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1.
Cell ; 187(9): 2117-2119, 2024 Apr 25.
Article in English | MEDLINE | ID: mdl-38670068

ABSTRACT

While some people pore over the textbook and train through the classics of the field, many scientists come to immunology when they discover it intersecting with their "first love" interests. Five of these "accidental immunologists" tell us how they found their way to a fascination with the immune system.


Subject(s)
Allergy and Immunology , Humans , History, 20th Century , History, 21st Century , Animals , Immune System
2.
Nat Immunol ; 24(8): 1318-1330, 2023 08.
Article in English | MEDLINE | ID: mdl-37308665

ABSTRACT

Immune checkpoint blockade (ICB) targeting PD-1 and CTLA-4 has revolutionized cancer treatment. However, many cancers do not respond to ICB, prompting the search for additional strategies to achieve durable responses. G-protein-coupled receptors (GPCRs) are the most intensively studied drug targets but are underexplored in immuno-oncology. Here, we cross-integrated large singe-cell RNA-sequencing datasets from CD8+ T cells covering 19 distinct cancer types and identified an enrichment of Gαs-coupled GPCRs on exhausted CD8+ T cells. These include EP2, EP4, A2AR, ß1AR and ß2AR, all of which promote T cell dysfunction. We also developed transgenic mice expressing a chemogenetic CD8-restricted Gαs-DREADD to activate CD8-restricted Gαs signaling and show that a Gαs-PKA signaling axis promotes CD8+ T cell dysfunction and immunotherapy failure. These data indicate that Gαs-GPCRs are druggable immune checkpoints that might be targeted to enhance the response to ICB immunotherapies.


Subject(s)
CD8-Positive T-Lymphocytes , Neoplasms , Mice , Animals , Signal Transduction , Mice, Transgenic , Immunotherapy , Tumor Microenvironment
3.
Cell ; 177(7): 1933-1947.e25, 2019 06 13.
Article in English | MEDLINE | ID: mdl-31160049

ABSTRACT

Heterotrimetic G proteins consist of four subfamilies (Gs, Gi/o, Gq/11, and G12/13) that mediate signaling via G-protein-coupled receptors (GPCRs), principally by receptors binding Gα C termini. G-protein-coupling profiles govern GPCR-induced cellular responses, yet receptor sequence selectivity determinants remain elusive. Here, we systematically quantified ligand-induced interactions between 148 GPCRs and all 11 unique Gα subunit C termini. For each receptor, we probed chimeric Gα subunit activation via a transforming growth factor-α (TGF-α) shedding response in HEK293 cells lacking endogenous Gq/11 and G12/13 proteins, and complemented G-protein-coupling profiles through a NanoBiT-G-protein dissociation assay. Interrogation of the dataset identified sequence-based coupling specificity features, inside and outside the transmembrane domain, which we used to develop a coupling predictor that outperforms previous methods. We used the predictor to engineer designer GPCRs selectively coupled to G12. This dataset of fine-tuned signaling mechanisms for diverse GPCRs is a valuable resource for research in GPCR signaling.


Subject(s)
Heterotrimeric GTP-Binding Proteins/metabolism , Models, Biological , Receptors, G-Protein-Coupled/metabolism , Signal Transduction , Female , HEK293 Cells , Heterotrimeric GTP-Binding Proteins/genetics , Humans , Male , PC-3 Cells , Receptors, G-Protein-Coupled/genetics
4.
Article in English | MEDLINE | ID: mdl-39270681

ABSTRACT

The advent of cancer immunotherapy based on PD-1 and CTLA-4 immune checkpoint blockade (ICB) has revolutionized cancer treatment. However, many cancers do not respond to ICB, highlighting the urgent need for additional approaches to achieve durable cancer remission. The large family of G protein-coupled receptors (GPCRs) is the target of more than 30% of all approved drugs, but GPCRs have been underexploited in cancer immunotherapy. In this review, we discuss the central role of GPCRs in immune cell migration and function and describe how single-cell transcriptomic studies are illuminating the complexity of the human tumor immune GPCRome. These receptors include multiple GPCRs expressed in CD8 T cells that are activated by inflammatory mediators, protons, neurotransmitters, and metabolites that accumulate in the tumor microenvironment, thereby promoting T cell dysfunction. We also discuss new opportunities to target GPCRs as a multimodal approach to enhance the response to ICB for a myriad of human malignancies.

6.
Proc Natl Acad Sci U S A ; 119(47): e2213835119, 2022 11 22.
Article in English | MEDLINE | ID: mdl-36395141

ABSTRACT

Somatic copy number alterations (SCNAs), generally (1) losses containing interferons and interferon-pathway genes, many on chromosome 9p, predict immune-cold, immune checkpoint therapy (ICT)-resistant tumors (2); however, genomic regions mediating these effects are unclear and probably tissue specific. Previously, 9p21.3 loss was found to be an early genetic driver of human papillomavirus-negative (HPV-) head and neck squamous cancer (HNSC), associated with an immune-cold tumor microenvironment (TME) signal, and recent evidence suggested that this TME-cold phenotype was greatly enhanced with 9p21 deletion size, notably encompassing band 9p24.1 (3). Here, we report multi-omic, -threshold and continuous-variable dissection of 9p21 and 9p24 loci (including depth and degree of somatic alteration of each band at each locus, and each gene at each band) and TME of four HPV- HNSC cohorts. Preferential 9p24 deletion, CD8 T-cell immune-cold associations were observed, driven by 9p24.1 loss, and in turn by an essential telomeric regulatory gene element, JAK2-CD274. Surprisingly, same genetic region gains were immune hot. Related 9p21-TME analyses were less evident. Inherent 9p-band-level influences on anti-PD1 ICT survival rates, coincident with TME patterns, were also observed. At a 9p24.1 whole-transcriptome expression threshold of 60th percentile, ICT survival rate exceeded that of lower expression percentiles and of chemotherapy; below this transcript threshold, ICT survival was inferior to chemotherapy, the latter unaffected by 9p24.1 expression level (P-values < 0.01, including in a PD-L1 immunohistochemistry-positive patient subgroup). Whole-exome analyses of 10 solid-tumor types suggest that these 9p-related ICT findings could be relevant to squamous cancers, in which 9p24.1 gain/immune-hot associations exist.


Subject(s)
Carcinoma, Squamous Cell , Head and Neck Neoplasms , Papillomavirus Infections , Humans , Tumor Microenvironment/genetics , Immune Checkpoint Inhibitors , Papillomavirus Infections/genetics , Squamous Cell Carcinoma of Head and Neck/drug therapy , Squamous Cell Carcinoma of Head and Neck/genetics , Head and Neck Neoplasms/drug therapy , Head and Neck Neoplasms/genetics
7.
J Biol Chem ; 299(2): 102866, 2023 02.
Article in English | MEDLINE | ID: mdl-36596361

ABSTRACT

G proteins and G protein-coupled receptors activate a diverse array of signal transduction pathways that promote cell growth and survival. Indeed, hot spot-activating mutations in GNAQ/GNA11, encoding Gαq proteins, are known to be driver oncogenes in uveal melanoma (UM), for which there are limited effective therapies currently available. Focal adhesion kinase (FAK) has been recently shown to be a central mediator of Gαq-driven signaling in UM, and as a result, is being explored clinically as a therapeutic target for UM, both alone and in combination therapies. Despite this, the repertoire of Gαq/FAK-regulated signaling mechanisms have not been fully elucidated. Here, we used a whole-genome CRISPR screen in GNAQ-mutant UM cells to identify mechanisms that, when overactivated, lead to reduced sensitivity to FAK inhibition. In this way, we found that the PI3K/AKT signaling pathway represented a major resistance driver. Our dissection of the underlying mechanisms revealed that Gαq promotes PI3K/AKT activation via a conserved signaling circuitry mediated by FAK. Further analysis demonstrated that FAK activates PI3K through the association and tyrosine phosphorylation of the p85 regulatory subunit of PI3K and that UM cells require PI3K/AKT signaling for survival. These findings establish a novel link between Gαq-driven signaling and the stimulation of PI3K as well as demonstrate aberrant activation of signaling networks underlying the growth and survival of UM and other Gαq-driven malignancies.


Subject(s)
Carcinogenesis , Focal Adhesion Protein-Tyrosine Kinases , GTP-Binding Protein alpha Subunits, Gq-G11 , Phosphatidylinositol 3-Kinases , Proto-Oncogene Proteins c-akt , Clustered Regularly Interspaced Short Palindromic Repeats , Focal Adhesion Protein-Tyrosine Kinases/genetics , Focal Adhesion Protein-Tyrosine Kinases/metabolism , GTP-Binding Protein alpha Subunits/metabolism , GTP-Binding Protein alpha Subunits, Gq-G11/genetics , GTP-Binding Protein alpha Subunits, Gq-G11/metabolism , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction , Humans , Carcinogenesis/genetics
8.
J Biol Chem ; 299(11): 105293, 2023 11.
Article in English | MEDLINE | ID: mdl-37774973

ABSTRACT

ß-arrestins play a key role in G protein-coupled receptor (GPCR) internalization, trafficking, and signaling. Whether ß-arrestins act independently of G protein-mediated signaling has not been fully elucidated. Studies using genome-editing approaches revealed that whereas G proteins are essential for mitogen-activated protein kinase activation by GPCRs., ß-arrestins play a more prominent role in signal compartmentalization. However, in the absence of G proteins, GPCRs may not activate ß-arrestins, thereby limiting the ability to distinguish G protein from ß-arrestin-mediated signaling events. We used ß2-adrenergic receptor (ß2AR) and its ß2AR-C tail mutant expressed in human embryonic kidney 293 cells wildtype or CRISPR-Cas9 gene edited for Gαs, ß-arrestin1/2, or GPCR kinases 2/3/5/6 in combination with arrestin conformational sensors to elucidate the interplay between Gαs and ß-arrestins in controlling gene expression. We found that Gαs is not required for ß2AR and ß-arrestin conformational changes, ß-arrestin recruitment, and receptor internalization, but that Gαs dictates the GPCR kinase isoforms involved in ß-arrestin recruitment. By RNA-Seq analysis, we found that protein kinase A and mitogen-activated protein kinase gene signatures were activated by stimulation of ß2AR in wildtype and ß-arrestin1/2-KO cells but absent in Gαs-KO cells. These results were validated by re-expressing Gαs in the corresponding KO cells and silencing ß-arrestins in wildtype cells. These findings were extended to cellular systems expressing endogenous levels of ß2AR. Overall, our results support that Gs is essential for ß2AR-promoted protein kinase A and mitogen-activated protein kinase gene expression signatures, whereas ß-arrestins initiate signaling events modulating Gαs-driven nuclear transcriptional activity.


Subject(s)
GTP-Binding Proteins , Gene Expression Regulation , Receptors, Adrenergic, beta-2 , beta-Arrestins , Humans , beta-Arrestin 1/genetics , beta-Arrestin 1/metabolism , beta-Arrestin 2/genetics , beta-Arrestin 2/metabolism , beta-Arrestins/genetics , beta-Arrestins/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Gene Expression Regulation/genetics , GTP-Binding Proteins/metabolism , Mitogen-Activated Protein Kinases/metabolism , Phosphorylation , Receptors, Adrenergic, beta-2/chemistry , Receptors, Adrenergic, beta-2/genetics , Receptors, Adrenergic, beta-2/metabolism , HEK293 Cells , GTP-Binding Protein alpha Subunits/genetics , GTP-Binding Protein alpha Subunits/metabolism , Protein Structure, Tertiary , Protein Isoforms , Enzyme Activation/genetics
9.
J Biol Chem ; 299(12): 105418, 2023 Dec.
Article in English | MEDLINE | ID: mdl-37923138

ABSTRACT

Most uveal melanoma cases harbor activating mutations in either GNAQ or GNA11. Despite activation of the mitogen-activated protein kinase (MAPK) signaling pathway downstream of Gαq/11, there are no effective targeted kinase therapies for metastatic uveal melanoma. The human genome encodes numerous understudied kinases, also called the "dark kinome". Identifying additional kinases regulated by Gαq/11 may uncover novel therapeutic targets for uveal melanoma. In this study, we treated GNAQ-mutant uveal melanoma cell lines with a Gαq/11 inhibitor, YM-254890, and conducted a kinase signaling proteomic screen using multiplexed-kinase inhibitors followed by mass spectrometry. We observed downregulated expression and/or activity of 22 kinases. A custom siRNA screen targeting these kinases demonstrated that knockdown of microtubule affinity regulating kinase 3 (MARK3) and serine/threonine kinase 10 (STK10) significantly reduced uveal melanoma cell growth and decreased expression of cell cycle proteins. Additionally, knockdown of MARK3 but not STK10 decreased ERK1/2 phosphorylation. Analysis of RNA-sequencing and proteomic data showed that Gαq signaling regulates STK10 expression and MARK3 activity. Our findings suggest an involvement of STK10 and MARK3 in the Gαq/11 oncogenic pathway and prompt further investigation into the specific roles and targeting potential of these kinases in uveal melanoma.


Subject(s)
Melanoma , Protein Serine-Threonine Kinases , Uveal Neoplasms , Humans , Cell Line, Tumor , GTP-Binding Protein alpha Subunits, Gq-G11/genetics , GTP-Binding Protein alpha Subunits, Gq-G11/metabolism , Melanoma/drug therapy , Melanoma/enzymology , Melanoma/genetics , Mutation , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Proteomics , Uveal Neoplasms/drug therapy , Uveal Neoplasms/enzymology , Uveal Neoplasms/genetics
10.
Mol Ther ; 31(7): 2154-2168, 2023 07 05.
Article in English | MEDLINE | ID: mdl-36869589

ABSTRACT

Immune checkpoint blockade (ICB) treatment has demonstrated excellent medical effects in oncology, and it is one of the most sought after immunotherapies for tumors. However, there are several issues with ICB therapy, including low response rates and a lack of effective efficacy predictors. Gasdermin-mediated pyroptosis is a typical inflammatory death mode. We discovered that increased expression of gasdermin protein was linked to a favorable tumor immune microenvironment and prognosis in head and neck squamous cell carcinoma (HNSCC). We used the mouse HNSCC cell lines 4MOSC1 (responsive to CTLA-4 blockade) and 4MOSC2 (resistant to CTLA-4 blockade) orthotopic models and demonstrated that CTLA-4 blockade treatment induced gasdermin-mediated pyroptosis of tumor cells, and gasdermin expression positively correlated to the effectiveness of CTLA-4 blockade treatment. We found that CTLA-4 blockade activated CD8+ T cells and increased the levels of interferon γ (IFN-γ) and tumor necrosis factor α (TNF-α) cytokines in the tumor microenvironment. These cytokines synergistically activated the STAT1/IRF1 axis to trigger tumor cell pyroptosis and the release of large amounts of inflammatory substances and chemokines. Collectively, our findings revealed that CTLA-4 blockade triggered tumor cells pyroptosis via the release of IFN-γ and TNF-α from activated CD8+ T cells, providing a new perspective of ICB.


Subject(s)
CD8-Positive T-Lymphocytes , Head and Neck Neoplasms , Mice , Animals , Squamous Cell Carcinoma of Head and Neck/therapy , Squamous Cell Carcinoma of Head and Neck/metabolism , CTLA-4 Antigen , Tumor Necrosis Factor-alpha/metabolism , Pyroptosis , Gasdermins , Cytokines/metabolism , Interferon-gamma/metabolism , Head and Neck Neoplasms/metabolism , Tumor Microenvironment
11.
Nucleic Acids Res ; 50(W1): W598-W610, 2022 07 05.
Article in English | MEDLINE | ID: mdl-35639758

ABSTRACT

In this study we show that protein language models can encode structural and functional information of GPCR sequences that can be used to predict their signaling and functional repertoire. We used the ESM1b protein embeddings as features and the binding information known from publicly available studies to develop PRECOGx, a machine learning predictor to explore GPCR interactions with G protein and ß-arrestin, which we made available through a new webserver (https://precogx.bioinfolab.sns.it/). PRECOGx outperformed its predecessor (e.g. PRECOG) in predicting GPCR-transducer couplings, being also able to consider all GPCR classes. The webserver also provides new functionalities, such as the projection of input sequences on a low-dimensional space describing essential features of the human GPCRome, which is used as a reference to track GPCR variants. Additionally, it allows inspection of the sequence and structural determinants responsible for coupling via the analysis of the most important attention maps used by the models as well as through predicted intramolecular contacts. We demonstrate applications of PRECOGx by predicting the impact of disease variants (ClinVar) and alternative splice forms from healthy tissues (GTEX) of human GPCRs, revealing the power to dissect system biasing mechanisms in both health and disease.


Subject(s)
Machine Learning , Receptors, G-Protein-Coupled , Signal Transduction , Software , Humans , Receptors, G-Protein-Coupled/chemistry , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Internet , beta-Arrestins/chemistry , beta-Arrestins/metabolism , Heterotrimeric GTP-Binding Proteins/chemistry , Heterotrimeric GTP-Binding Proteins/metabolism , Computers , Genetic Predisposition to Disease/genetics , Alternative Splicing/genetics
12.
Pharmacol Rev ; 73(4): 155-197, 2021 10.
Article in English | MEDLINE | ID: mdl-34663687

ABSTRACT

Many of the fundamental concepts of signal transduction and kinase activity are attributed to the discovery and crystallization of cAMP-dependent protein kinase, or protein kinase A. PKA is one of the best-studied kinases in human biology, with emphasis in biochemistry and biophysics, all the way to metabolism, hormone action, and gene expression regulation. It is surprising, however, that our understanding of PKA's role in disease is largely underappreciated. Although genetic mutations in the PKA holoenzyme are known to cause diseases such as Carney complex, Cushing syndrome, and acrodysostosis, the story largely stops there. With the recent explosion of genomic medicine, we can finally appreciate the broader role of the Gαs-PKA pathway in disease, with contributions from aberrant functioning G proteins and G protein-coupled receptors, as well as multiple alterations in other pathway components and negative regulators. Together, these represent a broad family of diseases we term the Gαs-PKA pathway signalopathies. The Gαs-PKA pathway signalopathies encompass diseases caused by germline, postzygotic, and somatic mutations in the Gαs-PKA pathway, with largely endocrine and neoplastic phenotypes. Here, we present a signaling-centric review of Gαs-PKA-driven pathophysiology and integrate computational and structural analysis to identify mutational themes commonly exploited by the Gαs-PKA pathway signalopathies. Major mutational themes include hotspot activating mutations in Gαs, encoded by GNAS, and mutations that destabilize the PKA holoenzyme. With this review, we hope to incite further study and ultimately the development of new therapeutic strategies in the treatment of a wide range of human diseases. SIGNIFICANCE STATEMENT: Little recognition is given to the causative role of Gαs-PKA pathway dysregulation in disease, with effects ranging from infectious disease, endocrine syndromes, and many cancers, yet these disparate diseases can all be understood by common genetic themes and biochemical signaling connections. By highlighting these common pathogenic mechanisms and bridging multiple disciplines, important progress can be made toward therapeutic advances in treating Gαs-PKA pathway-driven disease.


Subject(s)
Cyclic AMP-Dependent Protein Kinases , Genomic Medicine , Cyclic AMP-Dependent Protein Kinases/metabolism , GTP-Binding Protein alpha Subunits, Gs/genetics , GTP-Binding Protein alpha Subunits, Gs/metabolism , Humans , Mutation , Signal Transduction
13.
J Biol Chem ; 298(8): 102209, 2022 08.
Article in English | MEDLINE | ID: mdl-35779635

ABSTRACT

Trio is a large and highly conserved metazoan signaling scaffold that contains two Dbl family guanine nucleotide exchange factor (GEF) modules, TrioN and TrioC, selective for Rac and RhoA GTPases, respectively. The GEF activities of TrioN and TrioC are implicated in several cancers, especially uveal melanoma. However, little is known about how these modules operate in the context of larger fragments of Trio. Here we show via negative stain electron microscopy that the N-terminal region of Trio is extended and could thus serve as a rigid spacer between the N-terminal putative lipid-binding domain and TrioN, whereas the C-terminal half of Trio seems globular. We found that regions C-terminal to TrioN enhance its Rac1 GEF activity and thus could play a regulatory role. We went on to characterize a minimal, well-behaved Trio fragment with enhanced activity, Trio1284-1959, in complex with Rac1 using cryo-electron microscopy and hydrogen-deuterium exchange mass spectrometry and found that the region conferring enhanced activity is disordered. Deletion of two different strongly conserved motifs in this region eliminated this enhancement, suggesting that they form transient intramolecular interactions that promote GEF activity. Because Dbl family RhoGEF modules have been challenging to directly target with small molecules, characterization of accessory Trio domains such as these may provide alternate routes for the development of therapeutics that inhibit Trio activity in human cancer.


Subject(s)
Guanine Nucleotide Exchange Factors/chemistry , Protein Serine-Threonine Kinases/chemistry , Rho Guanine Nucleotide Exchange Factors/chemistry , Animals , Cryoelectron Microscopy , Guanine Nucleotide Exchange Factors/metabolism , Humans , Protein Binding , Protein Serine-Threonine Kinases/metabolism , Rho Guanine Nucleotide Exchange Factors/metabolism , Signal Transduction , Uveal Neoplasms , rac1 GTP-Binding Protein/genetics , rac1 GTP-Binding Protein/metabolism
14.
Nat Immunol ; 12(1): 86-95, 2011 Jan.
Article in English | MEDLINE | ID: mdl-21131965

ABSTRACT

The molecular mechanisms that direct transcription of the gene encoding the transcription factor Foxp3 in CD4(+) T cells remain ill-defined. We show here that deletion of the DNA-binding inhibitor Id3 resulted in the defective generation of Foxp3(+) regulatory T cells (T(reg) cells). We identify two transforming growth factor-ß1 (TGF-ß1)-dependent mechanisms that were vital for activation of Foxp3 transcription and were defective in Id3(-/-) CD4(+) T cells. Enhanced binding of the transcription factor E2A to the Foxp3 promoter promoted Foxp3 transcription. Id3 was required for relief of inhibition by the transcription factor GATA-3 at the Foxp3 promoter. Furthermore, Id3(-/-) T cells showed greater differentiation into the T(H)17 subset of helper T cells in vitro and in a mouse asthma model. Therefore, a network of factors acts in a TGF-ß-dependent manner to control Foxp3 expression and inhibit the development of T(H)17 cells.


Subject(s)
Asthma/metabolism , Forkhead Transcription Factors/metabolism , Inhibitor of Differentiation Proteins/metabolism , T-Lymphocytes, Regulatory/metabolism , Th17 Cells/metabolism , Animals , Asthma/chemically induced , Asthma/genetics , Asthma/immunology , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Differentiation/genetics , Cells, Cultured , Disease Models, Animal , Forkhead Transcription Factors/genetics , Inhibitor of Differentiation Proteins/genetics , Inhibitor of Differentiation Proteins/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Promoter Regions, Genetic/genetics , Protein Binding/genetics , Sequence Deletion/genetics , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/pathology , Th17 Cells/immunology , Th17 Cells/pathology , Transcriptional Activation/genetics , Transforming Growth Factor beta1/metabolism
15.
EMBO Rep ; 22(9): e51872, 2021 09 06.
Article in English | MEDLINE | ID: mdl-34324787

ABSTRACT

Epithelial plasticity, or epithelial-to-mesenchymal transition (EMT), is a well-recognized form of cellular plasticity, which endows tumor cells with invasive properties and alters their sensitivity to various agents, thus representing a major challenge to cancer therapy. It is increasingly accepted that carcinoma cells exist along a continuum of hybrid epithelial-mesenchymal (E-M) states and that cells exhibiting such partial EMT (P-EMT) states have greater metastatic competence than those characterized by either extreme (E or M). We described recently a P-EMT program operating in vivo by which carcinoma cells lose their epithelial state through post-translational programs. Here, we investigate the underlying mechanisms and report that prolonged calcium signaling induces a P-EMT characterized by the internalization of membrane-associated E-cadherin (ECAD) and other epithelial proteins as well as an increase in cellular migration and invasion. Signaling through Gαq-associated G-protein-coupled receptors (GPCRs) recapitulates these effects, which operate through the downstream activation of calmodulin-Camk2b signaling. These results implicate calcium signaling as a trigger for the acquisition of hybrid/partial epithelial-mesenchymal states in carcinoma cells.


Subject(s)
Calcium Signaling , Epithelial-Mesenchymal Transition , Cadherins/genetics , Cadherins/metabolism , Cell Line, Tumor , Cell Movement , Cell Plasticity
16.
Int J Mol Sci ; 24(24)2023 Dec 15.
Article in English | MEDLINE | ID: mdl-38139359

ABSTRACT

The serine-threonine kinase Akt plays a fundamental role in cell survival, metabolism, proliferation, and migration. To keep these essential processes under control, Akt activity and stability must be tightly regulated; otherwise, life-threatening conditions might prevail. Although it is well understood that phosphorylation regulates Akt activity, much remains to be known about how its stability is maintained. Here, we characterize BAG5, a chaperone regulator, as a novel Akt-interactor and substrate that attenuates Akt stability together with Hsp70. BAG5 switches monoubiquitination to polyubiquitination of Akt and increases its degradation caused by Hsp90 inhibition and Hsp70 overexpression. Akt interacts with BAG5 at the linker region that joins the first and second BAG domains and phosphorylates the first BAG domain. The Akt-BAG5 complex is formed in serum-starved conditions and dissociates in response to HGF, coincident with BAG5 phosphorylation. BAG5 knockdown attenuated Akt degradation and facilitated its activation, whereas the opposite effect was caused by BAG5 overexpression. Altogether, our results indicate that Akt stability and signaling are dynamically regulated by BAG5, depending on growth factor availability.


Subject(s)
Molecular Chaperones , Proto-Oncogene Proteins c-akt , HSP70 Heat-Shock Proteins/metabolism , Molecular Chaperones/metabolism , Phosphorylation , Proto-Oncogene Proteins c-akt/metabolism , Ubiquitination , HEK293 Cells , Humans , Animals , Mice
17.
J Biol Chem ; 295(50): 16920-16928, 2020 12 11.
Article in English | MEDLINE | ID: mdl-33023908

ABSTRACT

Gα proteins promote dynamic adjustments of cell shape directed by actin-cytoskeleton reorganization via their respective RhoGEF effectors. For example, Gα13 binding to the RGS-homology (RH) domains of several RH-RhoGEFs allosterically activates these proteins, causing them to expose their catalytic Dbl-homology (DH)/pleckstrin-homology (PH) regions, which triggers downstream signals. However, whether additional Gα proteins might directly regulate the RH-RhoGEFs was not known. To explore this question, we first examined the morphological effects of expressing shortened RH-RhoGEF DH/PH constructs of p115RhoGEF/ARHGEF1, PDZ-RhoGEF (PRG)/ARHGEF11, and LARG/ARHGEF12. As expected, the three constructs promoted cell contraction and activated RhoA, known to be downstream of Gα13 Intriguingly, PRG DH/PH also induced filopodia-like cell protrusions and activated Cdc42. This pathway was stimulated by constitutively active Gαs (GαsQ227L), which enabled endogenous PRG to gain affinity for Cdc42. A chemogenetic approach revealed that signaling by Gs-coupled receptors, but not by those coupled to Gi or Gq, enabled PRG to bind Cdc42. This receptor-dependent effect, as well as CREB phosphorylation, was blocked by a construct derived from the PRG:Gαs-binding region, PRG-linker. Active Gαs interacted with isolated PRG DH and PH domains and their linker. In addition, this construct interfered with GαsQ227L's ability to guide PRG's interaction with Cdc42. Endogenous Gs-coupled prostaglandin receptors stimulated PRG binding to membrane fractions and activated signaling to PKA, and this canonical endogenous pathway was attenuated by PRG-linker. Altogether, our results demonstrate that active Gαs can recognize PRG as a novel effector directing its DH/PH catalytic module to gain affinity for Cdc42.


Subject(s)
Cell Movement , GTP-Binding Protein alpha Subunits, G12-G13/metabolism , Pleckstrin Homology Domains/genetics , Pseudopodia/metabolism , Rho Guanine Nucleotide Exchange Factors/metabolism , Signal Transduction , cdc42 GTP-Binding Protein/metabolism , Animals , Cell Line , Humans , Mice , Phosphorylation
18.
Exp Cell Res ; 386(1): 111684, 2020 01 01.
Article in English | MEDLINE | ID: mdl-31654625

ABSTRACT

Vulvar squamous cell carcinoma associated with lichen sclerosus (VLS-VSCC) are rare tumors but with higher recurrence and worse prognosis than other types of VSCC. Lack of experimental models has limited the search for better understanding of the biology and development of treatment modalities. In this study, we isolated and characterized primary cells from VSCC (n = 7) and normal vulvar tissue adjacent to tumor (n = 7). Detailed characterization of the novel spontaneously immortalized cell line, VCC1 revealed a characteristic epithelial morphology in vitro and a well-differentiated keratinizing SCC histology in vivo, closely resembling the tumor of origin. VCC1 expressed higher levels of epithelial-mesenchymal transition markers and higher clonogenic properties as compared to other established non VLS-VSCC cell lines. In vitro 3D organotypic assays and in vivo xenografts revealed a prominent role of cancer-associated fibroblasts in VCC1 invasion and tumor formation. In conclusion, VCC1 mirrored several major VLS-VSCC features and provided a robust experimental tool for further elucidation of VLS-related oncogenesis and drug testing.


Subject(s)
Carcinoma, Squamous Cell/pathology , Cell Culture Techniques/methods , Vulvar Lichen Sclerosus/pathology , Vulvar Neoplasms/pathology , Animals , Carcinogenesis , Carcinoma, Squamous Cell/metabolism , Cell Line, Tumor , Cells, Cultured , Female , Fibroblasts/metabolism , Fibroblasts/pathology , Humans , Mice , Mice, Inbred NOD , Mice, SCID , Vulvar Lichen Sclerosus/metabolism , Vulvar Neoplasms/metabolism , Xenograft Model Antitumor Assays/methods
19.
Mol Cell ; 49(1): 94-108, 2013 Jan 10.
Article in English | MEDLINE | ID: mdl-23177739

ABSTRACT

Activating mutations in GNAQ and GNA11, encoding members of the Gα(q) family of G protein α subunits, are the driver oncogenes in uveal melanoma, and mutations in Gq-linked G protein-coupled receptors have been identified recently in numerous human malignancies. How Gα(q) and its coupled receptors transduce mitogenic signals is still unclear because of the complexity of signaling events perturbed upon Gq activation. Using a synthetic-biology approach and a genome-wide RNAi screen, we found that a highly conserved guanine nucleotide exchange factor, Trio, is essential for activating Rho- and Rac-regulated signaling pathways acting on JNK and p38, and thereby transducing proliferative signals from Gα(q) to the nucleus independently of phospholipase C-ß. Indeed, whereas many biological responses elicited by Gq depend on the transient activation of second-messenger systems, Gq utilizes a hard-wired protein-protein-interaction-based signaling circuitry to achieve the sustained stimulation of proliferative pathways, thereby controlling normal and aberrant cell growth.


Subject(s)
Guanine Nucleotide Exchange Factors/physiology , Mitosis , Protein Serine-Threonine Kinases/physiology , Receptors, G-Protein-Coupled/metabolism , Signal Transduction , Transcription Factor AP-1/metabolism , rho GTP-Binding Proteins/metabolism , Animals , Cell Line, Tumor , Cell Proliferation , Clozapine/analogs & derivatives , Clozapine/pharmacology , Drosophila/genetics , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Enzyme Activation , Female , GTP-Binding Protein alpha Subunits/metabolism , GTP-Binding Protein alpha Subunits, Gq-G11 , Gene Knockdown Techniques , Guanine Nucleotide Exchange Factors/metabolism , Humans , Mice , Mice, Nude , Mitogen-Activated Protein Kinases/metabolism , Mitogens/pharmacology , NIH 3T3 Cells , Neoplasm Transplantation , Neoplasms/pathology , Protein Serine-Threonine Kinases/metabolism , RNA Interference , Receptors, G-Protein-Coupled/genetics
20.
Nucleic Acids Res ; 47(W1): W395-W401, 2019 07 02.
Article in English | MEDLINE | ID: mdl-31143927

ABSTRACT

G-protein coupled receptors (GPCRs) control multiple physiological states by transducing a multitude of extracellular stimuli into the cell via coupling to intra-cellular heterotrimeric G-proteins. Deciphering which G-proteins couple to each of the hundreds of GPCRs present in a typical eukaryotic organism is therefore critical to understand signalling. Here, we present PRECOG (precog.russelllab.org): a web-server for predicting GPCR coupling, which allows users to: (i) predict coupling probabilities for GPCRs to individual G-proteins instead of subfamilies; (ii) visually inspect the protein sequence and structural features that are responsible for a particular coupling; (iii) suggest mutations to rationally design artificial GPCRs with new coupling properties based on predetermined coupling features.


Subject(s)
GTP-Binding Proteins/chemistry , Receptors, G-Protein-Coupled/chemistry , Recombinant Fusion Proteins/chemistry , Software , Amino Acid Sequence , Animals , Binding Sites , GTP-Binding Proteins/genetics , GTP-Binding Proteins/metabolism , Humans , Internet , Models, Molecular , Mutation , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Engineering , Protein Interaction Domains and Motifs , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Signal Transduction
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