ORCID Profile
0000-0003-4377-1836
Current Organisation
University of New South Wales - Randwick Campus
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Biochemistry and Cell Biology | Signal Transduction | Cell Metabolism
Expanding Knowledge in the Biological Sciences | Expanding Knowledge in the Medical and Health Sciences |
Publisher: Elsevier BV
Date: 06-2013
Publisher: American Chemical Society (ACS)
Date: 30-03-2012
DOI: 10.1021/PR300072J
Abstract: A key step in the analysis of mass spectrometry (MS)-based proteomics data is the inference of proteins from identified peptide sequences. Here we describe Re-Fraction, a novel machine learning algorithm that enhances deterministic protein identification. Re-Fraction utilizes several protein physical properties to assign proteins to expected protein fractions that comprise large-scale MS-based proteomics data. This information is then used to appropriately assign peptides to specific proteins. This approach is sensitive, highly specific, and computationally efficient. We provide algorithms and source code for the current version of Re-Fraction, which accepts output tables from the MaxQuant environment. Nevertheless, the principles behind Re-Fraction can be applied to other protein identification pipelines where data are generated from s les fractionated at the protein level. We demonstrate the utility of this approach through reanalysis of data from a previously published study and generate lists of proteins deterministically identified by Re-Fraction that were previously only identified as members of a protein group. We find that this approach is particularly useful in resolving protein groups composed of splice variants and homologues, which are frequently expressed in a cell- or tissue-specific manner and may have important biological consequences.
Publisher: Wiley
Date: 19-10-2006
Publisher: Impact Journals, LLC
Date: 02-2017
Publisher: Public Library of Science (PLoS)
Date: 16-09-2021
DOI: 10.1371/JOURNAL.PCBI.1008513
Abstract: The PI3K/MTOR signalling network regulates a broad array of critical cellular processes, including cell growth, metabolism and autophagy. The mechanistic target of rapamycin (MTOR) kinase functions as a core catalytic subunit in two physically and functionally distinct complexes mTORC1 and mTORC2, which also share other common components including MLST8 (also known as GβL) and DEPTOR. Despite intensive research, how mTORC1 and 2 assembly and activity are coordinated, and how they are functionally linked remain to be fully characterized. This is due in part to the complex network wiring, featuring multiple feedback loops and intricate post-translational modifications. Here, we integrate predictive network modelling, in vitro experiments and -omics data analysis to elucidate the emergent dynamic behaviour of the PI3K/MTOR network. We construct new mechanistic models that encapsulate critical mechanistic details, including mTORC1/2 coordination by MLST8 (de)ubiquitination and the Akt-to-mTORC2 positive feedback loop. Model simulations validated by experimental studies revealed a previously unknown biphasic, threshold-gated dependence of mTORC1 activity on the key mTORC2 subunit SIN1, which is robust against cell-to-cell variation in protein expression. In addition, our integrative analysis demonstrates that ubiquitination of MLST8, which is reversed by OTUD7B, is regulated by IRS1/2. Our results further support the essential role of MLST8 in enabling both mTORC1 and 2’s activity and suggest MLST8 as a viable therapeutic target in breast cancer. Overall, our study reports a new mechanistic model of PI3K/MTOR signalling incorporating MLST8-mediated mTORC1/2 formation and unveils a novel regulatory linkage between mTORC1 and mTORC2.
Publisher: Springer Science and Business Media LLC
Date: 02-12-2021
DOI: 10.1038/S41587-021-01099-9
Abstract: Protein phosphorylation dynamically integrates environmental and cellular information to control biological processes. Identifying functional phosphorylation amongst the thousands of phosphosites regulated by a perturbation at a global scale is a major challenge. Here we introduce 'personalized phosphoproteomics', a combination of experimental and computational analyses to link signaling with biological function by utilizing human phenotypic variance. We measure in idual subject phosphoproteome responses to interventions with corresponding phenotypes measured in parallel. Applying this approach to investigate how exercise potentiates insulin signaling in human skeletal muscle, we identify both known and previously unidentified phosphosites on proteins involved in glucose metabolism. This includes a cooperative relationship between mTOR and AMPK whereby the former directly phosphorylates the latter on S377, for which we find a role in metabolic regulation. These results establish personalized phosphoproteomics as a general approach for investigating the signal transduction underlying complex biology.
Publisher: American Chemical Society (ACS)
Date: 22-05-2018
DOI: 10.1021/ACS.BIOCHEM.8B00361
Abstract: Trafficking regulator of GLUT4 1 (TRARG1) was recently identified to localize to glucose transporter type 4 (GLUT4) storage vesicles (GSVs) and to positively regulate GLUT4 trafficking. Our knowledge of TRARG1 structure and membrane topology is limited to predictive models, h ering efforts to further our mechanistic understanding of how it carries out its functions. Here, we use a combination of bioinformatics prediction tools and biochemical assays to define the membrane topology of the 173-amino acid mouse TRARG1. These analyses revealed that, contrary to the consensus prediction, the N-terminus is cytosolic and that a short segment at the C-terminus resides in the luminal/extracellular space. Based on our biochemical analyses including membrane association and antibody accessibility assays, we conclude that TRARG1 has one transmembrane domain (TMD) (145-172) and a re-entrant loop between residues 101 and 127.
Publisher: EMBO
Date: 14-12-2018
Publisher: Proceedings of the National Academy of Sciences
Date: 06-05-2022
Abstract: Both the mTORC2 and Ras-ERK pathways respond to growth factor stimulation and play critical roles in cell growth and proliferation, disarray of these pathways leads to many diseases, especially cancer. These two signaling pathways crosstalk at many levels recently it's become clear that the SIN1 component of mTORC2 could interact with Ras family small GTPases, but how these two proteins interact at the molecular level and the functional outcomes of this interaction remain to be addressed. In this work we determined the high-resolution structure of Ras-SIN1 complexes and revealed the detailed interaction mechanism. We also showed that Ras-SIN1 association inhibits insulin-induced ERK activation. Insights from this work could improve our understanding of the disease-causing mechanism of errant mTORC2 or Ras proteins.
Publisher: Wiley
Date: 27-10-2009
Abstract: C-terminal Src kinase (Csk) that functions as an essential negative regulator of Src family tyrosine kinases (SFKs) interacts with tyrosine-phosphorylated molecules through its Src homology 2 (SH2) domain, allowing it targeting to the sites of SFKs and concomitantly enhancing its kinase activity. Identification of additional Csk-interacting proteins is expected to reveal potential signaling targets and previously undescribed functions of Csk. In this study, using a direct proteomic approach, we identified 151 novel potential Csk-binding partners, which are associated with a wide range of biological functions. Bioinformatics analysis showed that the majority of identified proteins contain one or several Csk-SH2 domain-binding motifs, indicating a potentially direct interaction with Csk. The interactions of Csk with four proteins (partitioning defective 3 (Par3), DDR1, SYK and protein kinase C iota) were confirmed using biochemical approaches and phosphotyrosine 1127 of Par3 C-terminus was proved to directly bind to Csk-SH2 domain, which was consistent with predictions from in silico analysis. Finally, immunofluorescence experiments revealed co-localization of Csk with Par3 in tight junction (TJ) in a tyrosine phosphorylation-dependent manner and overexpression of Csk, but not its SH2-domain mutant lacking binding to phosphotyrosine, promoted the TJ assembly in Madin-Darby canine kidney cells, implying the involvement of Csk-SH2 domain in regulating cellular TJs. In conclusion, the newly identified potential interacting partners of Csk provided new insights into its functional ersity in regulation of numerous cellular events, in addition to controlling the SFK activity.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 21-09-2021
DOI: 10.1126/SCISIGNAL.ABE0161
Abstract: RapaLink1 enables differentiation between functions mediated by the closely related mTORC1 and mTORC2.
Publisher: Springer Science and Business Media LLC
Date: 02-2007
Abstract: The partitioning-defective 3 (Par3), a key component in the conserved Par3/Par6/aPKC complex, plays fundamental roles in cell polarity. Herein we report the identification of Ku70 and Ku80 as novel Par3-interacting proteins through an in vitro binding assay followed by liquid chromatography-tandem mass spectrometry. Ku70/Ku80 proteins are two key regulatory subunits of the DNA-dependent protein kinase (DNA-PK), which plays an essential role in repairing double-strand DNA breaks (DSBs). We determined that the nuclear association of Par3 with Ku70/Ku80 was enhanced by gamma-irradiation (IR), a potent DSB inducer. Furthermore, DNA-PKcs, the catalytic subunit of DNA-PK, interacted with the Par3/Ku70/Ku80 complex in response to IR. Par3 over-expression or knockdown was capable of up- or downregulating DNA-PK activity, respectively. Moreover, the Par3 knockdown cells were found to be defective in random plasmid integration, defective in DSB repair following IR, and radiosensitive, phenotypes similar to that of Ku70 knockdown cells. These findings identify Par3 as a novel component of the DNA-PK complex and implicate an unexpected link of cell polarity to DSB repair.
Publisher: China Science Publishing & Media Ltd.
Date: 08-2013
DOI: 10.1093/ABBS/GMT055
Abstract: The plant homeodomain (PHD) finger is identified in many chromatin-binding proteins, and functions as a 'reader' that recognizes specific epigenetic marks on histone tails, bridging transcription factors and their associated complexes to chromatin, and regulating gene expression. PHD finger-containing proteins perform many biological functions and are involved in many human diseases including cancer. PHF14 is predicted to code for a protein with multiple PHD fingers. However, its function is unidentified. The aim of this study is to characterize PHF14 and investigate its biological significance by employing multiple approaches including mouse gene-targeting knockout, and molecular cloning and characterization. Three transcripts of PHF14 in human cell lines were identified by reverse transcriptase polymerase chain reaction. Two isoforms of PHF14 (PHF14α and PHF14β) were cloned in this study. It was found that PHF14 was ubiquitously expressed in mouse tissues and human cell lines. PHF14α, the major isoform of PHF14, was localized in the nucleus and also bound to chromatin during cell ision. Interestingly, co-immunoprecipitation results suggested that PHF14α bound to histones via its PHD fingers. Strikingly, gene-targeting knockout of PHF14 in mice resulted in a neonatal lethality due to respiratory failure. Pathological analysis revealed severe disorders of tissue and cell structures in multiple organs, particularly in the lungs. These results indicated that PHF14 might be an epigenetic regulator and play an important role in the development of multiple organs in mouse.
Publisher: Springer Science and Business Media LLC
Date: 02-12-2019
DOI: 10.1038/S41467-019-13114-4
Abstract: Protein oxidation sits at the intersection of multiple signalling pathways, yet the magnitude and extent of crosstalk between oxidation and other post-translational modifications remains unclear. Here, we delineate global changes in adipocyte signalling networks following acute oxidative stress and reveal considerable crosstalk between cysteine oxidation and phosphorylation-based signalling. Oxidation of key regulatory kinases, including Akt, mTOR and AMPK influences the fidelity rather than their absolute activation state, highlighting an unappreciated interplay between these modifications. Mechanistic analysis of the redox regulation of Akt identified two cysteine residues in the pleckstrin homology domain (C60 and C77) to be reversibly oxidized. Oxidation at these sites affected Akt recruitment to the plasma membrane by stabilizing the PIP 3 binding pocket. Our data provide insights into the interplay between oxidative stress-derived redox signalling and protein phosphorylation networks and serve as a resource for understanding the contribution of cellular oxidation to a range of diseases.
Publisher: Elsevier BV
Date: 08-2015
DOI: 10.1016/J.CELREP.2015.07.016
Abstract: The mechanistic target of rapamycin complex 2 (mTORC2) regulates cell survival and cytoskeletal organization by phosphorylating its AGC kinase substrates however, little is known about the regulation of mTORC2 itself. It was previously reported that Akt phosphorylates the mTORC2 subunit SIN1 at T86, activating mTORC2 through a positive feedback loop, though another study reported that S6K phosphorylates SIN1 at the same site, inhibiting mTORC2 activity. We performed extensive analysis of SIN1 phosphorylation upon inhibition of Akt, S6K, and mTOR under erse cellular contexts, and we found that, in all cell lines and conditions studied, Akt is the major kinase responsible for SIN1 phosphorylation. These findings refine the activation mechanism of the Akt-mTORC2 signaling branch as follows: PDK1 phosphorylates Akt at T308, increasing Akt kinase activity. Akt phosphorylates SIN1 at T86, enhancing mTORC2 kinase activity, which leads to phosphorylation of Akt S473 by mTORC2, thereby catalyzing full activation of Akt.
Publisher: eLife Sciences Publications, Ltd
Date: 13-07-2021
DOI: 10.7554/ELIFE.66942
Abstract: The phosphoinositide 3-kinase (PI3K)-Akt network is tightly controlled by feedback mechanisms that regulate signal flow and ensure signal fidelity. A rapid overshoot in insulin-stimulated recruitment of Akt to the plasma membrane has previously been reported, which is indicative of negative feedback operating on acute timescales. Here, we show that Akt itself engages this negative feedback by phosphorylating insulin receptor substrate (IRS) 1 and 2 on a number of residues. Phosphorylation results in the depletion of plasma membrane-localised IRS1/2, reducing the pool available for interaction with the insulin receptor. Together these events limit plasma membrane-associated PI3K and phosphatidylinositol (3,4,5)-trisphosphate (PIP3) synthesis. We identified two Akt-dependent phosphorylation sites in IRS2 at S306 (S303 in mouse) and S577 (S573 in mouse) that are key drivers of this negative feedback. These findings establish a novel mechanism by which the kinase Akt acutely controls PIP3 abundance, through post-translational modification of the IRS scaffold.
Publisher: Cold Spring Harbor Laboratory
Date: 30-11-2020
DOI: 10.1101/2020.11.30.403774
Abstract: The PI3K/mTOR signalling network critically regulates a broad array of important biological processes, including cell growth, metabolism and autophagy. Dysregulation of PI3K/mTOR signalling is associated with a variety of human diseases, including cancer and metabolic disorders. The mechanistic target of rapamycin (mTOR) is a kinase that functions as a core catalytic subunit in two physically and functionally distinct complexes termed mTOR complex 1 (mTORC1) and mTORC2, which also share other common components such as mLTS8 (also known as GβL) and DEPTOR. Despite being the subject of intensive research, a full picture of how mTORC1/2 assembly and activity are coordinated, and how they are functionally connected remain to be fully characterised. This is due primarily to the complex network wiring, featuring a growing number of intricate feedback loops and post-translational modifications, which require quantitative systems-level approaches to decipher. Here, we integrate predictive computational modelling, in vitro experiments and -omics data analysis to elucidate the dynamic and emergent features of the PI3K/mTOR network behavior. We construct new mechanistic models of the network that encapsulate novel critical mechanistic details, including mTORC1/2 coordination by mLTS8 (de)ubiquitination, and Akt-to-mTORC2 positive feedback loop. Model simulations subsequently confirmed by experimental validation revealed a previously unknown biphasic, threshold-gated dependence of mTORC1 activity on the key mTORC2 subunit Sin1, which is robust against cell-to-cell variation in protein expression. Furthermore, our results support the essential role of mLST8 in both mTORC1 and 2 activity, and suggest mLST8 could serve as a viable therapeutic target in breast cancer. Overall, our integrated analyses provide fresh systems-level insights into the dynamic behavior of PI3K/mTOR signalling and shed new light on the complexity of this important network. Signalling networks are the key information-processing machineries that underpin the ability of living cells to respond proportionately to extra- (and intra-) cellular cues. The PI3K/mTOR signalling network is one of the most important signalling networks in human cells that regulates cellular response to hormones such as insulin, yet our understanding of the network behaviour remains far from complete. Here, we employed a highly integrative approach that combines predictive mathematical modelling, biological experimentation, and data analysis to gain novel systems-level insights into PI3K/mTOR signalling. We constructed new mathematical models of this complex network incorporating important regulatory mechanisms. In contrary to commonly held views that mTORC2 lies upstream and is a positive regulator of mTORC1, we found that their relationship is highly nonlinear and dose dependent. This finding has major implications for mTORC2-directed anti-cancer strategies as depending on the cellular contexts, blocking mTORC2 may reduce or even enhance mTORC1 activation, the latter could inadvertently blunt the effect of mTORC2 blockade. Furthermore, our results demonstrate that mLST8 is required for the assembly and activity of both mTOR complexes, and suggest mLST8 is a viable therapeutic target in breast cancer, notably breast cancer.
Publisher: Springer Science and Business Media LLC
Date: 15-12-2020
DOI: 10.1038/S41421-020-00216-3
Abstract: Calcium/calmodulin-dependent protein serine kinase (CASK) is a key player in vesicle transport and release in neurons. However, its precise role, particularly in nonneuronal systems, is incompletely understood. We report that CASK functions as an important regulator of insulin secretion. CASK depletion in mouse islets/β cells substantially reduces insulin secretion and vesicle docking/fusion. CASK forms a ternary complex with Mint1 and Munc18-1, and this event is regulated by glucose stimulation in β cells. The crystal structure of the CASK/Mint1 complex demonstrates that Mint1 exhibits a unique “whip”-like structure that wraps tightly around the CASK-CaMK domain, which contains dual hydrophobic interaction sites. When triggered by CASK binding, Mint1 modulates the assembly of the complex. Further investigation revealed that CASK-Mint1 binding is critical for ternary complex formation, thereby controlling Munc18-1 membrane localization and insulin secretion. Our work illustrates the distinctive molecular basis underlying CASK/Mint1/Munc18-1 complex formation and reveals the importance of the CASK-Mint1-Munc18 signaling axis in insulin secretion.
Publisher: S. Karger AG
Date: 13-11-2015
DOI: 10.1159/000442205
Abstract: b i Background: /i /b Despite the success in treating some cancers, the efficacy of the mTOR inhibitors rapalogs as anti-cancer therapeutics has been limited. b i Aims: /i /b We undertook to examine the effects of Torin1, a second-generation selective ATP-competitive mTOR inhibitor, in non-functioning pituitary tumor cells. During characterization of the molecular mechanisms that mediate Torin1 actions, there seemed to be a rationale for combining it with rapalogs. b i Methods: /i /b Proliferation assays, flow cytometry and Western blotting were applied to assess the effects of Torin1, RAD001 and their combination on an MtT/E pituitary cell line and human-derived non-functioning pituitary tumor cells. b i Results: /i /b Combined long treatments of Torin1 and RAD001 induced a pronounced reduction in cell growth and viability of both MtT/E pituitary cells and human-derived non-functioning pituitary tumor cells, superior to each drug alone. This was remarkable in the 10 n smlcap M /smlcap combination and was reflected in a triggered decrease of cyclin D3 and p21/CIP expression. Interestingly, Akt-Thr308 and SIN1-Thr86 phosphorylations were robustly elevated in the combined treatment, accompanied by a reduction in PTEN expression. Phosphorylation of p70S6K was abolished in all in idual and combined treatments. Akt-Ser473 phosphorylation, induced by RAD001, was reduced by the combined treatment to the same extent as when treated by Torin1 alone. b i Conclusions: /i /b Our results suggest that the differential signaling mechanisms induced by these compounds eventually converge to lead to an efficient blockade of the PI3K/Akt/mTOR pathway in pituitary tumor cells and may facilitate a reduction in treatment dosage.
Publisher: Springer Science and Business Media LLC
Date: 10-2008
Abstract: Many well-represented domains recognize primary sequences usually less than 10 amino acids in length, called Short Linear Motifs (SLiMs). Accurate prediction of SLiMs has been difficult because they are short (often 10 amino acids) and highly degenerate. In this study, we combined scoring matrixes derived from peptide library and conservation analysis to identify protein classes enriched of functional SLiMs recognized by SH2, SH3, PDZ and S/T kinase domains. Our combined approach revealed that SLiMs are highly conserved in proteins from functional classes that are known to interact with a specific domain, but that they are not conserved in most other protein groups. We found that SLiMs recognized by SH2 domains were highly conserved in receptor kinases hosphatases, adaptor molecules, and tyrosine kinases hosphatases, that SLiMs recognized by SH3 domains were highly conserved in cytoskeletal and cytoskeletal-associated proteins, that SLiMs recognized by PDZ domains were highly conserved in membrane proteins such as channels and receptors, and that SLiMs recognized by S/T kinase domains were highly conserved in adaptor molecules, S/T kinases hosphatases, and proteins involved in transcription or cell cycle control. We studied Tyr-SLiMs recognized by SH2 domains in more detail, and found that SH2-recognized Tyr-SLiMs on the cytoplasmic side of membrane proteins are more highly conserved than those on the extra-cellular side. Also, we found that SH2-recognized Tyr-SLiMs that are associated with SH3 motifs and a tyrosine kinase phosphorylation motif are more highly conserved. The interactome of protein domains is reflected by the evolutionary conservation of SLiMs recognized by these domains. Combining scoring matrixes derived from peptide libraries and conservation analysis, we would be able to find those protein groups that are more likely to interact with specific domains.
Publisher: American Association for Cancer Research (AACR)
Date: 15-05-2009
DOI: 10.1158/1078-0432.CCR-08-2078
Abstract: Purpose: Lethal giant larvae functions as a cell polarity regulator and a tumor suppressor in Drosophila. Its evolutionary conservation implies a tumor suppressor role for its human homologue, Hugl-1. The aims of this study were to characterize Hugl-1 and to determine the clinical significance of Hugl-1 alterations in hepatocellular carcinoma (HCC). Experimental Design: Sequence alterations of Hugl-1 from 80 HCC specimens and 5 HCC cell lines were characterized by reverse transcription-PCR and sequence analysis. Western blot was used for determining Hugl-1 expression. The biological activities of Hugl-1 and its aberrant variants were examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, wound healing assay, Boyden chamber assay, and tumorigenicity assay. Results: In 32.5% (26 of 80) of the specimens and 20.0% (one of five) of HCC cell lines, 23 unique aberrant Hugl-1 transcripts were identified, most of which resulted from skipping part of and/or entire exon or insertion of intron sequences. The majority of these aberrant Hugl-1 transcripts encoded truncated proteins lacking one or more conserved WD-40 repeat motifs. Two truncated Hugl-1 proteins were found exclusively in HCC tissues. Aberrant Hugl-1 transcripts (78.3%, 20 of 23) had a short “direct repeat” sequence flanking their deleted regions. The abnormal Hugl-1 was significantly correlated with poor differentiation and large tumor size of HCC. Overexpression of two representative HCC-derived aberrant Hugl-1 variants promoted HCC cell migration, invasion, and tumorigenicity in nude mice. Conclusions: We provide the first evidence that Hugl-1 mRNA is frequently mutated by aberrant splicing exclusively in HCC, which may be involved in HCC progression.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 09-06-2015
DOI: 10.1126/SCISIGNAL.AAA3139
Abstract: A mass spectrometry method may enable the simultaneous and rapid quantification of multiple phosphorylation events in multiple s les.
Publisher: Elsevier BV
Date: 09-2014
Publisher: Springer Science and Business Media LLC
Date: 06-2007
DOI: 10.1038/CR.2007.50
Publisher: Wiley
Date: 18-01-2011
DOI: 10.1002/PROT.22955
Start Date: 2012
End Date: 2013
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2021
End Date: 2023
Funder: Australian Research Council
View Funded ActivityStart Date: 2021
End Date: 12-2023
Amount: $545,000.00
Funder: Australian Research Council
View Funded Activity