and guide future research towards inventive treatment options.
Comprehensive profiling of genomic alterations and integration of this data with other molecular and clinical information in recent years has substantially advanced our understanding of the genetic basis underlying the disparate disease courses of neuroblastoma. While the overall mutational burden in neuroblastoma is low, as in other pediatric tumors, it was found that genomic alterations affecting key regulators of telomere maintenance occur in the vast majority of high-risk cases. Amplification of the transcription factor MYCN or genomic rearrangements of the TERT locus are found in most high-risk tumors, both leading to induction of telomerase expression. In another substantial fraction of high-risk neuroblastoma, telomeres are maintained by the telomerase-independent Alternative Lengthening of Telomeres (ALT) pathway, which is associated with inactivating mutations of the ATRX gene. By contrast, such alterations are invariably lacking in low-risk neuroblastoma, suggesting that absence of telomere maintenance and consecutive lack of cellular immortality may cause spontaneous regression and differentiation. In addition, oncogenic mutations have been found in the gene ALK, encoding for a receptor tyrosine kinase, in roughly 10% of neuroblastoma cases, and ALK inhibitory therapies are currently implemented in neuroblastoma treatment. Recurrent alterations are also detected in genes related to the RAS/MAPK and the p53 pathways. Integration of the diverse genomic information revealed that activation of telomere maintenance is the primary determinant of the clinical phenotype, whereas other alterations increase tumor aggressiveness only if telomere maintenance is present. This chapter summarizes the genomic alterations underlying the divergent courses of neuroblastoma, highlighting the pivotal role of telomere maintenance in this disease.
B cell receptor (BCR) signaling is required for the survival and maturation of B cells and is deregulated in B cell lymphomas. While proximal BCR signaling is well studied, little is known about the crosstalk of downstream effector pathways, and a comprehensive quantitative network analysis of BCR signaling is missing. Here, we semi-quantitatively modelled BCR signaling in Burkitt lymphoma (BL) cells using systematically perturbed phosphorylation data of BL-2 and BL-41 cells. The models unveiled feedback and crosstalk structures in the BCR signaling network, including a negative crosstalk from p38 to MEK/ERK. The relevance of the crosstalk was verified for BCR and CD40 signaling in different BL cells and confirmed by global phosphoproteomics on ERK itself and known ERK target sites. Compared to the starting network, the trained network for BL-2 cells was better transferable to BL-41 cells. Moreover, the BL-2 network was also suited to model BCR signaling in Diffuse large B cell lymphoma cells lines with aberrant BCR signaling (HBL-1, OCI-LY3), indicating that BCR aberration does not cause a major downstream rewiring.
At least 40% of human cancers are associated with aberrant ERK pathway activity (ERKp). Inhibitors targeting various effectors within the ERKp have been developed and explored for over two decades. Conversely, a substantial body of evidence suggests that both normal human cells and, notably to a greater extent, cancer cells exhibit susceptibility to hyperactivation of ERKp. However, this vulnerability of cancer cells remains relatively unexplored. In this review, we reexamine the evidence on the selective lethality of highly elevated ERKp activity in human cancer cells of varying backgrounds. We synthesize the insights proposed for harnessing this vulnerability of ERK-associated cancers for therapeutical approaches and contextualize these insights within established pharmacological cancer-targeting models. Moreover, we compile the intriguing preclinical findings of ERK pathway agonism in diverse cancer models. Lastly, we present a conceptual framework for target discovery regarding ERKp agonism, emphasizing the utilization of mutual exclusivity among oncogenes to develop novel targeted therapies for precision oncology.
Introduction: Melanoma, the most lethal and aggressive variant of skin cancer, poses a concerning health issue due to existing therapeutic deficiencies, necessitating the development of novel treatments. Tumour microenvironment (TME) is a crucial player in cancer progression and treatment responses. TME, a heterogeneous and dynamic ecosystem, is actively deployed by cancer to orchestrate disease-permissive influences via a complex network of spatial interaction and communication. This thesis proposes two innovative therapeutic approaches concerted around re-sculpting the pro-tumorigenic microenvironment.
Aims: To assess the therapeutic efficacy of the proposed drug combination of vemurafenib and the in-house CSF1R inhibitor SN38114 or cannabidiol (CBD) in both in vitro and in vivo settings.
Methods: Therapeutic efficacy was evaluated via transplantation of patient-derived melanoma tumour xenografts into homozygous NIH-III mice. Immunohistochemistry analyses of xenograft tumours were conducted to virtualise the effect of the combinations. In vitro investigations of the cytotoxicity of single agents and combination treatments were performed on a panel of colorectal cancer cell lines, melanoma cell lines, and tumour microenvironment cell lines.
Results: A synergetic tumour growth inhibitory effect on BRAF V600E mutated NZM 20 human melanoma xenograft tumours was observed with the combination of vemurafenib and CBD, while in vitro cell viability assays revealed ineffectiveness. Significant attenuation of Ki-67 and CD31 expression was observed in vivo tumours treated with vemurafenib and CBD. Furthermore, we observed decreased expression of type one collagen and alpha-smooth muscle actin in the BRAF wild type NZM 40 human melanoma xenograft model treated with CBD.
Conclusion: We demonstrate the potential synergetic anti-tumour effect of a combination of mitogen-activated protein kinases inhibitor vemurafenib and cannabidiol on a patient-derived melanoma tumour xenograft model. We propose the immune-modulating role of cannabidiol in human melanoma and reveal enhanced anti-proliferative and anti-angiogenesis effects of cannabidiol with vemurafenib in vivo. We further suggest CBD's potential dual anti-invasiveness impact in the BRAF wild-type human melanoma xenograft model. This study advocated the medical use of CBD as a viable adjunctive option in combination with other therapeutic agents.
Cancer remains one of the most complex and challenging diseases in mankind. To address the need for a personalized treatment approach for particularly complex tumor cases, molecular tumor boards (MTBs) have been initiated. MTBs are interdisciplinary teams that perform in‐depth molecular diagnostics to cooperatively and interdisciplinarily advise on the best therapeutic strategy. Current molecular diagnostics are routinely performed on the transcriptomic and genomic levels, aiming to identify tumor‐driving mutations. However, these approaches can only partially capture the actual phenotype and the molecular key players of tumor growth and progression. Thus, direct investigation of the expressed proteins and activated signaling pathways provide valuable complementary information on the tumor‐driving molecular characteristics of the tissue. Technological advancements in mass spectrometry‐based proteomics enable the robust, rapid, and sensitive detection of thousands of proteins in minimal sample amounts, paving the way for clinical proteomics and the probing of oncogenic signaling activity. Therefore, proteomics is currently being integrated into molecular diagnostics within MTBs and holds promising potential in aiding tumor classification and identifying personalized treatment strategies. This review introduces MTBs and describes current clinical proteomics, its potential in precision oncology, and highlights the benefits of multi‐omic data integration.
Abnormal Extracellular Regulated Kinase 1/2 (ERK1/2) signaling is linked to multiple neurodevelopmental diseases, especially the RASopathies, which typically exhibit ERK1/2 hyperactivation in neurons and non-neuronal cells. To better understand how excitatory neuron-autonomous ERK1/2 activity regulates the development of the mouse motor cortex, we conditionally expressed a hyperactive MEK1 S217/221E variant using Nex/NeuroD6:Cre . Our results show that MEK1 S217/221E expression led to persistent hyperactivation of ERK1/2 in neocortical axons, but not excitatory neuron somas or nuclei. We noted reduced axonal arborization of multiple subcortical target domains in mutants and reduced cortical expression of the activity dependent gene, ARC. These changes did not coincide with significant differences in voluntary locomotor activity or motor performance in the accelerating rotarod task. However, motor learning in a single-pellet retrieval task was significantly diminished in Nex/NeuroD6:Cre ; MEK1 S217/221E mutants. Restriction of MEK1 S217/221E expression to layer V cortical neurons recapitulated axonal outgrowth deficits, however, had no effect on motor learning. Collectively, these results indicate that within the cortex, glutamatergic neuron-autonomous hyperactivation of MEK1 is sufficient to drive deficits in axon outgrowth, activity dependent gene expression, and skilled motor learning.
Summary statement
MEK-ERK1/2 hyperactivation in developing cortical excitatory neurons is sufficient to decrease long-range axonal outgrowth, which coincides with reduced Arc expression and deficits in aspects of skilled motor learning by adulthood.
The RAS-ERK pathway is a fundamental signaling cascade crucial for many biological processes including proliferation, cell cycle control, growth, and survival; common across all cell types. Notably, ERK1/2 are implicated in specific processes in a context-dependent manner as in stem cells and pancreatic β-cells. Alterations in the different components of this cascade result in dysregulation of the effector kinases ERK1/2 which communicate with hundreds of substrates. Aberrant activation of the pathway contributes to a range of disorders, including cancer. This review provides an overview of the structure, activation, regulation, and mutational frequency of the different tiers of the cascade; with a particular focus on ERK1/2. We highlight the importance of scaffold proteins that contribute to kinase localization and coordinate interaction dynamics of the kinases with substrates, activators, and inhibitors. Additionally, we explore innovative therapeutic approaches emphasizing promising avenues in this field.
Tumor necrosis factor-alpha (TNFα) is a pleiotropic pro-inflammatory cytokine of the TNF superfamily. It regulates key cellular processes such as death, and proliferation besides its well-known role in immune response through activation of various intracellular signaling pathways (such as MAPK, Akt, NF-κB, etc.) via complex formation by ligand-activated TNFα receptors. TNFα tightly regulates the activity of key signaling proteins via their phosphorylation and/or ubiquitination which culminate in specific cellular responses. Deregulated TNFα signaling is implicated in inflammatory diseases, neurological disorders, and cancer. TNFα has been shown to exert opposite effects on cancer cells since it activates pro-survival as well as anti-survival pathways depending on various contexts such as cell type, concentration, cell density, etc. A detailed understanding of TNFα signaling phenomena is crucial for understanding its pleiotropic role in malignancies and its potential as a drug target or an anticancer therapeutic. This review enlightens complex cellular signaling pathways activated by TNFα and further discusses its role in various cancers.
Autologous micrografting is a technique currently applied within skin wound healing, however, the potential use for surgical correction of other organs with epithelial lining, including the urinary bladder, remains largely unexplored. Currently, little is known about the micrograft expansion potential and the micromolecular events that occur in micrografted urothelial cells. In this study, we aimed to evaluate the proliferative potential of different porcine urothelial micrograft sizes in vitro, and, furthermore, to explore how urothelial micrografts communicate and which microcellular events are triggered. We demonstrated that increased tissue fragmentation subsequently potentiated the yield of proliferative cells and the cellular expansion potential, which confirms, that the micrografting principles of skin epithelium also apply to uroepithelium. Furthermore, we targeted the expression of the extracellular signal-regulated kinase (ERK) pathway and demonstrated that ERK activation occurred predominately at the micrograft borders and that ERK inhibition led to decreased urothelial migration and proliferation. Finally, we successfully isolated extracellular vesicles from the micrograft culture medium and evaluated their contents and relevance within various enriched biological processes. Our findings substantiate the potential of applying urothelial micrografting in future tissue-engineering models for reconstructive urological surgery, and, furthermore, highlights certain mechanisms as potential targets for future wound healing treatments.
RAF kinases play major roles in cancer. BRAFV600E mutants drive ~6% of human cancers. Potent kinase inhibitors exist but show variable effects in different cancer types, sometimes even inducing paradoxical RAF kinase activation. Both paradoxical activation and drug resistance are frequently due to enhanced dimerization between RAF1 and BRAF, which maintains or restores the activity of the downstream MEK-ERK pathway. Here, using quantitative proteomics we mapped the interactomes of RAF1 monomers, RAF1-BRAF and RAF1-BRAFV600E dimers identifying and quantifying >1,000 proteins. In addition, we examined the effects of vemurafenib and sorafenib, two different types of clinically used RAF inhibitors. Using regression analysis to compare different conditions we found a large overlapping core interactome but also distinct condition specific differences. Given that RAF proteins have kinase independent functions such dynamic interactome changes could contribute to their functional diversification. Analysing this dataset may provide a deeper understanding of RAF signalling and mechanisms of resistance to RAF inhibitors.
Different tissues and organs coordinate to keep glucose levels at homeostasis. However, excessive gluconeogenesis in patients with type 2 diabetes mellitus increases blood glucose and causes dysregulation of homeostasis, which easily induces various diseases. Therefore, inhibiting excessive gluconeogenesis is a potential direction to regulate the disorder of blood glucose homeostasis and treat diabetes. The process of gluconeogenesis can be pretranslationally or posttranslationally regulated, and some natural small molecules can be involved in the pretranslational phase of gluconeogenesis, regulating glucose homeostasis by inhibiting gene expression, interfering with RNA transcription, and antagonizing related proteins. This paper will classify and review the natural small molecules with pretranslational regulation of gluconeogenesis according to the action sites and signaling pathways.
Small molecules targeting aberrant RAF activity, like Vemurafenib (PLX4032), are highly effective against cancers harboring the V600E BRAF mutation, and are now approved for clinical use against metastatic melanoma. However, in tissues showing elevated RAS activity and in RAS-mutant tumors, these inhibitors stimulate RAF dimerization, resulting in inhibitor resistance and downstream paradoxical ERK activation. To understand the global signaling response of cancer cells to RAF inhibitors, we profiled the temporal changes of the phosphoproteome of two colon cancer cell lines (Colo205 and HCT116) that respond differently to Vemurafenib. Comprehensive data mining and filtering identified a total of 37910 phosphorylation sites, 660 of which were dynamically modulated upon treatment with Vemurafenib. We established that 83% of these dynamic phosphorylation sites were modulated in accordance with the phospho-ERK profile of the two cell lines. Accordingly, kinase-substrate prediction algorithms linked most of these dynamic sites to direct ERK1/2-mediated phosphorylation, supporting a low off-target rate for Vemurafenib. Functional classification of target proteins indicated the enrichment of known (nuclear pore, transcription factors, RAS-RTK signaling) and novel (Rho GTPases signaling, actin cytoskeleton) ERK-controlled functions. Our phosphoproteomic data combined with experimental validation established novel dynamic connections between ERK signaling and the transcriptional regulators TEAD3 (Hippo pathway), MKL1 and MKL2 (Rho-SRF pathway). We also confirm that an ERK docking site found in MKL1 is directly antagonized by overlapping actin binding, defining a novel mechanism of actin-modulated phosphorylation. Altogether, time-resolved phosphoproteomics further documented Vemurafenib selectivity and identified novel ERK downstream substrates.
Functional enrichment analysis is a key step in interpreting gene lists discovered in diverse high-throughput experiments.
g:Profiler studies flat and ranked gene lists and finds statistically significant Gene Ontology terms, pathways and other
gene function related terms. Translation of hundreds of gene identifiers is another core feature of g:Profiler. Since its
first publication in 2007, our web server has become a popular tool of choice among basic and translational researchers. Timeliness
is a major advantage of g:Profiler as genome and pathway information is synchronized with the Ensembl database in quarterly
updates. g:Profiler supports 213 species including mammals and other vertebrates, plants, insects and fungi. The 2016 update
of g:Profiler introduces several novel features. We have added further functional datasets to interpret gene lists, including
transcription factor binding site predictions, Mendelian disease annotations, information about protein expression and complexes
and gene mappings of human genetic polymorphisms. Besides the interactive web interface, g:Profiler can be accessed in computational
pipelines using our R package, Python interface and BioJS component. g:Profiler is freely available at http://biit.cs.ut.ee/gprofiler/.
Mitogen-activated protein kinases (MAPK) are broadly used regulators of cellular signaling. However, how these enzymes can be involved in such a broad spectrum of physiological functions is not understood. Systematic discovery of MAPK networks both experimentally and in silico has been hindered because MAPKs bind to other proteins with low affinity and mostly in less-characterized disordered regions. We used a structurally consistent model on kinase-docking motif interactions to facilitate the discovery of short functional sites in the structurally flexible and functionally under-explored part of the human proteome and applied experimental tools specifically tailored to detect low-affinity protein-protein interactions for their validation invitro and in cell-based assays. The combined computational and experimental approach enabled the identification of many novel MAPK-docking motifs that were elusive for other large-scale protein-protein interaction screens. The analysis produced an extensive list of independently evolved linear binding motifs from a functionally diverse set of proteins. These all target, with characteristic binding specificity, an ancient protein interaction surface on evolutionarily related but physiologically clearly distinct three MAPKs (JNK, ERK, and p38). This inventory of human protein kinase binding sites was compared with that of other organisms to examine how kinase-mediated partnerships evolved over time. The analysis suggests that most human MAPK-binding motifs are surprisingly new evolutionarily inventions and newly found links highlight (previously hidden) roles of MAPKs. We propose that short MAPK-binding stretches are created in disordered protein segments through a variety of ways and they represent a major resource for ancient signaling enzymes to acquire new regulatory roles.
Cancer is often considered a genetic disease. However, much of the enormous plasticity of cancer cells to evolve different phenotypes, to adapt to challenging microenvironments and to withstand therapeutic assaults is encoded by the structure and spatiotemporal dynamics of signal transduction networks. In this Review, we discuss recent concepts concerning how the rich signalling dynamics afforded by these networks are regulated and how they impinge on cancer cell proliferation, survival, invasiveness and drug resistance. Understanding this dynamic circuitry by mathematical modelling could pave the way to new therapeutic approaches and personalized treatments.
Most biological processes are influenced by protein post-translational modifications (PTMs). Identifying novel PTM sites in different organisms, including humans and model organisms, has expedited our understanding of key signal transduction mechanisms. However, with increasing availability of deep, quantitative datasets in diverse species, there is a growing need for tools to facilitate cross-species comparison of PTM data. This is particularly important because functionally important modification sites are more likely to be evolutionarily conserved; yet cross-species comparison of PTMs is difficult since they often lie in structurally disordered protein domains. Current tools that address this can only map known PTMs between species based on known orthologous phosphosites, and do not enable the cross-species mapping of newly identified modification sites. Here, we addressed this by developing a web-based software tool, PhosphOrtholog ( www.phosphortholog.com ) that accurately maps protein modification sites between different species. This facilitates the comparison of datasets derived from multiple species, and should be a valuable tool for the proteomics community.
Here we describe PhosphOrtholog, a web-based application for mapping known and novel orthologous PTM sites from experimental data obtained from different species. PhosphOrtholog is the only generic and automated tool that enables cross-species comparison of large-scale PTM datasets without relying on existing PTM databases. This is achieved through pairwise sequence alignment of orthologous protein residues. To demonstrate its utility we apply it to two sets of human and rat muscle phosphoproteomes generated following insulin and exercise stimulation, respectively, and one publicly available mouse phosphoproteome following cellular stress revealing high mapping and coverage efficiency. Although coverage statistics are dataset dependent, PhosphOrtholog increased the number of cross-species mapped sites in all our example data sets by more than double when compared to those recovered using existing resources such as PhosphoSitePlus.
PhosphOrtholog is the first tool that enables mapping of thousands of novel and known protein phosphorylation sites across species, accessible through an easy-to-use web interface. Identification of conserved PTMs across species from large-scale experimental data increases our knowledgebase of functional PTM sites. Moreover, PhosphOrtholog is generic being applicable to other PTM datasets such as acetylation, ubiquitination and methylation.
The treatment of melanoma by targeted inhibition of the mutated kinase BRAF with small molecules only temporarily suppresses metastatic disease. In the face of chemical inhibition tumor plasticity, both innate and adaptive, promotes survival through the biochemical and genetic reconfiguration of cellular pathways that can engage proliferative and migratory systems. To investigate this process, high-resolution mass spectrometry was used to characterize the phosphoproteome of this transition in vitro. A simple and accurate, label-free quantitative method was used to localize and quantitate thousands of phosphorylation events. We also correlated changes in the phosphoproteome with the proteome to more accurately determine changes in the activity of regulatory kinases determined by kinase landscape profiling. The abundance of phosphopeptides with sites that function in cytoskeletal regulation, GTP/GDP exchange, protein kinase C, IGF signaling, and melanosome maturation were highly divergent after transition to a drug resistant phenotype.
Inhibitors of oncogenic B-RAF(V600E) and MKK1/2 have yielded remarkable responses in B-RAF(V600E)-positive melanoma patients. However, the efficacy of these inhibitors is limited by the inevitable onset of resistance. Despite the fact that these inhibitors target the same pathway, combination treatment with B-RAF(V600E) and MKK1/2 inhibitors has been shown to improve both response rates and progression-free survival in B-RAF(V600E) melanoma patients. To provide insight into the molecular nature of the combinatorial response, we used quantitative mass spectrometry to characterize the inhibitor-dependent phosphoproteome of human melanoma cells treated with the B-RAF(V600E) inhibitor PLX4032 (vemurafenib) or the MKK1/2 inhibitor AZD6244 (selumetinib). In three replicate experiments, we quantified changes at a total of 23,986 phosphosites on 4,784 proteins. This included 1,317 phosphosites that reproducibly decreased in response to at least one inhibitor. Phosphosites that responded to both inhibitors grouped into networks that included the nuclear pore complex, growth factor signaling, and transcriptional regulators. While the majority of phosphosites were responsive to both inhibitors, we identified 16 sites that decreased only in response to PLX4032, suggesting rare instances where oncogenic B-RAF signaling occurs in an MKK1/2-independent manner. Only two phosphosites were identified that appeared to be uniquely responsive to AZD6244. When cells were treated with the combination of AZD6244 and PLX4032 at subsaturating concentrations (30 nM), responses at nearly all phosphosites were additive. We conclude that AZD6244 does not substantially widen the range of phosphosites inhibited by PLX4032 and that the benefit of the drug combination is best explained by their additive effects on suppressing ERK1/2 signaling. Comparison of our results to another recent ERK1/2 phosphoproteomics study revealed a surprising degree of variability in the sensitivity of phosphosites to MKK1/2 inhibitors in human cell lines, revealing unexpected cell specificity in the molecular responses to pathway activation.
Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
Kinase mediated phosphorylation signaling is extensively involved in cellular functions and human diseases, and unraveling phosphorylation networks requires the identification of substrates targeted by kinases, which has remained challenging. We report here a novel proteomic strategy to identify the specificity and direct substrates of kinases by coupling phosphoproteomics with a sensitive stable isotope labeled kinase reaction. A whole cell extract was moderately dephosphorylated and subjected to in vitro kinase reaction under the condition in which 18O-ATP is the phosphate donor. The phosphorylated proteins are then isolated and identified by mass spectrometry, in which the heavy phosphate (+85.979 Da) labeled phosphopeptides reveal the kinase specificity. The in vitro phosphorylated proteins with heavy phosphates are further overlapped with in vivo kinase-dependent phosphoproteins for the identification of direct substrates with high confidence. The strategy allowed us to identify 46 phosphorylation sites on 38 direct substrates of extracellular signal-regulated kinase 1, including multiple known substrates and novel substrates, highlighting the ability of this high throughput method for direct kinase substrate screening.
A major constituent of the nuclear basket region of the NPC, nucleoporin Tpr plays a multi-dimensional role in regulating important processes. We have previously established that Tpr is phosphorylated in both, MAP kinase dependent and independent manner, and found that Tpr acts as both a substrate and as a scaffold for ERK2. Here, we report the identification of S2059 and S2094 as the major novel ERK-independent phosphorylation sites and T1677, S2020, S2023 and S2034 as the minor ERK independent phosphorylation sites found in the Tpr protein in vivo. Our results suggest that Protein Kinase A phosphorylates the S2094 residue, and the site is hyperphosphorylated during mitosis. Further, we find that Tpr is phosphorylated at the S2059 residue by CDK1 and the phosphorylated form distinctly localizes with chromatin during the telophase. Abrogation of S2059 phosphorylation abolishes its interaction with Mad1, thus compromising the localization of both Mad1 and Mad2 proteins, and results in cell cycle defects. The identification of novel phosphorylation sites on Tpr and the observations presented in this study open fresh avenues for the better understanding of Tpr functions.
The ERK1/2 MAP kinase pathway is an evolutionarily conserved signaling module that controls many fundamental physiological processes. Deregulated activity of ERK1/2 MAP kinases is associated with developmental syndromes and several human diseases. Despite the importance of this pathway, a comprehensive picture of the natural substrate repertoire and biochemical mechanisms regulated by ERK1/2 is still lacking. In this study, we used large-scale quantitative phosphoproteomics and bioinformatics analyses to identify novel candidate ERK1/2 substrates based on their phosphorylation signature and kinetic profiles in epithelial cells. We identified a total of 7936 phosphorylation sites within 1861 proteins, of which 155 classify as candidate ERK1/2 substrates, including 128 new targets. Candidate ERK1/2 substrates are involved in diverse cellular processes including transcriptional regulation, chromatin remodeling, RNA splicing, cytoskeleton dynamics, cellular junctions and cell signaling. Detailed characterization of one newly identified substrate, the transcriptional regulator JunB, revealed that ERK1/2 phosphorylate JunB on a serine adjacent to the DNA-binding domain, resulting in increased DNA-binding affinity and transcriptional activity. Our study expands the spectrum of cellular functions controlled by ERK1/2 kinases.
Cancer often arises when normal cellular growth goes awry due to defects in critical signal transduction pathways. A growing number of inhibitors that target specific components of these pathways are in clinical use, but the success of these agents has been limited by the resistance to inhibitor therapy that ultimately develops. Studies have now shown that cancer cells respond to chronic drug treatment by adapting their signaling circuitry, taking advantage of pathway redundancy and routes of feedback and cross-talk to maintain their function. This review focuses on the compensatory signaling mechanisms highlighted by the use of targeted inhibitors in cancer therapy.
Proteomic studies of post-translational modifications by metal affinity or antibody-based methods often employ data-dependent analysis, providing rich data sets that consist of randomly sampled identified peptides because of the dynamic response of the mass spectrometer. This can complicate the primary goal of programs for drug development, mutational analysis, and kinase profiling studies, which is to monitor how multiple nodes of known, critical signaling pathways are affected by a variety of treatment conditions. Cell Signaling Technology has developed an immunoaffinity-based LC-MS/MS method called PTMScan Direct for multiplexed analysis of these important signaling proteins. PTMScan Direct enables the identification and quantification of hundreds of peptides derived from specific proteins in signaling pathways or specific protein types. Cell lines, tissues, or xenografts can be used as starting material. PTMScan Direct is compatible with both SILAC and label-free quantification. Current PTMScan Direct reagents target key nodes of many signaling pathways (PTMScan Direct: Multipathway), serine/threonine kinases, tyrosine kinases, and the Akt/PI3K pathway. Validation of each reagent includes score filtering of MS/MS assignments, filtering by identification of peptides derived from expected targets, identification of peptides homologous to expected targets, minimum signal intensity of peptide ions, and dependence upon the presence of the reagent itself compared with a negative control. The Multipathway reagent was used to study sensitivity of human cancer cell lines to receptor tyrosine kinase inhibitors and showed consistent results with previously published studies. The Ser/Thr kinase reagent was used to compare relative levels of kinase-derived phosphopeptides in mouse liver, brain, and embryo, showing tissue-specific activity of many kinases including Akt and PKC family members. PTMScan Direct will be a powerful quantitative method for elucidation of changes in signaling in a wide array of experimental systems, combining the specificity of traditional biochemical methods with the high number of data points and dynamic range of proteomic methods.
PhosphoSitePlus (http://www.phosphosite.org) is an open, comprehensive, manually curated and interactive resource for studying experimentally observed post-translational modifications, primarily of human and mouse proteins. It encompasses 1,30,000 non-redundant modification sites, primarily phosphorylation, ubiquitinylation and acetylation. The interface is designed for clarity and ease of navigation. From the home page, users can launch simple or complex searches and browse high-throughput data sets by disease, tissue or cell line. Searches can be restricted by specific treatments, protein types, domains, cellular components, disease, cell types, cell lines, tissue and sequences or motifs. A few clicks of the mouse will take users to substrate pages or protein pages with sites, sequences, domain diagrams and molecular visualization of side-chains known to be modified; to site pages with information about how the modified site relates to the functions of specific proteins and cellular processes and to curated information pages summarizing the details from one record. PyMOL and Chimera scripts that colorize reactive groups on residues that are modified can be downloaded. Features designed to facilitate proteomic analyses include downloads of modification sites, kinase-substrate data sets, sequence logo generators, a Cytoscape plugin and BioPAX download to enable pathway visualization of the kinase-substrate interactions in PhosphoSitePlus®.
The mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase 2 (ERK2) is ubiquitously expressed in mammalian tissues and is involved in a wide range of biological processes. Although MAPKs have been intensely studied, identification of their substrates remains challenging. We have optimized a chemical genetic system using analog-sensitive ERK2, a form of ERK2 engineered to use an analog of adenosine 5'-triphosphate (ATP), to tag and isolate ERK2 substrates in vitro. This approach identified 80 proteins phosphorylated by ERK2, 13 of which are known ERK2 substrates. The 80 substrates are associated with diverse cellular processes, including regulation of transcription and translation, mRNA processing, and regulation of the activity of the Rho family guanosine triphosphatases. We found that one of the newly identified substrates, ETV3 (a member of the E twenty-six family of transcriptional regulators), was extensively phosphorylated on sites within canonical and noncanonical ERK motifs. Phosphorylation of ETV3 regulated transcription by preventing its binding to DNA at promoters for several thousand genes, including some involved in negative feedback regulation of itself and of upstream signals.
Ten years after the publication of the position paper "The hallmarks of cancer" (Hanahan and Weinberg Cell 100:57-70, 2000), it has become increasingly clear that mutated cells on their way to giving rise to a tumor have also to learn how to thrive in a chronically inflamed microenvironment, evade immune recognition, and suppress immune reactivity. Genetic and molecular definition of these three immune hallmarks of cancer offers the opportunity to learn how to deploy specific countermeasures to reverse the situation in favor of the immune system and, eventually, the patient. This new information could be channeled to address what seem to be the three major hallmarks for the immune control of cancer progression: effective procedures to activate immune reactivity; characterization of not-disposable oncoantigens; and counteraction of immune suppression.
High accuracy mass spectrometry has proven to be a powerful technology for the large scale identification of serine/threonine/tyrosine phosphorylation in the living cell. However, despite many described phosphoproteomes, there has been no comparative study of the extent of phosphorylation and its evolutionary conservation in all domains of life. Here we analyze the results of phosphoproteomics studies performed with the same technology in a diverse set of organisms. For the most ancient organisms, the prokaryotes, only a few hundred proteins have been found to be phosphorylated. Applying the same technology to eukaryotic species resulted in the detection of thousands of phosphorylation events. Evolutionary analysis shows that prokaryotic phosphoproteins are preferentially conserved in all living organisms, whereas-site specific phosphorylation is not. Eukaryotic phosphosites are generally more conserved than their non-phosphorylated counterparts (with similar structural constraints) throughout the eukaryotic domain. Yeast and Caenorhabditis elegans are two exceptions, indicating that the majority of phosphorylation events evolved after the divergence of higher eukaryotes from yeast and reflecting the unusually large number of nematode-specific kinases. Mitochondria present an interesting intermediate link between the prokaryotic and eukaryotic domains. Applying the same technology to this organelle yielded 174 phosphorylation sites mapped to 74 proteins. Thus, the mitochondrial phosphoproteome is similarly sparse as the prokaryotic phosphoproteomes. As expected from the endosymbiotic theory, phosphorylated as well as non-phosphorylated mitochondrial proteins are significantly conserved in prokaryotes. However, mitochondrial phosphorylation sites are not conserved throughout prokaryotes, consistent with the notion that serine/threonine phosphorylation in prokaryotes occurred relatively recently in evolution. Thus, the phosphoproteome reflects major events in the evolution of life.
Cancer is now appreciated as not only a highly heterogenous pathology with respect to cell type and tissue origin but also as a disease involving dysregulation of multiple pathways governing fundamental cell processes such as death, proliferation, differentiation and migration. Thus, the activities of molecular networks that execute metabolic or cytoskeletal processes, or regulate these by signal transduction, are altered in a complex manner by diverse genetic mutations in concert with the environmental context. A major challenge therefore is how to develop actionable understanding of this multivariate dysregulation, with respect both to how it arises from diverse genetic mutations and to how it may be ameliorated by prospective treatments. While high-throughput experimental platform technologies ranging from genomic sequencing to transcriptomic, proteomic and metabolomic profiling are now commonly used for molecular-level characterization of tumor cells and surrounding tissues, the resulting data sets defy straightforward intuitive interpretation with respect to potential therapeutic targets or the effects of perturbation. In this review article, we will discuss how significant advances can be obtained by applying computational modeling approaches to elucidate the pathways most critically involved in tumor formation and progression, impact of particular mutations on pathway operation, consequences of altered cell behavior in tissue environments and effects of molecular therapeutics.
Many extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase substrates have been identified, but the diversity of ERK-mediated processes suggests the existence of additional targets. Using a phosphoproteomic approach combining the steroid receptor fusion system, IMAC, 2D-DIGE and phosphomotif-specific antibodies, we detected 38 proteins showing reproducible phosphorylation changes between ERK-activated and ERK-inhibited samples, including 24 new candidate ERK targets. ERK directly phosphorylated at least 13 proteins in vitro. Of these, Nup50 was verified as a bona fide ERK substrate. Notably, ERK phosphorylation of the FG repeat region of Nup50 reduced its affinity for importin-beta family proteins, importin-beta and transportin. Other FG nucleoporins showed a similar functional change after ERK-mediated phosphorylation. Nuclear migration of importin-beta and transportin was impaired in ERK-activated, digitonin-permeabilized cells, as a result of ERK phosphorylation of Nup50. Thus, we propose that ERK phosphorylates various nucleoporins to regulate nucleocytoplasmic transport.
Aberrant signaling causes many diseases, and manipulating signaling pathways with kinase inhibitors has emerged as a promising area of drug research. Most kinase inhibitors target the conserved ATP-binding pocket; therefore specificity is a major concern. Proteomics has previously been used to identify the direct targets of kinase inhibitors upon affinity purification from cellular extracts. Here we introduce a complementary approach to evaluate the effects of kinase inhibitors on the entire cell signaling network. We used triple labeling SILAC (stable isotope labeling by amino acids in cell culture) to compare cellular phosphorylation levels for control, epidermal growth factor stimulus, and growth factor combined with kinase inhibitors. Of thousands of phosphopeptides, less than 10% had a response pattern indicative of targets of U0126 and SB202190, two widely used MAPK inhibitors. Interestingly, 83% of the growth factor-induced phosphorylation events were affected by either or both inhibitors, showing quantitatively that early signaling processes are predominantly transmitted through the MAPK cascades. In contrast to MAPK inhibitors, dasatinib, a clinical drug directed against BCR-ABL, which is the cause of chronic myelogenous leukemia, affected nearly 1,000 phosphopeptides. In addition to the proximal effects on ABL and its immediate targets, dasatinib broadly affected the downstream MAPK pathways. Pathway mapping of regulated sites implicated a variety of cellular functions, such as chromosome remodeling, RNA splicing, and cytoskeletal organization, some of which have been described in the literature before. Our assay is streamlined and generic and could become a useful tool in kinase drug development.
Identifying direct substrates of mitogen-activated protein kinases (MAPKs) and understanding how those substrates are selected
is central to understanding how these ubiquitously activated enzymes generate diverse biological responses. In previous work,
we identified several new candidate substrates for the MAPK ERK2 (extracellular signal-regulated kinase 2), including the nuclear pore complex protein Tpr (translocated promoter region). In this report, we identify sites on Tpr for ERK2 phosphorylation and binding and demonstrate their functional interaction.
ERK2 phosphorylation and dimerization are necessary for ERK2-Tpr binding, and this occurs through a DEF (docking site for ERK2, FXF) domain on Tpr. Surprisingly, the DEF domain and the phosphorylation sites displayed positive cooperativity to promote
ERK2 binding to Tpr, in contrast to substrates where phosphorylation reduces binding. Ectopic expression or depletion of Tpr
resulted in decreased movement of activated ERK2 from the cytoplasm to the nucleus, implying a role for Tpr in ERK2 translocation.
Collectively, the data provide direct evidence that a component of the nuclear pore complex is a bona fide substrate of ERK2
in vivo and that activated ERK2 stably associates with this substrate after phosphorylation, where it could play a continuing
role in nuclear pore function. We propose that Tpr is both a substrate and a scaffold for activated ERKs.
Mass spectrometry-based proteomics can reveal protein-protein interactions on a large scale, but it has been difficult to separate background binding from functionally important interactions and still preserve weak binders. To investigate the epidermal growth factor receptor (EGFR) pathway, we employ stable isotopic amino acids in cell culture (SILAC) to differentially label proteins in EGF-stimulated versus unstimulated cells. Combined cell lysates were affinity-purified over the SH2 domain of the adapter protein Grb2 (GST-SH2 fusion protein) that specifically binds phosphorylated EGFR and Src homologous and collagen (Shc) protein. We identified 228 proteins, of which 28 were selectively enriched upon stimulation. EGFR and Shc, which interact directly with the bait, had large differential ratios. Many signaling molecules specifically formed complexes with the activated EGFR-Shc, as did plectin, epiplakin, cytokeratin networks, histone H3, the glycosylphosphatidylinositol (GPI)-anchored molecule CD59, and two novel proteins. SILAC combined with modification-based affinity purification is a useful approach to detect specific and functional protein-protein interactions.
Post-translational phosphorylation is one of the most common protein modifications. Phosphoserine, threonine and tyrosine residues play critical roles in the regulation of many cellular processes. The fast growing number of research reports on protein phosphorylation points to a general need for an accurate database dedicated to phosphorylation to provide easily retrievable information on phosphoproteins.
Phospho.ELM http://phospho.elm.eu.org is a new resource containing experimentally verified phosphorylation sites manually curated from the literature and is developed as part of the ELM (Eukaryotic Linear Motif) resource. Phospho.ELM constitutes the largest searchable collection of phosphorylation sites available to the research community. The Phospho.ELM entries store information about substrate proteins with the exact positions of residues known to be phosphorylated by cellular kinases. Additional annotation includes literature references, subcellular compartment, tissue distribution, and information about the signaling pathways involved as well as links to the molecular interaction database MINT. Phospho.ELM version 2.0 contains 1703 phosphorylation site instances for 556 phosphorylated proteins.
Phospho.ELM will be a valuable tool both for molecular biologists working on protein phosphorylation sites and for bioinformaticians developing computational predictions on the specificity of phosphorylation reactions.
Overexpression and enhanced activation of the epidermal growth factor (EGF) receptor are frequent events in human cancers that correlate with poor prognosis. Anti-phosphotyrosine and anti-EGFr affinity chromatography, isotope-coded muLC-MS/MS, and immunoblot methods were combined to describe and measure signaling networks associated with EGF receptor activation and pharmacological inhibition. The squamous carcinoma cell line HN5, which overexpresses EGF receptor and displays sustained receptor kinase activation, was used as a model system, where pharmacological inhibition of EGF receptor kinase by erlotinib markedly reduced auto and substrate phosphorylation, Src family phosphorylation at EGFR Y845, while increasing total EGF receptor protein. Diverse sets of known and poorly described functional protein classes were unequivocally identified by affinity selection, comprising either proteins tyrosine phosphorylated or complexed therewith, predominantly through EGF receptor and Src family kinases, principally 1) immediate EGF receptor signaling complexes (18%); 2) complexes involved in adhesion and cell-cell contacts (34%); and 3) receptor internalization and degradation signals. Novel and known phosphorylation sites could be located despite the complexity of the peptide mixtures. In addition to interactions with multiple signaling adaptors Grb2, SHC, SCK, and NSP2, EGF receptors in HN5 cells were shown to form direct or indirect physical interactions with additional kinases including ACK1, focal adhesion kinase (FAK), Pyk2, Yes, EphA2, and EphB4. Pharmacological inhibition of EGF receptor kinase activity by erlotinib resulted in reduced phosphorylation of downstream signaling, for example through Cbl/Cbl-B, phospholipase Cgamma (PLCgamma), Erk1/2, PI-3 kinase, and STAT3/5. Focal adhesion proteins, FAK, Pyk2, paxillin, ARF/GIT1, and plakophillin were down-regulated by transient EGF stimulation suggesting a complex balance between growth factor induced kinase and phosphatase activities in the control of cell adhesion complexes. The functional interactions between IGF-1 receptor, lysophosphatidic acid (LPA) signaling, and EGF receptor were observed, both direct and/or indirectly on phospho-Akt, phospho-Erk1/2, and phospho-ribosomal S6.
Ligand binding to cell surface receptors initiates a cascade of signaling events regulated by dynamic phosphorylation events on a multitude of pathway proteins. Quantitative features, including intensity, timing, and duration of phosphorylation of particular residues, may play a role in determining cellular response, but experimental data required for analysis of these features have not previously been available. To understand the dynamic operation of signaling cascades, we have developed a method enabling the simultaneous quantification of tyrosine phosphorylation of specific residues on dozens of key proteins in a time-resolved manner, downstream of epidermal growth factor receptor (EGFR) activation. Tryptic peptides from four different EGFR stimulation time points were labeled with four isoforms of the iTRAQ reagent to enable downstream quantification. After mixing of the labeled samples, tyrosine-phosphorylated peptides were immunoprecipitated with an anti-phosphotyrosine antibody and further enriched by IMAC before LC/MS/MS analysis. Database searching and manual confirmation of peptide phosphorylation site assignments led to the identification of 78 tyrosine phosphorylation sites on 58 proteins from a single analysis. Replicate analyses of a separate biological sample provided both validation of this first data set and identification of 26 additional tyrosine phosphorylation sites and 18 additional proteins. iTRAQ fragment ion ratios provided time course phosphorylation profiles for each site. The data set of quantitative temporal phosphorylation profiles was further characterized by self-organizing maps, which resulted in identification of several cohorts of tyrosine residues exhibiting self-similar temporal phosphorylation profiles, operationally defining dynamic modules in the EGFR signaling network consistent with particular cellular processes. The presence of novel proteins and associated tyrosine phosphorylation sites within these modules indicates additional components of this network and potentially localizes the topological action of these proteins. Additional analysis and modeling of the data generated in this study are likely to yield more sophisticated models of receptor tyrosine kinase-initiated signal transduction, trafficking, and regulation.
MAPK (mitogen-activated protein kinase) signalling pathways contribute to the regulation of diverse responses, including normal and pathological aspects of cell growth, division, differentiation and death. Their ubiquity and versatility raise the issue of how they achieve specific coupling of signal with cellular response. How do the kinases in the cascade distinguish their correct substrates from the vast excess of incorrect substrates? Furthermore, how do different signals elicit distinct responses when they are transmitted by the same components? This short review highlights several mechanisms that can promote specificity in MAPK signalling, including tethering interactions between MAPKs and their substrates and regulators mediated by docking sites, feedback loops and cross-pathway regulatory circuits, and the selective activation of scaffold proteins.
The RAS-RAF-MEK-ERK (MAPK) pathway is prevalently perturbed in cancer. Recent large-scale sequencing initiatives profiled thousands of tumors providing insight into alterations at the DNA and RNA levels. These efforts confirmed that key nodes of the MAPK pathway, in particular KRAS and BRAF, are among the most frequently altered proteins in cancer. The establishment of targeted therapies, however, has proven difficult. To decipher the underlying challenges, it is essential to decrypt the phosphorylation network spanned by the MAPK core axis. Using mass spectrometry we identified 2241 phosphorylation sites on 1020 proteins, and measured their responses to inhibition of MEK or ERK. Multiple phosphorylation patterns revealed previously undetected feedback, as upstream signaling nodes, including receptor kinases, showed changes at the phosphorylation level. We provide a dataset rich in potential therapeutic targets downstream of the MAPK cascade. By integrating TCGA (The Cancer Genome Atlas) data we highlight some downstream phosphoproteins that are frequently altered in cancer. This article is protected by copyright. All rights reserved.
Overexpression of stathmin (STMN1) is closely linked to tumor metastases and poor prognosis in endometrial carcinoma (EC). However, the underlying mechanism is little known. In the present study, we investigated the expression of STMN1 in EC. Subsequently, we assessed the role of STMN1 in EC cell proliferation and migration. Our data show that STMN1 is upregulated in EC, and elevated expression of STMN1 is correlated positively with tumor stage and lymph node metastasis. In vitro, forced expression of STMN1 promoted cell invasion and migration. In contrast, knockdown of STMN1 inhibited cell aggressive behaviors. Moreover, the expression and the activity changes of matrix metalloproteinases (MMP)-2/9 were observed in EC cells after the cells being silenced or overexpression of STMN1. In conclusion, STMN1 is an oncogene and it enhances the growth and invasion of EC possibly by mediating the secretion and activation of MMP2 and MMP9 protein.
Cell signaling pathways control cells' responses to their environment through an intricate network of proteins and small molecules partitioned by intracellular structures, such as the cytoskeleton and nucleus. Our understanding of these pathways has been revised recently with the advent of more advanced experimental techniques; no longer are signaling pathways viewed as linear cascades of information flowing from membrane-bound receptors to the nucleus. Instead, such pathways must be understood in the context of networks, and studying such networks requires an integration of computational and experimental approaches. This understanding is becoming more important in designing novel therapies for diseases such as cancer. Using the MAPK (mitogen-activated protein kinase) and PI3K (class I phosphoinositide-3' kinase) pathways as case studies of cellular signaling, we give an overview of these pathways and their functions. We then describe, using a number of case studies, how computational modeling has aided in understanding these pathways' deregulation in cancer, and how such understanding can be used to optimally tailor current therapies or help design new therapies against cancer. Expected final online publication date for the Annual Review of Biomedical Engineering Volume 17 is July 11, 2015. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
Activating mutations in the MAPK pathway are prevalent drivers of several cancers. The chief consequence of these mutations is a hyperactive ERK1/2 MAP kinase able to promote cell proliferation, producing a critical hallmark of metastatic disease. The biochemistry of the ERK pathway is well characterized, however how the pathway achieves different outcomes in the face of genetic aberrations of cancer and subsequent treatment with chemical inhibitors is not clear. To investigate this we used mass spectrometry to complete a global phosphoproteomic analysis of a BRAFV600E thyroid cancer cell line (SW1736) after treatment with the mutation selective inhibitor vemurafenib (PLX4032) and MEK1/2 inhibitor selumetinib (AZD6244). We identified thousands of phosphorylation events orchestrated in BRAFV600E cells and performed kinase landscape analysis to identify putative kinases regulated in response to MAPK blockade. The abundance of phosphopeptides containing consensus motifs for acidophilic kinases increased after short-term inhibition with these compounds. We showed that co-inhibition of the pleiotropic acidophilic kinase CK2 and BRAFV600E synergistically reduced proliferation in patient-derived melanomas and thyroid cancer cells harboring the BRAF lesion. We investigated this mechanism and show a role for CK2 in controlling AKT activation that was not reliant on changes to PTEN or PDK1 phosphorylation. These findings highlight a role for CK2 blockade in potentiating the anti-proliferative effects of BRAF and MEK inhibition in BRAF cancers.
THE normal cellular homologue of the acutely transforming oncogene v-ra/is c-raf-l, which encodes a serine/threonine protein kinase that is activated by many extracellular stimuli1. The physiological substrates of the protein c-Raf-1 are unknown. The mitogen-activated protein (MAP) kinases ErkI and 2 are also activated by mitogens through phosphorylation of Erk tyrosine and threonine residues catalysed by a protein kinase of relative molecular mass 50,000, MAP kinase-kinase (MAPK-K)2-7. Here we report that MAPK-K as well as Erkl and 2 are constitutively active in v-raf-transformed cells. MAPK-K partially purified from v-raf-transformed cells or from mitogen-treated cells3 can be deactivated by phosphatase 2A. c-Raf-1 purified after mitogen stimulation can reactivate the phosphatase 2A-inactivated MAPK-K over 30-fold in vitro. c-Raf-1 reactivation of MAPK-K coincides with the selective phosphorylation at serine/threonine residues of a polypeptide with Mr 50,000 which coelutes precisely on cation-exchange chromatography with the MAPK-K activatable by c-Raf-1. These results indicate that c-Raf-1 is an immediate upstream activator of MAPK-K in vivo. To our knowledge, MAPK-K is the first physiological substrate of the c-raf-l protooncogene product to be identified.
The Web-based motif-x program provides a simple interface to extract statistically significant motifs from large data sets, such as MS/MS post-translational modification data and groups of proteins that share a common biological function. Users upload data files and download results using common Web browsers on essentially any Web-compatible computer. Once submitted, data analyses are performed rapidly on an associated high-speed computer cluster and they produce both syntactic and image-based motif results and statistics. The protocols presented demonstrate the use of motif-x in three common user scenarios.
Novel and improved computational tools are required to transform large-scale proteomics data into valuable information of biological relevance. To this end, we developed ProteoConnections, a bioinformatics platform tailored to address the pressing needs of proteomics analyses. The primary focus of this platform is to organize peptide and protein identifications, evaluate the quality of the acquired data set, profile abundance changes, and accelerate data interpretation. Peptide and protein identifications are stored into a relational database to facilitate data mining and to evaluate the quality of data sets using graphical reports. We integrated databases of known PTMs and other bioinformatics tools to facilitate the analysis of phosphoproteomics data sets and to provide insights for subsequent biological validation experiments. Phosphorylation sites are also annotated according to kinase consensus motifs, contextual environment, protein domains, binding motifs, and evolutionary conservation across different species. The practical application of ProteoConnections is further demonstrated for the analysis of the phosphoproteomics data sets from rat intestinal IEC-6 cells where we identified 9615 phosphorylation sites on 2108 phosphoproteins. Combined proteomics and bioinformatics analyses revealed valuable biological insights on the regulation of phosphoprotein functions via the introduction of new binding sites on scaffold proteins or the modulation of protein-protein, protein-DNA, or protein-RNA interactions. Quantitative proteomics data can be integrated into ProteoConnections to determine the changes in protein phosphorylation under different cell stimulation conditions or kinase inhibitors, as demonstrated here for the MEK inhibitor PD184352.
Post-transcriptional regulation of mRNA includes restriction mechanisms to prevent export and expression of mRNAs that are incompletely spliced. Here we present evidence that the mammalian protein Tpr is involved in this restriction. To study the role of Tpr in export of mRNA with retained introns, we used reporters in which the mRNA was exported either via the Nxf1/Nxt1 pathway using a CTE or via the Crm1 pathway using Rev/RRE. Our data show that even modest knockdown of Tpr using RNAi leads to a significant increase in export and translation from the mRNA containing the CTE. In contrast, Tpr perturbation has no effect on export of mRNA containing the RRE, either in the absence or presence of Rev. Also, no effects were observed on export of a completely spliced mRNA. Taken together, our results indicate that Tpr plays an important role in quality control of mRNA trafficked on the Nxf1 pathway.