ORCID Profile
0000-0001-9354-5162
Current Organisation
University of Queensland
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Biochemistry and Cell Biology | Receptors and Membrane Biology | Signal Transduction
Publisher: Elsevier BV
Date: 08-2015
DOI: 10.1016/J.CELLSIG.2015.04.007
Abstract: Much is known about the how Gβγ subunits regulate effectors in response to G protein-coupled receptor stimulation. However, there is still a lot we don't know about how specific combinations of Gβ and Gγ are wired into different signalling pathways. Here, using an siRNA screen for different Gβ and Gγ subunits, we examined an endogenous M3 muscarinic receptor signalling pathway in HEK 293 cells. We observed that Gβ(4) subunits were critical for calcium signalling and a downstream surrogate measured as ERK1/2 MAP kinase activity. A number of Gγ subunits could partner with Gβ(4) but the best coupling was seen via Gβ(4)γ(1). Intriguingly, knocking down Gβ(1) actually increased signalling through the M3-mAChR most likely via an increase in Gβ(4) levels. We noted that Gβ(1) occupies the promoter of Gβ(4) and may participate in maturation of its mRNA. This highlights a new role for Gβγ signalling beyond their canonical roles in cellular signalling.
Publisher: MDPI AG
Date: 30-09-2022
Abstract: Although breast cancer cells often exhibit both abnormal AKT signaling and calcium signaling, the association between these two pathways is unclear. Using a combination of pharmacological tools, siRNA and CRISPR/Cas9 gene silencing techniques, we investigated the association between PTEN, AKT phosphorylation and calcium signaling in a basal breast cancer cell line. We found that siRNA-mediated PTEN silencing promotes AKT phosphorylation and calcium influx in MDA-MB-231 cells. This increase in AKT phosphorylation and calcium influx was phenocopied by the pharmacological AKT activator, SC79. The increased calcium influx associated with SC79 is inhibited by silencing AKT2, but not AKT1. This increase in calcium influx is suppressed when the store-operated calcium channel, ORAI1 is silenced. The results from this study open a novel avenue for therapeutic targeting of cancer cells with increased AKT activation. Given the association between ORAI1 and breast cancer, ORAI1 is a possible therapeutic target in cancers with abnormal AKT signaling.
Publisher: The Endocrine Society
Date: 08-2013
DOI: 10.1210/ME.2013-1091
Publisher: Oxford University Press (OUP)
Date: 30-01-2019
DOI: 10.1093/NAR/GKZ010
Publisher: Springer Science and Business Media LLC
Date: 21-11-2016
DOI: 10.1038/NM.4219
Abstract: Forward genetic screens with CRISPR-Cas9 genome editing enable high-resolution detection of genetic vulnerabilities in cancer cells. We conducted genome-wide CRISPR-Cas9 screens in RNF43-mutant pancreatic ductal adenocarcinoma (PDAC) cells, which rely on Wnt signaling for proliferation. Through these screens, we discovered a unique requirement for a Wnt signaling circuit: engaging FZD5, one of the ten Frizzled receptors encoded in the human genome. Our results uncover an underappreciated level of context-dependent specificity at the Wnt receptor level. We further derived a panel of recombinant antibodies that reports the expression of nine FZD proteins and confirms that FZD5 functional specificity cannot be explained by protein expression patterns. Additionally, antibodies that specifically bind FZD5 and FZD8 robustly inhibited the growth of RNF43-mutant PDAC cells grown in vitro and as xenografts in vivo, providing orthogonal support for the functional specificity observed genetically. Proliferation of a patient-derived PDAC cell line harboring an RNF43 variant was also selectively inhibited by the FZD5 antibodies, further demonstrating their use as a potential targeted therapy. Tumor organoid cultures from colorectal carcinoma patients that carried RNF43 mutations were also sensitive to the FZD5 antibodies, highlighting the potential generalizability of these findings beyond PDAC. Our results show that CRIPSR-based genetic screens can be leveraged to identify and validate cell surface targets for antibody development and therapy.
Publisher: Humana Press
Date: 2011
DOI: 10.1007/978-1-61779-160-4_13
Abstract: Advances in imaging assays based on resonance energy transfer (RET) have made it possible to study protein rotein interactions in living cells under physiological conditions. It is now possible to measure the kinetics of changes in these interactions in response to ligand stimulation in real time. Here we describe protocols for these assays focusing on the basal and ligand-stimulated interaction between tagged Gβγ subunits and adenylyl cyclase II. We describe relevant positive and negative controls and various experimental considerations for optimization of these experiments.
Publisher: Elsevier BV
Date: 08-2010
DOI: 10.1016/J.CELLSIG.2010.04.005
Abstract: Following stimulation of G protein-coupled receptors (GPCRs) at the cell surface, heterotrimeric G proteins are activated. Both Galpha and Gbetagamma subunits regulate specific effectors to transmit signals received by the receptor. Recent data suggest potential nuclear localization or translocation of the Gbetagamma subunit. Here, we show that co-expression of the Gbetagamma dimer decreased phorbol 12-myristate 13-acetate (PMA)-stimulated AP-1 gene reporter activity in HEK293 cells as well as the AP-1 dependent gonadotropin-releasing hormone-stimulated human follicle-stimulating hormone beta reporter activity in LbetaT2 gonadotrope cells. Further, we identify Fos transcription factors as novel interactors of the Gbeta1 subunit, using protein fragment complementation assays, as well as co-immunoprecipitation in vivo and in vitro. Fos proteins dimerize with Jun proteins to form activator protein-1 (AP-1) transcription factor complexes, which regulate target gene expression. Gbetagamma did not interfere with the dimerization of Fos and Jun or their ability to bind DNA. Rather, Gbetagamma co-localized with the AP-1 complex in the nucleus and recruited histone deacetylases (HDACs) to inhibit AP-1 transcriptional activity. Our data indicate a novel role for Gbetagamma subunits as transcriptional regulators.
Publisher: Informa UK Limited
Date: 25-09-2018
Publisher: Humana Press
Date: 2009
DOI: 10.1007/978-1-60327-321-3_18
Abstract: Recent advances in imaging assays based on bioluminescence resonance energy transfer (BRET) have made it possible to study protein rotein interactions in living cells under physiological conditions. Here we describe protocols for these assays including relevant positive and negative controls, and we also show how they can be combined with protein complementation assays such as bimolecular fluorescence complementation (BiFC) to study three- and four-partner interactions. We also describe a BRET assay that uses SNAP-tagged proteins as a fluorescence acceptor molecule for the bioluminescent donor.
Publisher: MDPI AG
Date: 16-06-2021
DOI: 10.3390/BIOMEDICINES9060680
Abstract: Cancer-associated fibroblasts (CAFs) represent an important component of the tumour microenvironment and are implicated in disease progression. Two outstanding questions in cancer biology are how CAFs arise and how they might be targeted therapeutically. The calcium signal also has an important role in tumorigenesis. To date, the role of calcium signalling pathways in the induction of the CAF phenotype remains unexplored. A CAF model was generated through exogenous transforming growth factor beta 1 (TGFβ1) stimulation of the normal human mammary fibroblast cell line, HMF3S (HMF3S-CAF), and changes in calcium signalling were investigated. Functional changes in HMF3S-CAF calcium signalling pathways were assessed using a fluorescent indicator, gene expression, gene-silencing and pharmacological approaches. HMF3S-CAF cells demonstrated functionally altered calcium influx pathways with reduced store-operated calcium entry. In support of a calcium signalling switch, two voltage-gated calcium channel (VGCC) family members, CaV1.2 and CaV3.2, were upregulated in HMF3S-CAFs and a subset of patient-derived breast CAFs. Both siRNA-mediated silencing and pharmacological inhibition of CaV1.2 or CaV3.2 significantly impaired CAF activation in HMF3S cells. Our findings show that VGCCs contribute to TGFβ1-mediated induction of HMF3S-CAF cells and both transcriptional interference and pharmacological antagonism of CaV1.2 and CaV3.2 inhibit CAF induction. This suggests a potential therapeutic role for targeting calcium signalling in breast CAFs.
Publisher: Elsevier BV
Date: 05-2022
Publisher: Springer New York
Date: 20-09-2014
DOI: 10.1007/978-1-4939-1755-6_14
Abstract: It has become clear in recent years that the Gβγ subunits of heterotrimeric proteins serve broad roles in the regulation of cellular activity and interact with many proteins in different subcellular locations including the nucleus. Protein affinity purification is a common method to identify and confirm protein interactions. When used in conjugation with mass spectrometry it can be used to identify novel protein interactions with a given bait protein. The tandem affinity purification (TAP) technique identifies partner proteins bound to tagged protein bait. Combined with protocols to enrich the nuclear fraction of whole cell lysate through sucrose cushions, TAP allows for purification of interacting proteins found specifically in the nucleus. Here we describe the use of the TAP technique on cytosolic and nuclear lysates to identify candidate proteins, through mass spectrometry, that bind to Gβ1 subunits.
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 08-04-2009
Publisher: Elsevier BV
Date: 2023
Publisher: MDPI AG
Date: 11-02-2019
Abstract: The remodeling of specific calcium-permeable ion channels is a feature of some breast cancer subtypes. ORAI1 is a protein that forms a calcium-permeable ion channel responsible for store-operated calcium entry (SOCE) in a variety of cell types. ORAI3, a related isoform, is not a regulator of SOCE in most cell types. However, ORAI3 does control SOCE in many estrogen receptor-positive breast cancer cell lines, where it also controls proliferation. ORAI1 is a well-characterized regulator of the proliferation and migration of many basal breast cancer cells however, the role of ORAI3 in these types of breast cancer cells remains unclear. Here, we sought to define ORAI1 and ORAI3 expression in breast cancer cell lines of different molecular subtypes and assess the potential role and regulation of ORAI3 in basal breast cancer cells. Our study demonstrates that elevated ORAI1 is a feature of basal-like breast cancers, while elevated ORAI3 is a feature of luminal breast cancers. Intriguingly, we found that ORAI3 is over-expressed in the mesenchymal subtype of triple-negative breast cancer. Given this, we assessed ORAI3 levels in the presence of two inducers of the mesenchymal phenotype, hypoxia and epidermal growth factor (EGF). Hypoxia induced ORAI3 levels in basal breast cancer cell lines through a pathway involving hypoxia-inducible factor-1 alpha (HIF1α. The silencing of ORAI3 attenuated hypoxia-associated phosphorylation of the EGF receptor (EGFR) and the expression of genes associated with cell migration and inflammatory/immune responses in the MDA-MB-468 model of basal breast cancer. Although elevated ORAI3 levels were not associated with survival basal, estrogen receptor-negative and triple-negative breast cancers with high ORAI3 and low ORAI1 levels were associated with poorer clinical outcomes. This study defines ORAI3 as a potential fine-tuner for processes relevant to the progression of basal breast cancers.
Publisher: Wiley
Date: 11-11-2020
Publisher: Elsevier BV
Date: 06-2022
DOI: 10.1016/J.CECA.2022.102569
Abstract: Both matrix stiffening and remodeling of calcium signaling occur in breast cancers, with downstream consequences linked to the progression of the disease. However, the potential intersection between calcium signaling and matrix stiffness has not been fully assessed in models of cancer. Here, we describe the assessment of calcium signaling in breast cancer cells at high and low matrix stiffness using novel gel culture models (gelatin methacryloyl and polydimethylsiloxane) and MDA-MB-231 breast cancer cells expressing the calcium sensor GCaMP6m. Remodeling of ATP-stimulated cytosolic calcium responses in cells on different matrices was assessed using a high throughput fluorescence imaging plate reader. Our data reveal that matrices of higher stiffness attenuate ATP-induced sustained calcium influx in MDA-MB-231 breast cancer cells. This matrix-mediated attenuation of sustained calcium influx was dependent on the store-operated calcium channel component ORAI1. These studies suggest that calcium signaling in breast cancer cells can be altered as a consequence of matrix stiffness modulation of such pathways may represent a new mechanism to target calcium signaling to regulate tumor progression in breast cancer.
Publisher: Springer Science and Business Media LLC
Date: 04-01-2016
DOI: 10.1038/ONC.2015.491
Publisher: Elsevier BV
Date: 10-2023
Publisher: Elsevier BV
Date: 06-2018
DOI: 10.1016/J.CECA.2018.02.003
Abstract: Alterations in Ca
Publisher: Elsevier BV
Date: 11-2014
Publisher: The Company of Biologists
Date: 2013
DOI: 10.1242/JCS.132944
Abstract: We and others have shown that trafficking of G protein-coupled receptors is regulated by Rab GTPases. Cargo-mediated regulation of vesicular transport has received great attention lately. Rab GTPases, forming the largest branch of the Ras GTPase superfamily, regulate almost every step of vesicle-mediated trafficking. Rab GTPases are well-recognized targets of human diseases but their regulation and the mechanisms connecting them to cargo proteins are still poorly understood. Herein, we show by overexpression/depletion studies that HACE1, a HECT domain-containing ubiquitin ligase, promotes the recycling of the β2-adrenergic receptor (β2AR), a prototypical G protein-coupled receptor, through a Rab11a-dependent mechanism. Interestingly, the β2AR in conjunction with HACE1 triggered ubiquitination of Rab11a, as observed by Western blot analysis. LC-MS/MS experiments determined that Rab11a is ubiquitnatied on Lys145. A Rab11a-K145R mutant failed to undergo β2AR/HACE1-induced ubiquitination and inhibited the HACE1-mediated recycling of the β2AR. Rab11a, but not Rab11a-K145R, was activated by β2AR/HACE1 indicating that ubiquitination of Lys145 is involved in Rab11a activation. β2AR/HACE1 co-expression also potentiated ubiquitination of Rab6a and Rab8a, but not of other Rab GTPases that were tested. We report a novel regulatory mechanism of Rab GTPases by their ubiquitination with associated functional effects demonstrated on Rab11a. This partakes into a new pathway whereby a cargo protein, like a G protein-coupled receptor, can regulate its own trafficking by inducing the ubiquitination and activation of a Rab GTPase.
Publisher: Elsevier BV
Date: 07-2008
DOI: 10.1016/J.YMETH.2008.06.006
Abstract: A variety of fluorescent proteins with different spectral properties have been created by mutating green fluorescent protein. When these proteins are split in two, neither fragment is fluorescent per se, nor can a fluorescent protein be reconstituted by co-expressing the complementary N- and C-terminal fragments. However, when these fragments are genetically fused to proteins that associate with each other in cellulo, the N- and C-terminal fragments of the fluorescent protein are brought together and can reconstitute a fluorescent protein. A similar protein complementation assay (PCA) can be performed with two complementary fragments of various luciferase isoforms. This makes these assays useful tools for detecting the association of two proteins in living cells. Bioluminescence resonance energy transfer (BRET) or fluorescence resonance energy transfer (FRET) occurs when energy from, respectively, a luminescent or fluorescent donor protein is non-radiatively transferred to a fluorescent acceptor protein. This transfer of energy can only occur if the proteins are within 100A of each other. Thus, BRET and FRET are also useful tools for detecting the association of two proteins in living cells. By combining different protein fragment complementation assays (PCA) with BRET or FRET it is possible to demonstrate that three or more proteins are simultaneous parts of the same protein complex in living cells. As an ex le of the utility of this approach, we show that as many as four different proteins are simultaneously associated as part of a G protein-coupled receptor signalling complex.
Publisher: Wiley
Date: 08-04-2009
Publisher: Elsevier BV
Date: 05-2007
Publisher: Elsevier BV
Date: 11-2006
Publisher: Wiley
Date: 23-12-2022
Publisher: EDP Sciences
Date: 10-2009
DOI: 10.1051/MEDSCI/20092510821
Abstract: Extracellular signals received by G protein-coupled receptors (GPCRs) are transduced into intracellular responses following the activation of heterotrimeric G proteins. As their names suggests, they are composed of three subunits, Galpha and Gbetagamma, the latter being effectively treated as a single entity. The Gbetagamma dimer is assembled with the aid of a number of molecular chaperones in a tightly regulated process. The folding of nascent Gbeta1 is favoured by cellular chaperones such as PhLP-1 and CCT and the ER-resident protein DRiP78 plays an important role in the stability of nascent Ggamma2 subunits. However, much work remains to be done to completely understand the mechanisms underlying assembly of the heterotrimer.
Publisher: Elsevier BV
Date: 04-2008
DOI: 10.1016/J.CELLSIG.2008.11.011
Abstract: We have previously demonstrated that Kir3.1 channels and Gbeta1gamma2 subunits initially interact in the endoplasmic reticulum (ER). To elucidate the role that anterograde protein trafficking pathways may play in the formation of these complexes, we used dominant negative (DN) mutants of the small G proteins Sar 1 and various compartment-specific Rabs which impede anterograde protein trafficking at different steps. Sar 1 H79G and Rab 1 S25N mutants efficiently blocked the plasma membrane trafficking of the Kir3.1/Kir3.4 complex however they did not block the Gbeta1gamma2/Kir3.1 interaction as measured using bioluminescence resonance energy transfer (BRET). This interaction was also insensitive to the presence of DN Rabs 2, 6, 8, and 11. These results confirm that Gbetagamma/Kir3 complexes form early during channel biosynthesis and trafficking. Using a combination of BRET, protein complementation assays and co-immunoprecipitation, we demonstrate that Gbeta1-4 can interact with Kir3.1 in the absence of Kir3.4. Gbeta5 does not directly interact with the channel but can still be co-immunoprecipitated as part of a larger complex. The interaction between Gbeta and Kir3.1 was selectively fostered by co-expression with different Ggamma subunits. When Ggamma1 or Ggamma11 was co-expressed with eGFP-Gbeta3 or eGFP-Gbeta4, the interaction with the effector was lost. Kir3.2 was capable of interacting with Gbeta1-3 and not Gbeta4 or Gbeta5. These interactions were again fostered by co-expression with Ggamma and were also insensitive to DN Sar 1 or Rab 1. Taken together, our data show that these "precocious" channel/G protein interactions are specific and may have implications beyond their basic function in signalling events.
Publisher: Annual Reviews
Date: 02-2009
DOI: 10.1146/ANNUREV-PHARMTOX-061008-103038
Abstract: The role of Gβγ subunits in cellular signaling has become well established in the past 20 years. Not only do they regulate effectors once thought to be the sole targets of Gα subunits, but it has become clear that they also have a unique set of binding partners and regulate signaling pathways that are not always localized to the plasma membrane. However, this may be only the beginning of the story. Gβγ subunits interact with G protein–coupled receptors, Gα subunits, and several different effector molecules during assembly and trafficking of receptor-based signaling complexes and not simply in response to ligand stimulation at sites of receptor cellular activity. Gβγ assembly itself seems to be tightly regulated via the action of molecular chaperones and in turn may serve a similar role in the assembly of specific signaling complexes. We propose that specific Gβγ subunits have a broader role in controlling the architecture, assembly, and activity of cellular signaling pathways.
Publisher: Elsevier BV
Date: 2016
DOI: 10.1016/J.DEVCEL.2015.12.016
Abstract: Control of cell- ision orientation is integral to epithelial morphogenesis and asymmetric cell ision. Proper spatiotemporal localization of the evolutionarily conserved Gαi-LGN-NuMA protein complex is critical for mitotic spindle orientation, but how this is achieved remains unclear. Here we identify Suppressor APC domain containing 2 (SAPCD2) as a previously unreported LGN-interacting protein. We show that SAPCD2 is essential to instruct planar mitotic spindle orientation in both epithelial cell cultures and mouse retinal progenitor cells in vivo. Loss of SAPCD2 randomizes spindle orientation, which in turn disrupts cyst morphogenesis in three-dimensional cultures, and triples the number of terminal asymmetric cell isions in the developing retina. Mechanistically, we show that SAPCD2 negatively regulates the localization of LGN at the cell cortex, likely by competing with NuMA for its binding. These results uncover SAPCD2 as a key regulator of the ternary complex controlling spindle orientation during morphogenesis and asymmetric cell isions.
Publisher: Public Library of Science (PLoS)
Date: 15-06-2015
Publisher: Elsevier BV
Date: 07-2015
Publisher: MDPI AG
Date: 10-12-2020
DOI: 10.3390/IJMS21249417
Abstract: Epithelial to mesenchymal transition (EMT) in cancer is important in therapeutic resistance and invasiveness. Calcium signaling is key to the induction of EMT in breast cancer cells. Although inhibition of specific calcium-permeable ion channels regulates the induction of a sub-set of EMT markers in breast cancer cells, it is still unclear if activation of a specific calcium channel can be a driver for the induction of EMT events. In this study, we exploited the availability of a selective pharmacological activator of the calcium-permeable ion channel TRPV4 to assess the direct role of calcium influx in EMT marker induction. Gene association studies revealed a link between TRPV4 and gene-ontologies associated with EMT and poorer relapse-free survival in lymph node-positive basal breast cancers. TRPV4 was an important component of the calcium influx phase induced in MDA-MB-468 breast cancer cells by the EMT inducer epidermal growth factor (EGF). Pharmacological activation of TRPV4 then drove the induction of a variety of EMT markers in breast cancer cells. These studies demonstrate that calcium influx through specific pathways appears to be sufficient to trigger EMT events.
Publisher: The Company of Biologists
Date: 07-2006
DOI: 10.1242/JCS.03021
Abstract: Bioluminescence resonance energy transfer (BRET) and co-immunoprecipitation experiments revealed that heterotrimeric G proteins and their effectors were found in stable complexes that persisted during signal transduction. Adenylyl cyclase, Kir3.1 channel subunits and several G-protein subunits (Gαs, Gαi, Gβ1 and Gγ2) were tagged with luciferase (RLuc) or GFP, or the complementary fragments of YFP (specifically Gβ1-YFP1-158 and Gγ2-YFP159-238, which heterodimerize to produce fluorescent YFP-Gβ1γ2). BRET was observed between adenylyl-cyclase-RLuc or Kir3.1-RLuc and GFP-Gγ2, GFP-Gβ1 or YFP-Gβ1γ2. Gα subunits were also stably associated with both effectors regardless of whether or not signal transduction was initiated by a receptor agonist. Although BRET between effectors and Gβγ was increased by receptor stimulation, our data indicate that these changes are likely to be conformational in nature. Furthermore, receptor-sensitive G-protein-effector complexes could be detected before being transported to the plasma membrane, providing the first direct evidence for an intracellular site of assembly.
Start Date: 02-2022
End Date: 01-2025
Amount: $549,740.00
Funder: Australian Research Council
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