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Shelley B Hooks - One of the best experts on this subject based on the ideXlab platform.
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RGS10 physically and functionally interacts with stim2 and requires store operated calcium entry to regulate pro inflammatory gene expression in microglia
Cellular Signalling, 2021Co-Authors: Menbere Wendimu, Mohammed Alqinyah, Faris Almutairi, Stephen A Vella, Phillip Dean, Roseanne Davilarivera, Shima Rayatpisheh, James A Wohlschlegel, Silvia N J Moreno, Shelley B HooksAbstract:Chronic activation of microglia is a driving factor in the progression of neuroinflammatory diseases, and mechanisms that regulate microglial inflammatory signaling are potential targets for novel therapeutics. Regulator of G protein Signaling 10 is the most abundant RGS protein in microglia, where it suppresses inflammatory gene expression and reduces microglia-mediated neurotoxicity. In particular, microglial RGS10 downregulates the expression of pro-inflammatory mediators including cyclooxygenase 2 (COX-2) following stimulation with lipopolysaccharide (LPS). However, the mechanism by which RGS10 affects inflammatory signaling is unknown and is independent of its canonical G protein targeted mechanism. Here, we sought to identify non-canonical RGS10 interacting partners that mediate its anti-inflammatory mechanism. Through RGS10 co-immunoprecipitation coupled with mass spectrometry, we identified STIM2, an endoplasmic reticulum (ER) localized calcium sensor and a component of the store-operated calcium entry (SOCE) machinery, as a novel RGS10 interacting protein in microglia. Direct immunoprecipitation experiments confirmed RGS10-STIM2 interaction in multiple microglia and macrophage cell lines, as well as in primary cells, with no interaction observed with the homologue STIM1. We further determined that STIM2, Orai channels, and the calcium-dependent phosphatase calcineurin are essential for LPS-induced COX-2 production in microglia, and this pathway is required for the inhibitory effect of RGS10 on COX-2. Additionally, our data demonstrated that RGS10 suppresses SOCE triggered by ER calcium depletion and that ER calcium depletion, which induces SOCE, amplifies pro-inflammatory genes. In addition to COX-2, we also show that RGS10 suppresses the expression of pro-inflammatory cytokines in microglia in response to thrombin and LPS stimulation, and all of these effects require SOCE. Collectively, the physical and functional links between RGS10 and STIM2 suggest a complex regulatory network connecting RGS10, SOCE, and pro-inflammatory gene expression in microglia, with broad implications in the pathogenesis and treatment of chronic neuroinflammation.
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RGS10 regulates the expression of cyclooxygenase 2 and tumor necrosis factor alpha through a g protein independent mechanism
Molecular Pharmacology, 2018Co-Authors: Mohammed Alqinyah, Faris Almutairi, Menbere Wendimu, Shelley B HooksAbstract:The small regulator of G protein signaling protein RGS10 is a key regulator of neuroinflammation and ovarian cancer cell survival; however, the mechanism for RGS10 function in these cells is unknown and has not been linked to specific G protein pathways. RGS10 is highly enriched in microglia, and loss of RGS10 expression in microglia amplifies production of the inflammatory cytokine tumor necrosis factor α (TNFα) and enhances microglia-induced neurotoxicity. RGS10 also regulates cell survival and chemoresistance of ovarian cancer cells. Cyclooxygenase-2 (COX-2)-mediated production of prostaglandins such as prostaglandin E2 (PGE2) is a key factor in both neuroinflammation and cancer chemoresistance, suggesting it may be involved in RGS10 function in both cell types, but a connection between RGS10 and COX-2 has not been reported. To address these questions, we completed a mechanistic study to characterize RGS10 regulation of TNFα and COX-2 and to determine if these effects are mediated through a G protein-dependent mechanism. Our data show for the first time that loss of RGS10 expression significantly elevates stimulated COX-2 expression and PGE2 production in microglia. Furthermore, the elevated inflammatory signaling resulting from RGS10 loss was not affected by Gαi inhibition, and a RGS10 mutant that is unable to bind activated G proteins was as effective as wild type in inhibiting TNFα expression. Similarly, suppression of RGS10 in ovarian cancer cells enhanced TNFα and COX-2 expression, and this effect did not require Gi activity. Together, our data strongly indicate that RGS10 inhibits COX-2 expression by a G protein-independent mechanism to regulate inflammatory signaling in microglia and ovarian cancer cells.
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regulator of g protein signaling 10 RGS10 expression is transcriptionally silenced in activated microglia by histone deacetylase activity
Molecular Pharmacology, 2017Co-Authors: Mohammed Alqinyah, Ercan Cacan, Susanna F Greer, Mourad W. Ali, Nagini Maganti, Ruchi Yadav, Mei Gao, Hanrong Weng, Shelley B HooksAbstract:RGS10 has emerged as a key regulator of proinflammatory cytokine production in microglia, functioning as an important neuroprotective factor. Although RGS10 is normally expressed in microglia at high levels, expression is silenced in vitro following activation of TLR4 receptor. Given the ability of RGS10 to regulate inflammatory signaling, dynamic regulation of RGS10 levels in microglia may be an important mechanism to tune inflammatory responses. The goals of the current study were to confirm that RGS10 is suppressed in an in vivo inflammatory model of microglial activation and to determine the mechanism for activation-dependent silencing of RGS10 expression in microglia. We demonstrate that endogenous RGS10 is present in spinal cord microglia, and RGS10 protein levels are suppressed in the spinal cord in a nerve injury-induced neuropathic pain mouse model. We show that the histone deacetylase (HDAC) enzyme inhibitor trichostatin A blocks the ability of lipopolysaccharide (LPS) to suppress RGS10 transcription in BV-2 and primary microglia, demonstrating that HDAC enzymes are required for LPS silencing of RGS10 Furthermore, we used chromatin immunoprecipitation to demonstrate that H3 histones at the RGS10 proximal promoter are deacetylated in BV-2 microglia following LPS activation, and HDAC1 association at the RGS10 promoter is enhanced following LPS stimulation. Finally, we have shown that sphingosine 1-phosphate, an endogenous microglial signaling mediator that inhibits HDAC activity, enhances basal RGS10 expression in BV-2 microglia, suggesting that RGS10 expression is dynamically regulated in microglia in response to multiple signals.
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cellular deficiency in the RGS10 protein facilitates chemoresistant ovarian cancer
Future Medicinal Chemistry, 2015Co-Authors: Shelley B Hooks, Mandi M. MurphAbstract:More than 30 regulators of G protein signaling (RGS) proteins encompass the RGS protein superfamily of critical regulators essential to cellular homeostasis. There is enormous structural and functional diversity among the RGS superfamily, and as such they serve a wide range of functions in regulating cell biology and physiology. Recent evidence has suggested roles for multiple RGS proteins in cancer initiation and progression, which has prompted research toward the potential modulation of these proteins as a new approach in cancer therapy. This article will discuss basic RGS molecular pharmacology, summarize the cellular functions and epigenetic regulation of RGS10, review ovarian cancer chemotherapy and describe the role of RGS10 in ovarian cancer survival signaling.
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abstract poster biol 1317 releasing the brakes on ovarian cancer cell survival epigenetic suppression of RGS10 in ovarian cancer chemoresistance
Clinical Cancer Research, 2015Co-Authors: Shelley B HooksAbstract:G-protein signaling pathways promote cell survival, which is aberrantly enhanced in chemoresistant ovarian cancer. Regulator of G-protein Signaling (RGS) proteins limit the strength of G-protein signaling, and therefore indirectly control cell survival. We have previously shown that RGS10 suppresses ovarian cancer cell growth and limits survival in the presence of cytotoxic chemotherapeutic drugs. Further, we have shown that RGS10 expression is suppressed in cell models of ovarian cancer chemoresistance, suggesting that ovarian cancer cells silence RGS10 expression to release the brakes on their growth and survival. However, the mechanisms governing RGS10 expression in ovarian cancer cells are poorly understood. In this study, we fully investigate the molecular mechanism of RGS10 suppression in ovarian cancer acquired chemoresistance, revealing key roles for epigenetic regulation by DNA hypermethylation and histone deacetylation. Pharmacological inhibition of DNA methyl-transferases (DNMTs) increases RGS10 expression in chemoresistant ovarian cancer cells, suggesting potential regulation by DNA methylation. Further, bisulfite sequencing analysis identified a region of the RGS10-1 promoter with significantly enhanced DNA methylation in chemoresistant A2780-AD cells relative to parental A2780 cells. More marked differences are observed in histone acetylation of the RGS10-1 promoter. Acetylated histone H3 associates with the RGS10-1 promoter was significantly lower in A2780-AD cells compared to parental cells, with a corresponding increase in histone deacetylase (HDAC) enzyme association. Further, both HDAC1 and DNMT1 exhibit aberrant association with RGS10 promoters in chemoresistant ovarian cancer cells. Knockdown of HDAC1 or DNMT1 expression, and pharmacological inhibition of DNMT or HDAC enzymatic activity, significantly increases RGS10 expression and cisplatin-mediated cell death. Finally, DNMT1 knock down decreases HDAC1 binding to the RGS10 promoter in chemoresistant cells, suggesting HDAC1 recruitment to RGS10 promoters requires DNMT1 activity. Our results suggest that HDAC1 and DNMT1 cooperatively contribute to the suppression of RGS10 during acquired chemoresistance and support inhibition of HDAC1 and DNMT1 as an adjuvant therapeutic approach to overcome ovarian cancer chemoresistance. Citation Format: Shelley Hooks, PhD. Releasing the brakes on ovarian cancer cell survival: epigenetic suppression of RGS10 in ovarian cancer chemoresistance [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-BIOL-1317.
John R. Hepler - One of the best experts on this subject based on the ideXlab platform.
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rgs14 modulates locomotor behavior and erk signaling induced by environmental novelty and cocaine within discrete limbic structures
Psychopharmacology, 2021Co-Authors: Stephanie L Foster, John R. Hepler, Daniel Lustberg, Nicholas H Harbin, Sara N Bramlett, David WeinshenkerAbstract:In rodents, exposure to novel environments or psychostimulants promotes locomotion. Indeed, locomotor reactivity to novelty strongly predicts behavioral responses to psychostimulants in animal models of addiction. RGS14 is a plasticity-restricting protein with unique functional domains that enable it to suppress ERK-dependent signaling as well as regulate G protein activity. Although recent studies show that RGS14 is expressed in multiple limbic regions implicated in psychostimulant- and novelty-induced hyperlocomotion, its function has been examined mostly in the context of hippocampal physiology and memory. We investigated whether RGS14 modulates novelty- and cocaine-induced locomotion (NIL and CIL, respectively) and neuronal activity. We assessed Rgs14 knockout (RGS14 KO) mice and wild-type (WT) littermate controls using NIL and CIL behavioral tests, followed by quantification of c-fos and phosphorylated ERK (pERK) induction in limbic regions that normally express RGS14. RGS14 KO mice were less active than WT controls in the NIL test, driven by avoidance of the center of the novel environment. By contrast, RGS14 KO mice demonstrated augmented peripheral locomotion in the CIL test conducted in either a familiar or novel environment. RGS14 KO mice exhibited increased thigmotaxis, as well as greater c-fos and pERK induction in the central amygdala and dorsal hippocampus, when cocaine and novelty were paired. RGS14 KO mice exhibited anti-correlated locomotor responses to novelty and cocaine, but displayed increased thigmotaxis in response to either stimuli which was augmented by their combination. Our findings also suggest RGS14 may reduce neuronal activity in limbic subregions by inhibiting ERK-dependent signaling.
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rgs14 regulation of post synaptic signaling and spine plasticity in brain
International Journal of Molecular Sciences, 2021Co-Authors: Nicholas H Harbin, Sara N Bramlett, Carolina Montanezmiranda, Gizem Terzioglu, John R. HeplerAbstract:The regulator of G-protein signaling 14 (RGS14) is a multifunctional signaling protein that regulates post synaptic plasticity in neurons. RGS14 is expressed in the brain regions essential for learning, memory, emotion, and stimulus-induced behaviors, including the basal ganglia, limbic system, and cortex. Behaviorally, RGS14 regulates spatial and object memory, female-specific responses to cued fear conditioning, and environmental- and psychostimulant-induced locomotion. At the cellular level, RGS14 acts as a scaffolding protein that integrates G protein, Ras/ERK, and calcium/calmodulin signaling pathways essential for spine plasticity and cell signaling, allowing RGS14 to naturally suppress long-term potentiation (LTP) and structural plasticity in hippocampal area CA2 pyramidal cells. Recent proteomics findings indicate that RGS14 also engages the actomyosin system in the brain, perhaps to impact spine morphogenesis. Of note, RGS14 is also a nucleocytoplasmic shuttling protein, where its role in the nucleus remains uncertain. Balanced nuclear import/export and dendritic spine localization are likely essential for RGS14 neuronal functions as a regulator of synaptic plasticity. Supporting this idea, human genetic variants disrupting RGS14 localization also disrupt RGS14’s effects on plasticity. This review will focus on the known and unexplored roles of RGS14 in cell signaling, physiology, disease and behavior.
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rgs14 modulates locomotor behavior and erk signaling induced by environmental novelty and cocaine within discrete limbic structures
bioRxiv, 2021Co-Authors: Stephanie L Foster, John R. Hepler, Daniel Lustberg, Nicholas H Harbin, Sara N Bramlett, David WeinshenkerAbstract:Rationale: In rodents, exposure to novel environments or psychostimulants promotes locomotor activity. Indeed, locomotor reactivity to novelty strongly predicts behavioral responses to psychostimulants in animal models of addiction. RGS14 is a plasticity restricting protein with unique functional domains that enable it to suppress ERK-dependent signaling as well as regulate G protein activity. Although recent studies show that RGS14 is expressed in multiple limbic regions implicated in psychostimulant- and novelty-induced hyperlocomotion, its function has been studied almost entirely in the context of hippocampal physiology and hippocampus-dependent behaviors. Objective: We sought to determine whether RGS14 modulates novelty- and psychostimulant-induced locomotion and neuronal activity. Methods: We assessed Rgs14 knockout (RGS14 KO) mice and wild-type (WT) littermate controls using novelty-induced locomotion (NIL) and cocaine-induced locomotion (CIL) behavioral tests with subsequent quantification of c-fos and phosphorylated ERK (pERK) induction in limbic regions that express RGS14. Results: Compared to WT controls, RGS14 KO mice exhibited attenuated locomotor responses in the NIL test, driven by avoidance of the center of the novel environment. By contrast, RGS14 KO mice demonstrated augmented peripheral locomotion in the CIL test conducted in either a familiar or novel environment. The absence of RGS14 enhanced induction of c-fos and pERK in the central amygdala and hippocampus (areas CA1 and CA2) when cocaine was administered in a novel environment. Conclusions: RGS14 regulates novelty- and psychostimulant-induced hyperlocomotion, particularly with respect to thigmotaxis. Further, our findings suggest RGS14 may reduce neuronal activity in discrete limbic subregions by inhibiting ERK-dependent signaling and transcription.
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genetic variants disrupt human rgs14 binding to nherf1 and regulation of npt2a mediated phosphate transport
bioRxiv, 2019Co-Authors: Peter A Friedman, Katherine E. Squires, Tatyana Mamonova, Clara E Magyar, Bruce W Sneddon, David R Emlet, John R. HeplerAbstract:RGS14 is a multifunctional scaffolding protein that integrates G protein, MAPK, and Ca++/CaM signaling pathways. RGS14 contains an RGS domain, tandem Ras/Rap-binding domains, and a G protein regulatory motif. Human/primate RGS14 differ from rodent proteins by possessing a carboxy-terminal extension encoding a Type I PDZ ligand. RGS14 has been implicated in disordered phosphate metabolism. The human RGS14 gene is adjacent to SLC34A1 that encodes the NPT2A sodium-phosphate cotransporter. Hormone-regulated NPT2A requires the PDZ protein NHERF1 which contains two PDZ domains (PDZ1 and PDZ2). NHERF1 binds the PDZ ligand carboxy tail of NPT2A to regulate phosphate uptake, and this NPT2A:NHERF1 complex is inhibited by parathyroid hormone (PTH). Studies here define roles for RGS14 in NHERF1-dependent, PTH-sensitive phosphate transport. We found that RGS14 binds to NHERF1 via the PDZ2 domain. PTH inhibits NPT2A-mediated phosphate transport and RGS14 blocked this action. Several rare human mutations have been reported in the RGS14 PDZ ligand located at residues 563 (D563N, D563G) and 565 (A565S, A565V). D563N disrupted RGS14 binding to NHERF1 and did not interfere with PTH action, whereas D563G, A565S, and A565V bound NHERF1 and were functionally equivalent to wild-type RGS14. Computational analysis and molecular dynamics modeling of NHERF1 PDZ2 binding to the RGS14 C-terminal PDZ ligands refined the structural determinants of this interaction. Additional studies demonstrated that RGS14 is expressed in human kidney proximal and distal tubule cells. Together, our findings are consistent with the view that RGS14 contributes to PTH-sensitive phosphate transport in humans. RGS14 coding variants may cause disordered phosphate metabolism.
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14 3 3γ binds regulator of g protein signaling 14 rgs14 at distinct sites to inhibit the rgs14 gαi alf4 signaling complex and rgs14 nuclear localization
Journal of Biological Chemistry, 2018Co-Authors: Kyle J Gerber, Katherine E. Squires, John R. HeplerAbstract:Regulator of G protein signaling 14 (RGS14) is a multifunctional brain scaffolding protein that integrates G protein and Ras/ERK signaling pathways. It is also a nucleocytoplasmic shuttling protein. RGS14 binds active Gαi/o via its RGS domain, Raf and active H-Ras–GTP via its R1 Ras-binding domain (RBD), and inactive Gαi1/3 via its G protein regulatory (GPR) domain. RGS14 suppresses long-term potentiation (LTP) in the CA2 region of the hippocampus, thereby regulating hippocampally based learning and memory. The 14-3-3 family of proteins is necessary for hippocampal LTP and associative learning and memory. Here, we show direct interaction between RGS14 and 14-3-3γ at two distinct sties, one phosphorylation-independent and the other phosphorylation-dependent at Ser-218 that is markedly potentiated by signaling downstream of active H-Ras. Using bioluminescence resonance energy transfer (BRET), we show that the pSer-218–dependent RGS14/14-3-3γ interaction inhibits active Gαi1–AlF4− binding to the RGS domain of RGS14 but has no effect on active H-Ras and inactive Gαi1–GDP binding to RGS14. By contrast, the phosphorylation-independent binding of 14-3-3 has no effect on RGS14/Gαi interactions but, instead, inhibits (directly or indirectly) RGS14 nuclear import and nucleocytoplasmic shuttling. Together, our findings describe a novel mechanism of negative regulation of RGS14 functions, specifically interactions with active Gαi and nuclear import, while leaving the function of other RGS14 domains intact. Ongoing studies will further elucidate the physiological function of this interaction between RGS14 and 14-3-3γ, providing insight into the functions of both RGS14 and 14-3-3 in their roles in modulating synaptic plasticity in the hippocampus.
David P Siderovski - One of the best experts on this subject based on the ideXlab platform.
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A role for Regulator of G protein Signaling-12 (RGS12) in the balance between myoblast proliferation and differentiation.
PloS one, 2019Co-Authors: Adam B. Schroer, Melinda D. Willard, Emily A. Oestreich, Junaith S. Mohamed, Vincent Setola, David P SiderovskiAbstract:Regulators of G Protein Signaling (RGS proteins) inhibit G protein-coupled receptor (GPCR) signaling by accelerating the GTP hydrolysis rate of activated Gα subunits. Some RGS proteins exert additional signal modulatory functions, and RGS12 is one such protein, with five additional, functional domains: a PDZ domain, a phosphotyrosine-binding domain, two Ras-binding domains, and a Gα·GDP-binding GoLoco motif. RGS12 expression is temporospatially regulated in developing mouse embryos, with notable expression in somites and developing skeletal muscle. We therefore examined whether RGS12 is involved in the skeletal muscle myogenic program. In the adult mouse, RGS12 is expressed in the tibialis anterior (TA) muscle, and its expression is increased early after cardiotoxin-induced injury, suggesting a role in muscle regeneration. Consistent with a potential role in coordinating myogenic signals, RGS12 is also expressed in primary myoblasts; as these cells undergo differentiation and fusion into myotubes, RGS12 protein abundance is reduced. Myoblasts isolated from mice lacking Rgs12 expression have an impaired ability to differentiate into myotubes ex vivo, suggesting that RGS12 may play a role as a modulator/switch for differentiation. We also assessed the muscle regenerative capacity of mice conditionally deficient in skeletal muscle Rgs12 expression (via Pax7-driven Cre recombinase expression), following cardiotoxin-induced damage to the TA muscle. Eight days post-damage, mice lacking RGS12 in skeletal muscle had attenuated repair of muscle fibers. However, when mice lacking skeletal muscle expression of Rgs12 were cross-bred with mdx mice (a model of human Duchenne muscular dystrophy), no increase in muscle degeneration was observed over time. These data support the hypothesis that RGS12 plays a role in coordinating signals during the myogenic program in select circumstances, but loss of the protein may be compensated for within model syndromes of prolonged bouts of muscle damage and repair.
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the effect of rgs12 on pdgfβ receptor signalling to p42 p44 mitogen activated protein kinase in mammalian cells
Cellular Signalling, 2006Co-Authors: Balwinder Sambi, David P Siderovski, Francis S. Willard, Melinda D. Hains, Catherine M. Waters, Michelle Connell, Adam J. Kimple, Susan Pyne, Nigel J. PyneAbstract:We have previously shown that the PDGFβ receptor uses a classical GPCR-mediated pathway in order to induce efficient activation of p42/p44 MAPK in response to PDGF. We therefore, considered the possibility that GTPase accelerating proteins (RGS proteins), which regulate GPCR signalling, modulate PDGFβ receptor-mediated signal transmission. Several lines of evidence were obtained to support functional interaction between the PDGFβ receptor and RGS12 in HEK 293 and airway smooth muscle cells. Firstly, the over-expression of the RGS12 PDZ/PTB domain N-terminus or RGS12 PTB domain reduced the PDGF-induced activation of p42/p44 MAPK. Secondly, the RGS12 PDZ/PTB domain N-terminus and RGS12 PDZ domain can form a complex with the PDGFβ receptor. Therefore, the results presented here provide the first evidence to support the concept that the PDZ/PTB domain N-terminus and/or the PTB domain of RGS12 may modulate PDGFβ receptor signalling. In airway smooth muscle cells, over-expressed recombinant RGS12 and the isolated PDZ/PTB domain N-terminus co-localised with PDGFβ receptor in cytoplasmic vesicles. To provide additional evidence for a role of the PDZ/PTB domain N-terminus, we used RGS14. RGS14 has the same C-terminal domain architecture of an RGS box, tandem Ras-binding domains (RBDs) and GoLoco motif as RGS12, but lacks the PDZ/PTB domain N-terminus. In this regard, RGS14 exhibited a different sub-cellular distribution compared with RGS12, being diffusely distributed in ASM cells. These findings suggest that RGS12 via its PDZ/PTB domain N-terminus may regulate trafficking of the PDGFβ receptor in ASM cells.
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RGS14 is a Microtubule-Associated Protein
Cell cycle (Georgetown Tex.), 2005Co-Authors: Luke Martin-mccaffrey, David P Siderovski, Francis S. Willard, Agnieszka Pajak, Lina Dagnino, Sudhir J. A. D'souzaAbstract:Heterotrimeric G-proteins and their regulators are emerging as important players in modulating microtubule polymerization dynamics and in spindle force generation during cell division in C. elegans, D. melanogaster and mammals. We recently demonstrated that RGS14 is required for completion of the first mitotic division of the mouse embryo, and that it regulates microtubule organization in vivo. Here, we demonstrate that RGS14 is a microtubule-associated protein and a component of the mitotic spindle that may regulate microtubule polymerization and spindle organization. Taxol-stabilized tubulin, but not depolymerized tubulin coimmunoprecipitates with RGS14 from cell extracts. Furthermore, RGS14 copurifies with tubulin from porcine brain following multiple rounds of microtubule polymerization/depolymerization and binds directly to microtubules formed in vitro from pure tubulin (KD = 1.3 +/- 0.3 microM). Both RGS14 and Galpha(i1) in the presence of exogenous GTP promote tubulin polymerization, which is dependent on additional microtubule-associated proteins. However, preincubation of RGS14 with Galpha(i1)-GDP precludes either from promoting microtubule polymerization, suggesting that a functional GTP/GDP cycle is necessary. Finally, we show that RGS14 is a component of mitotic asters formed in vitro from HeLa cell extracts and that depletion of RGS14 from cell extracts blocks aster formation. Collectively, these results show that RGS14 is a microtubule-associated protein that may modulate microtubule dynamics and spindle formation.
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Return of the GDI: The GoLoco Motif in Cell Division
Annual review of biochemistry, 2004Co-Authors: Francis S. Willard, Randall J Kimple, David P SiderovskiAbstract:The GoLoco motif is a 19-amino-acid sequence with guanine nucleotide dissociation inhibitor activity against G-alpha subunits of the adenylyl-cyclase-inhibitory subclass. The GoLoco motif is present as an independent element within multidomain signaling regulators, such as Loco, RGS12, RGS14, and Rap1GAP, as well as in tandem arrays in proteins, such as AGS3, G18, LGN, Pcp-2/L7, and Partner of Inscuteable (Pins/Rapsynoid). Here we discuss the biochemical mechanisms of GoLoco motif action on G-alpha subunits in light of the recent crystal structure of G-alpha-i1 bound to the RGS14 GoLoco motif. Currently, there is sparse evidence for GoLoco motif regulation of canonical G-protein-coupled receptor signaling. Rather, studies of asymmetric cell division in Drosophila and Caenorhabditis elegans, as well as mammalian mitosis, implicate GoLoco proteins, such as Pins, GPR-1/GPR-2, LGN, and RGS14, in mitotic spindle organization and force generation. We discuss potential mechanisms by which GoLoco/Galpha complexes might modulate spindle dynamics.
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Purification and in vitro functional analyses of RGS12 and RGS14 GoLoco motif peptides
Methods in enzymology, 2004Co-Authors: Randall J Kimple, Francis S. Willard, David P SiderovskiAbstract:The GoLoco motif is a short polypeptide sequence that binds to heterotrimeric G-protein alpha subunits of the adenylyl cyclase-inhibitory (Galpha(i/o)) subclass in a nucleotide-dependent manner (i.e., solely to the GDP-bound ground state). This article describes methods used for the expression, purification, and in vitro evaluation of membrane-permeant tag fusion peptides derived from the GoLoco motif regions of "regulator of G-protein signaling" proteins type 12 (RGS12) and 14 (RGS14) and a consensus GoLoco sequence from the multiple GoLoco motif protein AGS3. Three different fluorescence-based assays are described for evaluating the in vitro function of these GoLoco peptides as guanine nucleotide dissociation inhibitors, including measurements of GTPgammaS binding and Galpha subunit activation by the planar ion aluminum tetrafluoride.
Mandi M. Murph - One of the best experts on this subject based on the ideXlab platform.
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suppression of the gtpase activating protein RGS10 increases rheb gtp and mtor signaling in ovarian cancer cells
Cancer Letters, 2015Co-Authors: Molly K Altman, Ali A Alshamrani, Wei Jia, Ha T Nguyen, Jada M Fambrough, Sterling K Tran, Mihir Patel, Pooya Hoseinzadeh, Aaron M Beedle, Mandi M. MurphAbstract:The regulator of G protein signaling 10 (RGS10) protein is a GTPase activating protein that accelerates the hydrolysis of GTP and therefore canonically inactivates G proteins, ultimately terminating signaling. Rheb is a small GTPase protein that shuttles between its GDP- and GTP-bound forms to activate mTOR. Since RGS10 suppression augments ovarian cancer cell viability, we sought to elucidate the molecular mechanism. Following RGS10 suppression in serum-free conditions, phosphorylation of mTOR, the eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1), p70S6K and S6 Ribosomal Protein appear. Furthermore, suppressing RGS10 increases activated Rheb, suggesting RGS10 antagonizes mTOR signaling via the small G-protein. The effects of RGS10 suppression are enhanced after stimulating cells with the growth factor, lysophosphatidic acid, and reduced with mTOR inhibitors, temsirolimus and INK-128. Suppression of RGS10 leads to an increase in cell proliferation, even in the presence of etoposide. In summary, the RGS10 suppression increases Rheb-GTP and mTOR signaling in ovarian cancer cells. Our results suggest that RGS10 could serve in a novel, and previously unknown, role by accelerating the hydrolysis of GTP from Rheb in ovarian cancer cells.
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cellular deficiency in the RGS10 protein facilitates chemoresistant ovarian cancer
Future Medicinal Chemistry, 2015Co-Authors: Shelley B Hooks, Mandi M. MurphAbstract:More than 30 regulators of G protein signaling (RGS) proteins encompass the RGS protein superfamily of critical regulators essential to cellular homeostasis. There is enormous structural and functional diversity among the RGS superfamily, and as such they serve a wide range of functions in regulating cell biology and physiology. Recent evidence has suggested roles for multiple RGS proteins in cancer initiation and progression, which has prompted research toward the potential modulation of these proteins as a new approach in cancer therapy. This article will discuss basic RGS molecular pharmacology, summarize the cellular functions and epigenetic regulation of RGS10, review ovarian cancer chemotherapy and describe the role of RGS10 in ovarian cancer survival signaling.
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transcriptional suppression dna methylation and histone deacetylation of the regulator of g protein signaling 10 RGS10 gene in ovarian cancer cells
PLOS ONE, 2013Co-Authors: Mourad Wagdy Ali, Mandi M. Murph, Ercan Cacan, Susanna F Greer, Yuying Liu, Jennifer Young Pierce, William T Creasman, Rajgopal Govindarajan, Scott T Eblen, Shelley B HooksAbstract:RGS10 regulates ovarian cancer cell growth and survival, and RGS10 expression is suppressed in cell models of ovarian cancer chemoresistance. However, the mechanisms governing RGS10 expression in ovarian cancer are poorly understood. Here we report RGS10 suppression in primary ovarian cancer and CAOV-3 ovarian cancer cells compared to immortalized ovarian surface epithelial (IOSE) cells, and in A2780-AD chemoresistant cells compared to parental A2780 cells. RGS10-1 and RGS10-2 transcripts are expressed in ovarian cancer cells, but only RGS10-1 is suppressed in A2780-AD and CAOV-3 cells, and the RGS10-1 promoter is uniquely enriched in CpG dinucleotides. Pharmacological inhibition of DNA methyl-transferases (DNMTs) increased RGS10 expression, suggesting potential regulation by DNA methylation. Bisulfite sequencing analysis identified a region of the RGS10-1 promoter with significantly enhanced DNA methylation in chemoresistant A2780-AD cells relative to parental A2780 cells. DNA methylation in CAOV-3 and IOSE cells was similar to A2780 cells. More marked differences were observed in histone acetylation of the RGS10-1 promoter. Acetylated histone H3 associated with the RGS10-1 promoter was significantly lower in A2780-AD cells compared to parental cells, with a corresponding increase in histone deacetylase (HDAC) enzyme association. Similarly, acetylated histone levels at the RGS10-1 promoter were markedly lower in CAOV-3 cells compared to IOSE cells, and HDAC1 binding was doubled in CAOV-3 cells. Finally, we show that pharmacological inhibition of DNMT or HDAC enzymes in chemoresistant A2780-AD cells increases RGS10 expression and enhances cisplatin toxicity. These data suggest that histone de-acetylation and DNA methylation correlate with RGS10 suppression and chemoresistance in ovarian cancer. Markers for loss of RGS10 expression may identify cancer cells with unique response to therapeutics.
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regulators of g protein signaling RGS10 and rgs17 regulate chemoresistance in ovarian cancer cells
Molecular Cancer, 2010Co-Authors: Shelley B Hooks, Phillip Callihan, Molly K Altman, Jillian H Hurst, Mandi M. MurphAbstract:A critical therapeutic challenge in epithelial ovarian carcinoma is the development of chemoresistance among tumor cells following exposure to first line chemotherapeutics. The molecular and genetic changes that drive the development of chemoresistance are unknown, and this lack of mechanistic insight is a major obstacle in preventing and predicting the occurrence of refractory disease. We have recently shown that Regulators of G-protein Signaling (RGS) proteins negatively regulate signaling by lysophosphatidic acid (LPA), a growth factor elevated in malignant ascites fluid that triggers oncogenic growth and survival signaling in ovarian cancer cells. The goal of this study was to determine the role of RGS protein expression in ovarian cancer chemoresistance. In this study, we find that RGS2, RGS5, RGS10 and RGS17 transcripts are expressed at significantly lower levels in cells resistant to chemotherapy compared with parental, chemo-sensitive cells in gene expression datasets of multiple models of chemoresistance. Further, exposure of SKOV-3 cells to cytotoxic chemotherapy causes acute, persistent downregulation of RGS10 and RGS17 transcript expression. Direct inhibition of RGS10 or RGS17 expression using siRNA knock-down significantly reduces chemotherapy-induced cell toxicity. The effects of cisplatin, vincristine, and docetaxel are inhibited following RGS10 and RGS17 knock-down in cell viability assays and phosphatidyl serine externalization assays in SKOV-3 cells and MDR-HeyA8 cells. We further show that AKT activation is higher following RGS10 knock-down and RGS 10 and RGS17 overexpression blocked LPA mediated activation of AKT, suggesting that RGS proteins may blunt AKT survival pathways. Taken together, our data suggest that chemotherapy exposure triggers loss of RGS10 and RGS17 expression in ovarian cancer cells, and that loss of expression contributes to the development of chemoresistance, possibly through amplification of endogenous AKT signals. Our results establish RGS10 and RGS17 as novel regulators of cell survival and chemoresistance in ovarian cancer cells and suggest that their reduced expression may be diagnostic of chemoresistance.
Malu G Tansey - One of the best experts on this subject based on the ideXlab platform.
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age related changes in regulator of g protein signaling rgs 10 expression in peripheral and central immune cells may influence the risk for age related degeneration
Neurobiology of Aging, 2015Co-Authors: George T Kannarkat, Jaekyung Lee, Jaegwon Chung, Chenere P Ramsey, Jianjun Chang, Isadora Porter, Danielle Oliver, Kennie R Shepherd, Malu G TanseyAbstract:Inflammation in the aging brain increases risk for neurodegenerative disease. In humans, the regulator of G-protein signaling-10 (RGS10) locus has been associated with age-related maculopathy. Chronic peripheral administration of lipopolysaccharide in the RGS10-null mice induces nigral dopaminergic (DA) degeneration, suggesting that RGS10 modulates neuroimmune interactions and may influence susceptibility to neurodegeneration. Because age is the strongest risk factor for neurodegenerative disease, we assessed whether RGS10 expression changes with age and whether aged RGS10-null mice have altered immune cell profiles. Loss of RGS10 in aged mice does not alter the regulation of nigral DA neurons but does alter B-cell, monocyte, microglial, and CD4+ T-cell populations and inflammatory cytokine levels in the cerebrospinal fluid. These results suggest that loss of RGS10 is associated with an age-dependent dysregulation of peripheral and central immune cells rather than dysregulation of DA neuron function.
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physiology of RGS10 in neurons and immune cells
Progress in Molecular Biology and Translational Science, 2015Co-Authors: Jaekyung Lee, Malu G TanseyAbstract:Regulator of G protein signaling-10 (RGS10), a GTPase-activating protein (GAP) for Gαi3, Gαq, and Gαz, belongs to the D/R12 subfamily based on the homology within the RGS domain and is one of the smallest RGS proteins, outside the RGS box. Although RGS10 lacks the flanking domains or motifs, RGS10 can be targeted to membrane by palmitoylation which markedly increases its net GAP activity. The cAMP-dependent protein kinase A phosphorylates RGS10 on serine 168 (Ser(168)) which promotes translocation of RGS10 from the cytosol to nucleus. In addition to its GAP function, RGS10 modulates adenylyl cyclase (AC) activity as well as the G protein-gated inwardly rectifying potassium channels. Although the roles of various RGS proteins have been well addressed in previous reviews, the function and mechanism of RGS10 in particular has not been reviewed in-depth. Moreover, recent arrays of studies implicate a role for RGS10 in immune and neuronal cells. RGS10 has been localized to various cell compartments including the cytoplasm and nucleus. In this chapter, we will review its role in neurons and immune cells.
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a novel function of RGS10 in effector t lymphocytes to augment mouse eae ba8p 126
Journal of Immunology, 2014Co-Authors: Jaekyung Lee, Jaegwon Chung, George T Kannarkat, Kareem L Graham, Malu G TanseyAbstract:A broad range of immune-based therapeutic drugs have been available for treatment of various autoimmune diseases but many have had limited success. Therefore, it is critical to identify additional cellular targets that can regulate the pathogenic response of immune cells. We hypothesize that GPCR modulator RGS proteins are important modulators of the immune responses involved in the development of multiple sclerosis (MS). Our hypothesis is based on reports that various SNPs in RGS proteins is highly correlated with the diagnosis of MS. Especially, an in-depth search of the GEO profiles database also revealed higher levels of RGS10 transcripts in MS patients’ PBMCs. Here, we showed RGS10-null mice displayed significantly milder clinical symptoms of EAE with reduced incidence and delayed disease onset. We observed that there were less numbers of CD45+ cells and CD4+ T cells in MOG35-55-immunized RGS10-null mice in the CNS. RGS10-null LN T cells were less proliferatative and produce less IFN-γ and IL-17 cytokines in response to MOG. Our data suggest a critical role for RGS10 in modulating disease by attenuating lymphocyte infiltration and/or peripheral immune responses during EAE. This is the first study ever conducted to elucidate the function of RGS10 in effector lymphocytes in the context of EAE. The identification of RGS10 as a central regulator of the inflammatory processes will open a possibility for developing more specific targeted therapy for the treatment of MS.
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critical role of regulator g protein signaling 10 RGS10 in modulating macrophage m1 m2 activation
PLOS ONE, 2013Co-Authors: Jaekyung Lee, Jaegwon Chung, George T Kannarkat, Malu G TanseyAbstract:Regulator of G protein signaling 10 (RGS10), a GTPase accelerating protein (GAP) for G alpha subunits, is a negative regulator of NF-κB in microglia. Here, we investigated the role of RGS10 in macrophages, a closely related myeloid-derived cell type. Features of classical versus alternative activation were assessed in RGS10-/- peritoneal and bone marrow-derived macrophages upon LPS or IL-4 treatments, respectively. Our results showed that RGS10-/- macrophages produced higher levels of pro-inflammatory cytokines including TNF, IL-1β and IL-12p70 in response to LPS treatment and exerted higher cytotoxicity on dopaminergic MN9D neuroblastoma cells. We also found that RGS10-/- macrophages displayed a blunted M2 phenotype upon IL-4 priming. Specifically, RGS10-/- macrophages displayed lower YM1 and Fizz1 mRNA levels as measured by QPCR compared to wild type macrophages upon IL-4 treatment and this response was not attributable to differences in IL-4 receptor expression. Importantly, phagocytic activities of RGS10-/- macrophages were blunted in response to IL-4 priming and/or LPS treatments. However, there was no difference in chemotaxis between RGS10-/- and WT macrophages. Our data indicate that RGS10-/- macrophages displayed dysregulated M1 responses along with blunted M2 alternative activation responses, suggesting that RGS10 plays an important role in determining macrophage activation responses.
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RGS10 exerts a neuroprotective role through the pka c amp response element creb pathway in dopaminergic neuron like cells
Journal of Neurochemistry, 2012Co-Authors: Jaekyung Lee, Kirk M Druey, Jaegwon Chung, Malu G TanseyAbstract:Regulator of G-protein signaling-10 (RGS10) is a GTPase activating protein for Gαi/q/z subunits that is highly expressed in the immune system and in a broad range of brain regions including the hippocampus, striatum, dorsal raphe, and ventral midbrain. Previously, we reported that RGS10-null mice display increased vulnerability to chronic systemic inflammation-induced degeneration of nigral dopaminergic (DA) neurons. Given that RGS10 is expressed in DA neurons, we investigated the extent to which RGS10 regulates cell survival under conditions of inflammatory stress. Because of the inherent limitations associated with use of primary DA neurons for biochemical analyses, we employed a well-characterized ventral mesencephalon DA neuroblastoma cell line (MN9D) for our studies. We found that stable over-expression of RGS10 rendered them resistant to TNF-induced cytotoxicity; whereas MN9D cells expressing mutant RGS10-S168A (which is resistant to phosphorylation by protein kinase A at a serine residue that promotes its nuclear translocation) showed similar sensitivity to TNF as the parental MN9D cells. Using biochemical and pharmacologic approaches, we identified protein kinase A and the downstream phospho-cAMP response element-binding signaling pathway (and ruled out ERK 1/2, JNK, and NFkB) as key mediators of the neuroprotective effect of RGS10 against inflammatory stress.