Spatiotemporal Control of Pathway Sensors and Cross-Pathway Feedback Regulate a Cell Differentiation MAPK Pathway in Yeast

ABSTRACTMitogen-Activated Protein Kinase (MAPK) pathways control cell differentiation and the response to stress. MAPK pathways can share components with other pathways yet induce specific responses through mechanisms that remain unclear. In Saccharomyces cerevisiae, the MAPK pathway that controls filamentous growth (fMAPK) shares components with the MAPK pathway that regulates the response to osmotic stress (HOG). By exploring temporal regulation of MAPK signaling, we show here that the two pathways exhibited different patterns of activity throughout the cell cycle. The different patterns resulted from different expression profiles of genes encoding the mucin sensors (MSB2 for fMAPK and HKR1 for HOG). We also show that positive feedback through the fMAPK pathway stimulated the HOG pathway, presumably to modulate fMAPK pathway activity. By exploring spatial regulation of MAPK signaling, we found that the shared tetraspan protein, Sho1p, which has a dynamic localization pattern, induced the fMAPK pathway at the mother-bud neck. A Sho1p-interacting protein, Hof1p, which also localizes to the mother-bud neck and regulates cytokinesis, also regulated the fMAPK pathway. Therefore, spatial and temporal regulation of pathway sensors, and cross-pathway feedback, regulate a MAPK pathway that controls a cell differentiation response in yeast.

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Spatiotemporal control of pathway sensors and cross-pathway feedback regulate a differentiation MAPK pathway in yeast

Journal of Cell Science ◽

10.1242/jcs.258341 ◽

2021 ◽

Author(s):

Aditi Prabhakar ◽

Beatriz Gonzalez ◽

Heather Dionne ◽

Sukanya Basu ◽

Paul J. Cullen

Keyword(s):

Cell Differentiation ◽

Mapk Pathway ◽

Expression Profiles ◽

Control Cell ◽

Mitogen Activated Protein Kinase ◽

Hog Pathway ◽

Interacting Protein ◽

Bud Neck ◽

Genes Encoding ◽

Pathway Regulation

Mitogen-Activated Protein Kinase (MAPK) pathways control cell differentiation and the response to stress. In Saccharomyces cerevisiae, the MAPK pathway that controls filamentous growth (fMAPK) shares components with the pathway that regulates the response to osmotic stress (HOG). Here, we show that the two pathways exhibit different patterns of activity throughout the cell cycle. The different patterns resulted from different expression profiles of genes encoding mucin sensors that regulate the pathways. Cross-pathway regulation from the fMAPK pathway stimulated the HOG pathway, presumably to modulate fMAPK pathway activity. We also show that the shared tetraspan protein, Sho1p, which has a dynamic localization pattern, induced the fMAPK pathway at the mother-bud neck. A Sho1p-interacting protein, Hof1p, which also localizes to the mother-bud neck and regulates cytokinesis, also regulated the fMAPK pathway. Therefore, spatial and temporal regulation of pathway sensors, and cross-pathway regulation, control a MAPK pathway that regulates cell differentiation in yeast.

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A Rab Escort Protein Regulates the MAPK Pathway That Controls Filamentous Growth in Yeast

10.1101/2020.06.02.130690 ◽

2020 ◽

Author(s):

Sheida Jamalzadeh ◽

Paul J. Cullen

Keyword(s):

Mapk Pathway ◽

Filamentous Growth ◽

Mapk Signaling ◽

Hog Pathway ◽

Mapk Pathways ◽

A Genome ◽

Dependent Signal ◽

Nutrient Signaling ◽

Pathway Regulation ◽

Yeast Genes

ABSTRACTMAPK pathways regulate different responses yet can share a subset of common components. In this study, a genome-wide screen was performed to identify genes that, when overexpressed, induce a growth reporter (FUS1-HIS3) that responds to ERK-type MAPK pathways (Mating/filamentous growth or fMAPK) but not p38-type MAPK pathways (HOG) in yeast. Approximately 4,500 plasmids overexpressing individual yeast genes were introduced into strains containing the FUS1-HIS3 reporter by high-throughput transformation. Candidate genes were identified by measuring the degree of growth, which was a reflection of reporter activity. Of fourteen genes identified and validated by re-testing, two were metabolic controls (HIS3 and ATR1), five had established roles in regulating ERK-type pathways (STE4, STE7, BMH1, BMH2, MIG2) and seven represent potentially new regulators of MAPK signaling (RRN6, CIN5, MRS6, KAR2, TFA1, RSC3, RGT2). MRS6 encodes a Rab escort protein and effector of the TOR pathway that plays an established role in nutrient signaling. MRS6 overexpression stimulated filamentous/invasive growth and phosphorylation of the ERK-type fMAPK, Kss1. Overexpression of MRS6 reduced the osmotolerance of cells and phosphorylation of the p38/HOG pathway MAPK, Hog1. Mrs6 interacted with the PAK kinase Ste20 and MAPKK Ste7 by two-hybrid analysis. Collectively, the data indicate that Mrs6 may function to selectively propagate an ERK-dependent signal. Generally speaking, the identification of new MAPK pathway regulators by genetic screening in yeast may be a useful resource for understanding signaling pathway regulation.

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Identification of RAS-Mitogen-Activated Protein Kinase Signaling Pathway Modulators in an ERF1 Redistribution® Screen

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057106287136 ◽

2006 ◽

Vol 11 (4) ◽

pp. 423-434 ◽

Cited By ~ 12

Author(s):

Charlotta Grånäs ◽

Betina Kerstin Lundholt ◽

Frosty Loechel ◽

Hans-Christian Pedersen ◽

Sara Petersen Bjørn ◽

...

Keyword(s):

Protein Kinase ◽

Signaling Pathway ◽

Kinase Inhibitors ◽

Mapk Pathway ◽

Mapk Signaling ◽

Mitogen Activated Protein Kinase ◽

Mitogen Activated Protein ◽

A Cell ◽

Protein Kinase Signaling

The RAS-mitogen-activated protein kinase (MAPK) signaling pathway has a central role in regulating the proliferation and survival of both normal and tumor cells. This pathway has been 1 focus area for the development of anticancer drugs, resulting in several compounds, primarily kinase inhibitors, in clinical testing. The authors have undertaken a cell-based, high-throughput screen using a novel ERF1 Redistribution® assay to identify compounds that modulate the signaling pathway. The hit compounds were subsequently tested for activity in a functional cell proliferation assay designed to selectively detect compounds inhibiting the proliferation of MAPK pathway-dependent cancer cells. The authors report the identification of 2 cell membrane-permeable compounds that exhibit activity in the ERF1 Redistribution® assay and selectively inhibit proliferation of MAPK pathway-dependent malignant melanoma cells at similar potencies (IC50 =< 5 μM). These compounds have drug-like structures and are negative in RAF, MEK, and ERK in vitro kinase assays. Drugs belonging to these compound classes may prove useful for treating cancers caused by excessive MAPK pathway signaling. The results also show that cell-based, high-content Redistribution® screens can detect compounds with different modes of action and reveal novel targets in a pathway known to be disease relevant.

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The MAPK hypothesis: immune-regulatory effects of MAPK-pathway genetic dysregulations and implications for breast cancer immunotherapy

Emerging Topics in Life Sciences ◽

10.1042/etls20170142 ◽

2017 ◽

Vol 1 (5) ◽

pp. 429-445 ◽

Cited By ~ 17

Author(s):

Davide Bedognetti ◽

Jessica Roelands ◽

Julie Decock ◽

Ena Wang ◽

Wouter Hendrickx

Keyword(s):

Breast Cancer ◽

Cell Function ◽

Checkpoint Inhibitors ◽

Mapk Pathway ◽

Treatment Resistance ◽

Mapk Signaling ◽

Mitogen Activated Protein Kinase ◽

Mapk Pathways ◽

Wide Range ◽

Regulatory Effects

With the advent of checkpoint inhibition, immunotherapy has revolutionized the clinical management of several cancers, but has demonstrated limited efficacy in mammary carcinoma. Transcriptomic profiling of cancer samples defined distinct immunophenotypic categories characterized by different prognostic and predictive connotations. In breast cancer, genomic alterations leading to the dysregulation of mitogen-activated protein kinase (MAPK) pathways have been linked to an immune-silent phenotype associated with poor outcome and treatment resistance. These aberrations include mutations of MAP3K1 and MAP2K4, amplification of KRAS, BRAF, and RAF1, and truncations of NF1. Anticancer therapies targeting MAPK signaling by BRAF and MEK inhibitors have demonstrated clear immunologic effects. These off-target properties could be exploited to convert the immune-silent tumor phenotype into an immune-active one. Preclinical evidence supports that MAPK-pathway inhibition can dramatically increase the efficacy of immunotherapy. In this review, we provide a detailed overview of the immunomodulatory impact of MAPK-pathway blockade through BRAF and MEK inhibitions. While BRAF inhibition might be relevant in melanoma only, MEK inhibition is potentially applicable to a wide range of tumors. Context-dependent similarities and differences of MAPK modulation will be dissected, in light of the complexity of the MAPK pathways. Therapeutic strategies combining the favorable effects of MAPK-oriented interventions on the tumor microenvironment while maintaining T-cell function will be presented. Finally, we will discuss recent studies highlighting the rationale for the implementation of MAPK-interference approaches in combination with checkpoint inhibitors and immune agonists in breast cancer.

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Association of MAPK signaling subtypes with prognostic benefit for bevacizumab in left-sided metastatic colorectal cancer (mCRC) patients treated with FOLFIRI + cetuximab / bevacizumab (FIRE-3 trial).

Journal of Clinical Oncology ◽

10.1200/jco.2019.37.15_suppl.3584 ◽

2019 ◽

Vol 37 (15_suppl) ◽

pp. 3584-3584

Author(s):

Arndt Stahler ◽

Sebastian Stintzing ◽

Dominik Paul Modest ◽

Ivan Jelas ◽

Kathrin Heinrich ◽

...

Keyword(s):

Mrna Expression ◽

Cox Regression ◽

Mapk Pathway ◽

Expression Profiles ◽

Mapk Signaling ◽

Gene Set Enrichment Analysis ◽

Rank Test ◽

Prognostic Information ◽

Mutational Status ◽

Gene Sets

3584 Background: We investigated the role of the MAPK pathway by mRNA expression profiles in microarrays of the FIRE-3 trial as it was formerly associated with prognosis. Methods: 451 patients provided eligible mRNA material for subsequent analyses of the MAPK pathway (295 genes). Non-negative matrix factorized (NMF) clustering for normalized mRNA microarray data (Almac Inc, Xcel Array) was performed for 2 to 6 ranks against randomized controls. Linear models with adjustment for multiple testing showed differential gene expression between groups. Single sample gene set enrichment analysis (ssGSEA) was used to compare differentially enriched hallmarks of cancer gene sets. Kaplan Meier method, log rank test and Cox regression analyses were performed to estimate overall (OS) and progression free survival (PFS) between MAPK subtypes. Results: NMF clustering built two groups of MAPK mRNA expression (coph: 0.91, silh: 1.00) without cohort-based bias in principal component analysis. Group MAPK1 (n = 238) was significantly associated with CMS2 (66.4 %), group MAPK2 (n = 213) with CMS4 (67.6 %, p < 0.0001). 5.551 of 23.561 genes were significantly differentially expressed between MAPK subtypes. 49 cancer hallmark gene sets were significantly differentially enriched in ssGSEA ( MAPK1: myc targets, DNA repair, cell cycle, PI3K- AKT- mTOR pathway upregulation; MAPK2: EMT-related signatures, TGFß pathway, angiogenesis upregulation among others). In overall analysis, MAPK1 showed slightly better outcome than MAPK2 (OS: HR: 0.80, 95% CI: 0.65 – 0.99, p = 0.049; PFS: HR: 0.81, 95% CI: 0.66 – 1.00, p = 0.05). However, MAPK1 was significantly more favourable for bevacizumab treatment in OS ( MAPK1: 30.8 m, MAPK2: 19.4 m, HR: 0.56, 95% CI: 0.39 – 0.81, p = 0.002) and PFS ( MAPK1: 11.7 m, MAPK2: 9.8 m, HR: 0.68, 95% CI: 0.48 – 0.98, p = 0.038) in left sided tumors, while no difference was seen for cetuximab treatment, RAS and BRAF status. Conclusions: mCRC subtypes by MAPK mRNA expression might contain prognostic information for the treatment with bevacizumab beyond mutational status in patients with left sided tumors of the FIRE-3 trial.

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Broad and thematic remodeling of the surface glycoproteome on isogenic cells transformed with driving proliferative oncogenes

10.1101/808139 ◽

2019 ◽

Author(s):

Kevin K. Leung ◽

Gary M. Wilson ◽

Lisa L. Kirkemo ◽

Nicholas M. Riley ◽

Joshua J. Coon ◽

...

Keyword(s):

Cell Surface ◽

Electron Transfer Dissociation ◽

Biomarker Discovery ◽

Mapk Pathway ◽

Strong Dependence ◽

Mapk Signaling ◽

Surface Proteins ◽

Breast Epithelial Cell ◽

Epithelial Cell Line ◽

A Cell

AbstractThe cell surface proteome, the surfaceome, is the interface for engaging the extracellular space in normal and cancer cells. Here we apply quantitative proteomics of N-linked glycoproteins to reveal how a collection of some 700 surface proteins is dramatically remodeled in an isogenic breast epithelial cell line stably expressing any of six of the most prominent proliferative oncogenes, including the receptor tyrosine kinases, EGFR and HER2, and downstream signaling partners such as KRAS, BRAF, MEK and AKT. We find that each oncogene has somewhat different surfaceomes but the functions of these proteins are harmonized by common biological themes including up-regulation of nutrient transporters, down-regulation of adhesion molecules and tumor suppressing phosphatases, and alteration in immune modulators. Addition of a potent MEK inhibitor that blocks MAPK signaling brings each oncogene-induced surfaceome back to a common state reflecting their strong dependence on the MAPK pathway to propagate signaling. Using a recently developed glyco-proteomics method of activated ion electron transfer dissociation (AI-ETD) we found massive oncogene-induced changes in 142 N-linked glycans and differential increases in complex hybrid glycans especially for KRAS and HER2 oncogenes. Overall, these studies provide a broad systems level view of how specific driver oncogenes remodel the surface glycoproteome in a cell autologous fashion, and suggest possible surface targets, and combinations thereof, for drug and biomarker discovery.Significant statementThe cell surface glycoproteome (surfaceome) mediates interactions between the cell and the extracellular environment, and is a major target for immunotherapy in cancer. Using state-of-the-art proteomics methods, we compared how six neighboring proliferative oncogenes cause large and bidirectional expression of some 700 surface proteins and the 142 different glycans that decorate them. While each oncogene induces large and somewhat unique glycoproteomes relative to non-transformed cells, we find common functional consequences that are massively reversed by small molecule inhibition of the MAPK pathway. This large-scale comparative study provides important insights for how oncogenes remodel isogenic cells in a cell autologous fashion, and suggest possible new opportunities for antibody drug discovery in more complex tumor settings.

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A Rab escort protein regulates the MAPK pathway that controls filamentous growth in yeast

Scientific Reports ◽

10.1038/s41598-020-78470-4 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Sheida Jamalzadeh ◽

Atindra N. Pujari ◽

Paul J. Cullen

Keyword(s):

Mapk Pathway ◽

Filamentous Growth ◽

Mapk Signaling ◽

Cellular Processes ◽

Mapk Pathways ◽

Genome Wide ◽

A Genome ◽

Dependent Signal ◽

Nutrient Signaling ◽

Yeast Genes

AbstractMAPK pathways regulate different responses yet can share common components. Although core regulators of MAPK pathways are well known, new pathway regulators continue to be identified. Overexpression screens can uncover new roles for genes in biological processes and are well suited to identify essential genes that cannot be evaluated by gene deletion analysis. In this study, a genome-wide screen was performed to identify genes that, when overexpressed, induce a reporter (FUS1-HIS3) that responds to ERK-type pathways (Mating and filamentous growth or fMAPK) but not p38-type pathways (HOG) in yeast. Approximately 4500 plasmids overexpressing individual yeast genes were introduced into strains containing the reporter by high-throughput transformation. Candidate genes were identified by measuring growth as a readout of reporter activity. Fourteen genes were identified and validated by re-testing: two were metabolic controls (HIS3, ATR1), five had established roles in regulating ERK-type pathways (STE4, STE7, BMH1, BMH2, MIG2) and seven represent potentially new regulators of MAPK signaling (RRN6, CIN5, MRS6, KAR2, TFA1, RSC3, RGT2). MRS6 encodes a Rab escort protein and effector of the TOR pathway that plays a role in nutrient signaling. MRS6 overexpression stimulated invasive growth and phosphorylation of the ERK-type fMAPK, Kss1. Overexpression of MRS6 reduced the osmotolerance of cells and phosphorylation of the p38/HOG MAPK, Hog1. Mrs6 interacted with the PAK kinase Ste20 and MAPKK Ste7 by two-hybrid analysis. Based on these results, Mrs6 may selectively propagate an ERK-dependent signal. Identifying new regulators of MAPK pathways may provide new insights into signal integration among core cellular processes and the execution of pathway-specific responses.

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Role of Phosphatidylinositol Phosphate Signaling in the Regulation of the Filamentous-Growth Mitogen-Activated Protein Kinase Pathway

Eukaryotic Cell ◽

10.1128/ec.00013-15 ◽

2015 ◽

Vol 14 (4) ◽

pp. 427-440 ◽

Cited By ~ 9

Author(s):

Hema Adhikari ◽

Paul J. Cullen

Keyword(s):

Protein Kinase ◽

Mapk Pathway ◽

Filamentous Growth ◽

Mapk Signaling ◽

Mitogen Activated Protein Kinase ◽

Hog Pathway ◽

Mapk Activity ◽

Conditional Allele ◽

Mitogen Activated Protein

ABSTRACTReversible phosphorylation of the phospholipid phosphatidylinositol (PI) is a key event in the determination of organelle identity and an underlying regulatory feature in many biological processes. Here, we investigated the role of PI signaling in the regulation of the mitogen-activated protein kinase (MAPK) pathway that controls filamentous growth in yeast. Lipid kinases that generate phosphatidylinositol 4-phosphate [PI(4)P] at the Golgi (Pik1p) or PI(4,5)P2 at the plasma membrane (PM) (Mss4p and Stt4p) were required for filamentous-growth MAPK pathway signaling. Introduction of a conditional allele ofPIK1(pik1-83) into the filamentous (Σ1278b) background reduced MAPK activity and caused defects in invasive growth and biofilm/mat formation. MAPK regulatory proteins that function at the PM, including Msb2p, Sho1p, and Cdc42p, were mislocalized in thepik1-83mutant, which may account for the signaling defects of the PI(4)P kinase mutants. Other PI kinases (Fab1p and Vps34p), and combinations of PIP (synaptojanin-type) phosphatases, also influenced the filamentous-growth MAPK pathway. Loss of these proteins caused defects in cell polarity, which may underlie the MAPK signaling defect seen in these mutants. In line with this possibility, disruption of the actin cytoskeleton by latrunculin A (LatA) dampened the filamentous-growth pathway. Various PIP signaling mutants were also defective for axial budding in haploid cells, cell wall construction, or proper regulation of the high-osmolarity glycerol response (HOG) pathway. Altogether, the study extends the roles of PI signaling to a differentiation MAPK pathway and other cellular processes.

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