18 results on '"Dougall M. Norris"'
Search Results
2. Global redox proteome and phosphoproteome analysis reveals redox switch in Akt
- Author
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Zhiduan Su, James G. Burchfield, Pengyi Yang, Sean J. Humphrey, Guang Yang, Deanne Francis, Sabina Yasmin, Sung-Young Shin, Dougall M. Norris, Alison L. Kearney, Miro A. Astore, Jonathan Scavuzzo, Kelsey H. Fisher-Wellman, Qiao-Ping Wang, Benjamin L. Parker, G. Gregory Neely, Fatemeh Vafaee, Joyce Chiu, Reichelle Yeo, Philip J. Hogg, Daniel J. Fazakerley, Lan K. Nguyen, Serdar Kuyucak, and David E. James
- Subjects
Science - Abstract
Crosstalk between protein oxidation and other post-translational modifications remains unexplored. Here, the authors map the phosphoproteome, cysteine redox proteome and total proteome of adipocytes under acute oxidative stress and reveal crosstalk between cysteine oxidation and phosphorylation-based signalling.
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- 2019
- Full Text
- View/download PDF
3. Akt phosphorylates insulin receptor substrate to limit PI3K-mediated PIP3 synthesis
- Author
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Alison L Kearney, Dougall M Norris, Milad Ghomlaghi, Martin Kin Lok Wong, Sean J Humphrey, Luke Carroll, Guang Yang, Kristen C Cooke, Pengyi Yang, Thomas A Geddes, Sungyoung Shin, Daniel J Fazakerley, Lan K Nguyen, David E James, and James G Burchfield
- Subjects
insulin ,phosphorylation ,plasma membrane ,signal transduction ,Akt ,PI3K ,Medicine ,Science ,Biology (General) ,QH301-705.5 - 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.
- Published
- 2021
- Full Text
- View/download PDF
4. Limited oxygen availability in standard cell culture alters metabolism and function in terminally-differentiated cells
- Author
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Joycelyn Tan, Sam Virtue, Dougall M. Norris, Olivia J. Conway, Ming Yang, Christopher Gribben, Fatima Lugtu, Ioannis Kamzolas, James R. Krycer, Richard J. Mills, Conceição Pereira, Martin Dale, Amber S. Shun-Shion, Harry J. M. Baird, James A. Horscroft, Alice P. Sowton, Marcella Ma, Stefania Carobbio, Evangelia Petsalaki, Andrew J. Murray, David C. Gershlick, James E. Hudson, Ludovic Vallier, Kelsey H Fisher-Wellman, Christian Frezza, Antonio Vidal-Puig, and Daniel J. Fazakerley
- Abstract
SUMMARYCell culture is generally considered to be hyperoxic. However, the importance of cellular oxygen consumption is often underappreciated, with rates of oxygen consumption often sufficient to cause hypoxia at cell monolayers. We initially focused on cultured adipocytes as a terminally differentiated cell-type with substantial oxygen consumption rates to support diverse cellular functions. Under standard conditions, cultured adipocytes are hypoxic and highly glycolytic. Increasing oxygen diverted glucose flux toward mitochondria and resulted in thousands of gene expression changes that pointed toward alleviated physiological transcriptional responses to hypoxia. Phenotypically, providing more oxygen increased adipokine secretion and rendered adipocytes more sensitive to insulin and lipolytic stimuli. The functional benefits of increasing pericellular oxygen were transferable to other cellular systems including hPSC-derived hepatocytes and cardiac organoids. Our findings suggest that oxygen is limiting in many terminally-differentiated cell culture systems, and that controlling oxygen availability can improve the quality and translatability of cell models.
- Published
- 2022
5. A high-content endogenous GLUT4 trafficking assay reveals new aspects of adipocyte biology
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Alexis Diaz-Vegas, Dougall M Norris, Sigrid Jall-Rogg, Kristen C Cooke, Olivia J Conway, Amber S Shun-Shion, Xiaowen Duan, Meg Potter, Julian van Gerwen, Harry JM Baird, Sean J Humphrey, David E James, Daniel J Fazakerley, James G Burchfield, Diaz-Vegas, Alexis [0000-0001-5227-4482], Cooke, Kristen C [0000-0002-3534-1660], Humphrey, Sean J [0000-0002-2666-9744], James, David E [0000-0001-5946-5257], Fazakerley, Daniel J [0000-0001-8241-2903], Burchfield, James G [0000-0002-6609-6151], and Apollo - University of Cambridge Repository
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Mice ,Glucose Transporter Type 4 ,Glucose ,Ecology ,rab GTP-Binding Proteins ,Health, Toxicology and Mutagenesis ,3T3-L1 Cells ,Adipocytes ,Animals ,Insulin ,Plant Science ,Biochemistry, Genetics and Molecular Biology (miscellaneous) ,Biology - Abstract
Funder: Wellcome-MRC, Institute of Metabolic Science, Metabolic Research Laboratories, Imaging Core, Insulin-induced GLUT4 translocation to the plasma membrane in muscle and adipocytes is crucial for whole-body glucose homeostasis. Currently, GLUT4 trafficking assays rely on overexpression of tagged GLUT4. Here we describe a high-content imaging platform for studying endogenous GLUT4 translocation in intact adipocytes. This method enables high fidelity analysis of GLUT4 responses to specific perturbations, multiplexing of other trafficking proteins and other features including lipid droplet morphology. Using this multiplexed approach we showed that Vps45 and Rab14 are selective regulators of GLUT4, but Trarg1, Stx6, Stx16, Tbc1d4 and Rab10 knockdown affected both GLUT4 and TfR translocation. Thus, GLUT4 and TfR translocation machinery likely have some overlap upon insulin-stimulation. In addition, we identified Kif13A, a Rab10 binding molecular motor, as a novel regulator of GLUT4 traffic. Finally, comparison of endogenous to overexpressed GLUT4 highlights that the endogenous GLUT4 methodology has an enhanced sensitivity to genetic perturbations and emphasises the advantage of studying endogenous protein trafficking for drug discovery and genetic analysis of insulin action in relevant cell types.
- Published
- 2022
- Full Text
- View/download PDF
6. Phosphoproteomics reveals rewiring of the insulin signaling network and multi-nodal defects in insulin resistance
- Author
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Daniel J. Fazakerley, Julian van Gerwen, Kristen C. Cooke, Xiaowen Duan, Elise J. Needham, Søren Madsen, Dougall M. Norris, Amber S. Shun-Shion, James R. Krycer, James G. Burchfield, Pengyi Yang, Mark R. Wade, Joseph T. Brozinick, David E. James, and Sean J. Humphrey
- Abstract
The failure of metabolic tissues to appropriately respond to insulin (“insulin resistance”) is an early marker in the pathogenesis of type 2 diabetes. Protein phosphorylation is central to the adipocyte insulin response, but how adipocyte signaling networks are dysregulated upon insulin resistance is unknown. Here we employed phosphoproteomics to delineate insulin signal transduction in adipocyte cells and adipose tissue. Across a range of insults triggering insulin resistance, we observed marked rewiring of the insulin signaling network. This included both attenuated insulin-responsive phosphorylation, and the emergence of phosphorylation uniquely insulin-regulated in insulin resistance. Identifying signaling changes common to multiple insults revealed subnetworks likely containing causal drivers of insulin resistance. Focusing on defective GSK3 signaling initially observed in a relatively small subset of well-characterized substrates, we employed a pipeline for identifying context-specific kinase substrates. This facilitated robust identification of widespread dysregulated GSK3 signaling. Pharmacological inhibition of GSK3 partially reversed insulin resistance in cells and tissue explants. These data highlight that insulin resistance is a multi-nodal signaling defect that encompasses dysregulated GSK3 activity.
- Published
- 2022
7. Trafficking regulator of GLUT4-1 (TRARG1) is a GSK3 substrate
- Author
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Xiaowen Duan, Dougall M. Norris, Sean J. Humphrey, Pengyi Yang, Kristen C. Cooke, Will P. Bultitude, Benjamin L. Parker, Olivia J. Conway, James G. Burchfield, James R. Krycer, Frances M. Brodsky, David E. James, Daniel J. Fazakerley, Fazakerley, Daniel J [0000-0001-8241-2903], and Apollo - University of Cambridge Repository
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animal structures ,Glucose Transporter Type 4 ,Cell Membrane ,glucose transport ,Cell Biology ,macromolecular substances ,trafficking regulator of GLUT4-1 ,insulin signalling ,Biochemistry ,glycogen synthase kinase ,Glycogen Synthase Kinase 3 ,Mice ,Phosphatidylinositol 3-Kinases ,Glucose ,Adipocytes ,Serine ,Animals ,Humans ,Insulin ,Phosphorylation ,Molecular Biology ,Proto-Oncogene Proteins c-akt - Abstract
Trafficking regulator of GLUT4-1, TRARG1, positively regulates insulin-stimulated GLUT4 trafficking and insulin sensitivity. However, the mechanism(s) by which this occurs remain(s) unclear. Using biochemical and mass spectrometry analyses we found that TRARG1 is dephosphorylated in response to insulin in a PI3K/Akt-dependent manner and is a novel substrate for GSK3. Priming phosphorylation of murine TRARG1 at serine 84 allows for GSK3-directed phosphorylation at serines 72, 76 and 80. A similar pattern of phosphorylation was observed in human TRARG1, suggesting that our findings are translatable to human TRARG1. Pharmacological inhibition of GSK3 increased cell surface GLUT4 in cells stimulated with a submaximal insulin dose, and this was impaired following Trarg1 knockdown, suggesting that TRARG1 acts as a GSK3-mediated regulator in GLUT4 trafficking. These data place TRARG1 within the insulin signaling network and provide insights into how GSK3 regulates GLUT4 trafficking in adipocytes.
- Published
- 2022
8. Serine 474 phosphorylation is essential for maximal Akt2 kinase activity in adipocytes
- Author
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Dougall M. Norris, Kristen C. Cooke, James G. Burchfield, Daniel J. Fazakerley, Armella Zadoorian, Alison L Kearney, David E. James, and James R. Krycer
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inorganic chemicals ,0301 basic medicine ,FOXO1 ,Mechanistic Target of Rapamycin Complex 2 ,mTORC1 ,Serine threonine protein kinase ,Mechanistic Target of Rapamycin Complex 1 ,environment and public health ,Biochemistry ,mTORC2 ,Mice ,03 medical and health sciences ,3T3-L1 Cells ,Tuberous Sclerosis Complex 2 Protein ,Adipocytes ,Serine ,Akt Inhibitor MK2206 ,Animals ,Insulin ,Phosphorylation ,Protein kinase A ,Molecular Biology ,Protein kinase B ,Cell Nucleus ,Glucose Transporter Type 4 ,030102 biochemistry & molecular biology ,Forkhead Box Protein O1 ,Chemistry ,Cell Biology ,Cell biology ,enzymes and coenzymes (carbohydrates) ,Glucose ,030104 developmental biology ,Protein Biosynthesis ,Mutagenesis, Site-Directed ,bacteria ,Heterocyclic Compounds, 3-Ring ,Proto-Oncogene Proteins c-akt ,hormones, hormone substitutes, and hormone antagonists ,Signal Transduction - Abstract
The Ser/Thr protein kinase Akt regulates essential biological processes such as cell survival, growth, and metabolism. Upon growth factor stimulation, Akt is phosphorylated at Ser(474); however, how this phosphorylation contributes to Akt activation remains controversial. Previous studies, which induced loss of Ser(474) phosphorylation by ablating its upstream kinase mTORC2, have implicated Ser(474) phosphorylation as a driver of Akt substrate specificity. Here we directly studied the role of Akt2 Ser(474) phosphorylation in 3T3-L1 adipocytes by preventing Ser(474) phosphorylation without perturbing mTORC2 activity. This was achieved by utilizing a chemical genetics approach, where ectopically expressed S474A Akt2 was engineered with a W80A mutation to confer resistance to the Akt inhibitor MK2206, and thus allow its activation independent of endogenous Akt. We found that insulin-stimulated phosphorylation of four bona fide Akt substrates (TSC2, PRAS40, FOXO1/3a, and AS160) was reduced by ∼50% in the absence of Ser(474) phosphorylation. Accordingly, insulin-stimulated mTORC1 activation, protein synthesis, FOXO nuclear exclusion, GLUT4 translocation, and glucose uptake were attenuated upon loss of Ser(474) phosphorylation. We propose a model where Ser(474) phosphorylation is required for maximal Akt2 kinase activity in adipocytes.
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- 2019
9. Akt phosphorylates insulin receptor substrate to limit PI3K-mediated PIP3 synthesis
- Author
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Sean J. Humphrey, James G. Burchfield, Milad Ghomlaghi, David E. James, Kristen C. Cooke, Thomas A Geddes, Luke Carroll, Guang Yang, Pengyi Yang, Lan K. Nguyen, Sung-Young Shin, Daniel J. Fazakerley, Dougall M. Norris, Martin Kin Lok Wong, Alison L Kearney, Kearney, Alison L [0000-0002-5736-3393], Ghomlaghi, Milad [0000-0001-9047-1049], Nguyen, Lan K [0000-0003-4040-7705], James, David E [0000-0001-5946-5257], Burchfield, James G [0000-0002-6609-6151], Apollo - University of Cambridge Repository, Humphrey, Sean J [0000-0002-2666-9744], and Fazakerley, Daniel [0000-0001-8241-2903]
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0301 basic medicine ,Mouse ,medicine.medical_treatment ,plasma membrane ,PI3K ,Mice ,Phosphatidylinositol 3-Kinases ,computational biology ,0302 clinical medicine ,Insulin receptor substrate ,Biology (General) ,biology ,Chemistry ,phosphorylation ,General Neuroscience ,systems biology ,General Medicine ,Cell biology ,030220 oncology & carcinogenesis ,Medicine ,Signal transduction ,signal transduction ,Research Article ,Computational and Systems Biology ,Human ,Cell signaling ,insulin ,QH301-705.5 ,Science ,Mechanistic Target of Rapamycin Complex 1 ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Antigens, CD ,Negative feedback ,medicine ,Animals ,Humans ,Protein kinase B ,PI3K/AKT/mTOR pathway ,General Immunology and Microbiology ,Insulin ,Akt ,Cell Membrane ,Cell Biology ,Receptor, Insulin ,Insulin receptor ,Glucose ,030104 developmental biology ,Insulin Receptor Substrate Proteins ,biology.protein ,Phosphatidylinositol 3-Kinase ,Proto-Oncogene Proteins c-akt - 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., eLife digest For the body to work properly, cells must constantly ‘talk’ to each other using signalling molecules. Receiving a chemical signal triggers a series of molecular events in a cell, a so-called ‘signal transduction pathway’ that connects a signal with a precise outcome. Disturbing cell signalling can trigger disease, and strict control mechanisms are therefore in place to ensure that communication does not break down or become erratic. For instance, just as a thermostat turns off the heater once the right temperature is reached, negative feedback mechanisms in cells switch off signal transduction pathways when the desired outcome has been achieved. The hormone insulin is a signal for growth that increases in the body following a meal to promote the storage of excess blood glucose (sugar) in muscle and fat cells. The hormone binds to insulin receptors at the cell surface and switches on a signal transduction pathway that makes the cell take up glucose from the bloodstream. If the signal is not engaged diseases such as diabetes develop. Conversely, if the signal cannot be adequately switched of cancer can develop. Determining exactly how insulin works would help to understand these diseases better and to develop new treatments. Kearney et al. therefore set out to examine the biochemical ‘fail-safes’ that control insulin signalling. Experiments using computer simulations of the insulin signalling pathway revealed a potential new mechanism for negative feedback, which centred on a molecule known as Akt. The models predicted that if the negative feedback were removed, then Akt would become hyperactive and accumulate at the cell’s surface after stimulation with insulin. Further manipulation of the ‘virtual’ insulin signalling pathway and studies of live cells in culture confirmed that this was indeed the case. The cell biology experiments also showed how Akt, once at the cell surface, was able to engage the negative feedback and shut down further insulin signalling. Akt did this by inactivating a protein required to pass the signal from the insulin receptor to the rest of the cell. Overall, this work helps to understand cell communication by revealing a previously unknown, and critical component of the insulin signalling pathway.
- Published
- 2021
10. Author response: Akt phosphorylates insulin receptor substrate to limit PI3K-mediated PIP3 synthesis
- Author
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Kristen C. Cooke, James G. Burchfield, Martin Kin Lok Wong, Sean J. Humphrey, David E. James, Daniel J. Fazakerley, Dougall M. Norris, Pengyi Yang, Lan K. Nguyen, Thomas A Geddes, Sung-Young Shin, Alison L Kearney, Luke Carroll, Milad Ghomlaghi, and Guang Yang
- Subjects
Chemistry ,Insulin receptor substrate ,Phosphorylation ,Limit (mathematics) ,Protein kinase B ,PI3K/AKT/mTOR pathway ,Cell biology - Published
- 2021
11. Signaling Heterogeneity is Defined by Pathway Architecture and Intercellular Variability in Protein Expression
- Author
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James G. Burchfield, Sung-Young Shin, Pengyi Yang, Lan K. Nguyen, Alison L Kearney, Daniel J. Fazakerley, Hani Jieun Kim, Alistair M. Senior, David E. James, Thomas A Geddes, Dougall M. Norris, Fazakerley, Daniel [0000-0001-8241-2903], and Apollo - University of Cambridge Repository
- Subjects
0301 basic medicine ,medicine.medical_treatment ,Glucose uptake ,Population ,Cell ,02 engineering and technology ,Experimental Models in Systems Biology ,Article ,03 medical and health sciences ,Mathematical Biosciences ,medicine ,education ,lcsh:Science ,Protein kinase B ,PI3K/AKT/mTOR pathway ,education.field_of_study ,Multidisciplinary ,biology ,Chemistry ,Insulin ,Systems Biology ,Cell Biology ,021001 nanoscience & nanotechnology ,Cell biology ,Insulin receptor ,030104 developmental biology ,medicine.anatomical_structure ,biology.protein ,lcsh:Q ,0210 nano-technology ,GLUT4 - Abstract
Summary Insulin's activation of PI3K/Akt signaling, stimulates glucose uptake by enhancing delivery of GLUT4 to the cell surface. Here we examined the origins of intercellular heterogeneity in insulin signaling. Akt activation alone accounted for ~25% of the variance in GLUT4, indicating that additional sources of variance exist. The Akt and GLUT4 responses were highly reproducible within the same cell, suggesting the variance is between cells (extrinsic) and not within cells (intrinsic). Generalized mechanistic models (supported by experimental observations) demonstrated that the correlation between the steady-state levels of two measured signaling processes decreases with increasing distance from each other and that intercellular variation in protein expression (as an example of extrinsic variance) is sufficient to account for the variance in and between Akt and GLUT4. Thus, the response of a population to insulin signaling is underpinned by considerable single-cell heterogeneity that is largely driven by variance in gene/protein expression between cells., Graphical Abstract, Highlights • Insulin signaling is heterogeneous between cells in the same population • The temporal response of signaling components within a cell is highly reproducible • Upstream responses (Akt) can only partially predict downstream response (GLUT4) • Protein expression variance is a driver of intercellular signaling heterogeneity, Cell Biology; Mathematical Biosciences; Systems Biology; Experimental Models in Systems Biology
- Published
- 2021
12. Trafficking regulator of GLUT4-1 (TRARG1) is a GSK3 substrate
- Author
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Kristen C. Cooke, Daniel J. Fazakerley, Xiaowen Duan, WP Bultitude, Frances M. Brodsky, James G. Burchfield, Pengyi Yang, Olivia J. Conway, James R. Krycer, David E. James, Dougall M. Norris, Benjamin L. Parker, and Sean J. Humphrey
- Subjects
biology ,Chemistry ,Insulin ,medicine.medical_treatment ,Regulator ,macromolecular substances ,Cell biology ,Serine ,Dephosphorylation ,Insulin receptor ,medicine ,biology.protein ,Phosphorylation ,GLUT4 ,PI3K/AKT/mTOR pathway - Abstract
Trafficking regulator of GLUT4-1, TRARG1, positively regulates insulin-stimulated GLUT4 trafficking and insulin sensitivity. However, the mechanism(s) by which this occurs remain(s) unclear. Using biochemical and mass spectrometry analyses we found that TRARG1 is dephosphorylated in response to insulin in a PI3K/Akt-dependent manner and is a novel substrate for GSK3. Priming phosphorylation of mouse TRARG1 at serine 84 allows for GSK3-directed phosphorylation at serines 72, 76 and 80. A similar pattern of phosphorylation was observed in human TRARG1, suggesting that our findings are translatable to human TRARG1. Pharmacological inhibition of GSK3 increased cell surface GLUT4 in cells stimulated with a submaximal insulin dose, and this was impaired following Trarg1 knockdown, suggesting that TRARG1 acts as a GSK3-mediated regulator in GLUT4 trafficking. TRARG1 dephosphorylation in response to insulin or GSK3 inhibition regulated its protein-protein interactions, decreasing the interaction between TRARG1 and BCL9L, JAGN1, PYGO2 and HSD17B12. Knock-down of Bcl9l promoted GLUT4 translocation to the plasma membrane. These data place TRARG1 within the insulin signaling network and provide insights into how TRARG1 regulates GLUT4 trafficking in adipocytes.
- Published
- 2020
13. Global redox proteome and phosphoproteome analysis reveals redox switch in Akt
- Author
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Reichelle X. Yeo, Philip J. Hogg, Deanne Francis, Sean J. Humphrey, Joyce Chiu, Miro A. Astore, Benjamin L. Parker, Dougall M. Norris, James G. Burchfield, Jonathan Scavuzzo, Sabina Yasmin, Sung-Young Shin, Zhiduan Su, Guang Yang, G. Gregory Neely, Daniel J. Fazakerley, Pengyi Yang, Lan K. Nguyen, David E. James, Fatemeh Vafaee, Kelsey H. Fisher-Wellman, Alison L Kearney, Qiao-Ping Wang, Serdar Kuyucak, Burchfield, James G [0000-0002-6609-6151], Yang, Pengyi [0000-0003-1098-3138], Humphrey, Sean J [0000-0002-2666-9744], Vafaee, Fatemeh [0000-0002-7521-2417], Hogg, Philip J [0000-0001-6486-2863], Fazakerley, Daniel J [0000-0001-8241-2903], and Apollo - University of Cambridge Repository
- Subjects
Proteomics ,0301 basic medicine ,Proteome ,Proto-Oncogene Proteins c-akt ,Science ,General Physics and Astronomy ,Protein oxidation ,Article ,General Biochemistry, Genetics and Molecular Biology ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Protein Domains ,Adipocytes ,Animals ,Humans ,Protein phosphorylation ,Cysteine ,Phosphorylation ,lcsh:Science ,Protein kinase B ,PI3K/AKT/mTOR pathway ,Multidisciplinary ,Chemistry ,TOR Serine-Threonine Kinases ,Insulin signalling ,General Chemistry ,Phosphoproteins ,Cell biology ,Pleckstrin homology domain ,Oxidative Stress ,Crosstalk (biology) ,030104 developmental biology ,lcsh:Q ,Oxidation-Reduction ,030217 neurology & neurosurgery ,Post-translational modifications ,Signal Transduction - 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 PIP3 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., Crosstalk between protein oxidation and other post-translational modifications remains unexplored. Here, the authors map the phosphoproteome, cysteine redox proteome and total proteome of adipocytes under acute oxidative stress and reveal crosstalk between cysteine oxidation and phosphorylation-based signalling.
- Published
- 2019
14. Phosphoproteomics reveals conserved exercise-stimulated signaling and AMPK regulation of store-operated calcium entry
- Author
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Erik A. Richter, Timur Naim, James G. Burchfield, David E. James, Marin E. Nelson, Lykke Sylow, Rima Chaudhuri, Nolan J. Hoffman, Kristen C. Cooke, Deanne Francis, Gordon S. Lynch, Elise J. Needham, Jonathan S. Oakhill, Naomi X.Y. Ling, Benjamin L. Parker, Jacqueline Stöckli, and Dougall M. Norris
- Subjects
AMPK ,Resource ,Male ,Proteomics ,STIM1 ,Protein Conformation ,Biology ,AMP-Activated Protein Kinases ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Mice ,0302 clinical medicine ,AMP-activated protein kinase ,medicine ,Animals ,Humans ,Protein phosphorylation ,Calcium Signaling ,Stromal Interaction Molecule 1 ,Phosphorylation ,Rats, Wistar ,Muscle, Skeletal ,Molecular Biology ,030304 developmental biology ,Calcium signaling ,0303 health sciences ,exercise ,General Immunology and Microbiology ,General Neuroscience ,Phosphoproteomics ,Skeletal muscle ,Membrane Proteins ,Store-operated calcium entry ,Cell biology ,Rats ,medicine.anatomical_structure ,biology.protein ,Calcium ,Drosophila ,Female ,Calcium Channels ,Corrigendum ,030217 neurology & neurosurgery ,Signal Transduction - Abstract
Exercise stimulates cellular and physiological adaptations that are associated with widespread health benefits. To uncover conserved protein phosphorylation events underlying this adaptive response, we performed mass spectrometry‐based phosphoproteomic analyses of skeletal muscle from two widely used rodent models: treadmill running in mice and in situ muscle contraction in rats. We overlaid these phosphoproteomic signatures with cycling in humans to identify common cross‐species phosphosite responses, as well as unique model‐specific regulation. We identified > 22,000 phosphosites, revealing orthologous protein phosphorylation and overlapping signaling pathways regulated by exercise. This included two conserved phosphosites on stromal interaction molecule 1 (STIM1), which we validate as AMPK substrates. Furthermore, we demonstrate that AMPK‐mediated phosphorylation of STIM1 negatively regulates store‐operated calcium entry, and this is beneficial for exercise in Drosophila. This integrated cross‐species resource of exercise‐regulated signaling in human, mouse, and rat skeletal muscle has uncovered conserved networks and unraveled crosstalk between AMPK and intracellular calcium flux.
- Published
- 2019
15. Before the Pump
- Author
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Dougall M, Norris and Massimo M, Santoro
- Subjects
Animals ,Brain ,Cilia ,Zebrafish Proteins ,Zebrafish ,Article - Abstract
OBJECTIVE: Endothelial cells (ECs) sense and respond to flow-induced mechanical stress, in part, via microtubule-based projections called primary cilia. However, many critical steps during vascular morphogenesis occur independent of flow. The involvement of cilia in regulating these stages of cranial vascular morphogenesis is poorly understood, as cilia have not been visualized in primary head vessels. The objective of this study was to investigate involvement of cilia in regulating the early stages of cranial vascular morphogenesis. APPROACH AND RESULTS: Using high-resolution imaging of the Tg(kdrl:mCherry-CAAX) (y171);(bactin::Arl13b:GFP) zebrafish line, we showed that cilia are enriched in the earliest formed cranial vessels that assemble via vasculogenesis and in angiogenic hindbrain capillaries. Cilia were more prevalent around the boundaries of putative intravascular spaces in primary and angiogenic vessels. Loss of cardiac contractility and blood flow, due to knockdown of cardiac troponin T type 2a (tnnt2a) expression, did not affect the distribution of cilia in primary head vasculature. In later stages of development, cilia were detected in retinal vasculature, areas of high curvature, vessel bifurcation points, and during vessel anastomosis. Loss of genes crucial for cilia biogenesis (ift172 and ift81) induced intracerebral hemorrhages in an EC-autonomous manner. Exposure to high shear stress induced premature cilia disassembly in brain ECs and was associated with intracerebral hemorrhages. CONCLUSION: Our study suggests a functional role for cilia in brain ECs, which is associated with the emergence and remodeling of the primary cranial vasculature. This cilia function is flow-independent, and cilia in ECs are required for cerebral vascular stability.
- Published
- 2018
16. Glucose Transport: Methods for Interrogating GLUT4 Trafficking in Adipocytes
- Author
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Dougall M, Norris, Tom A, Geddes, David E, James, Daniel J, Fazakerley, and James G, Burchfield
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Mice ,Protein Transport ,Glucose ,Glucose Transporter Type 4 ,3T3-L1 Cells ,Adipocytes ,Animals ,Fluorescent Antibody Technique ,Biological Transport ,Molecular Imaging - Abstract
In this chapter we detail methods for the systematic dissection of GLUT4 trafficking. The methods described have been optimized for cultured 3T3-L1 adipocytes, but can be readily adapted to other cell types.
- Published
- 2017
17. Before the Pump
- Author
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Massimo M. Santoro and Dougall M. Norris
- Subjects
0301 basic medicine ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Microtubule ,Cilium ,030204 cardiovascular system & hematology ,Biology ,Cardiology and Cardiovascular Medicine ,biology.organism_classification ,Zebrafish ,Cell biology - Published
- 2018
18. Improved Akt reporter reveals intra- and inter-cellular heterogeneity and oscillations in signal transduction
- Author
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David E. James, James G. Burchfield, Pengyi Yang, Dougall M. Norris, Daniel J. Fazakerley, and James R. Krycer
- Subjects
0301 basic medicine ,Recombinant Fusion Proteins ,medicine.medical_treatment ,Green Fluorescent Proteins ,AKT2 ,Biology ,Green fluorescent protein ,Mice ,03 medical and health sciences ,Genes, Reporter ,3T3-L1 Cells ,medicine ,Animals ,Tissue Distribution ,Protein kinase B ,PI3K/AKT/mTOR pathway ,Protein Stability ,Insulin ,Cell Biology ,Molecular Imaging ,Cell biology ,Protein Transport ,030104 developmental biology ,Signalling ,Biochemistry ,Phosphorylation ,Signal transduction ,Proto-Oncogene Proteins c-akt ,Signal Transduction - Abstract
Akt is a key node in a range of signal transduction cascades and play a critical role in diseases such as cancer and diabetes. Fluorescently-tagged Akt reporters have been used to discern Akt localisation, yet it has not been clear how well these tools recapitulate the behaviour of endogenous Akt proteins. Here, we observed that fusion of eGFP to Akt2 impaired both its insulin-stimulated plasma membrane recruitment and its phosphorylation. Endogenous-like responses were restored by replacing eGFP with TagRFP-T. The improved response magnitude and sensitivity afforded by TagRFP-T–Akt2 over eGFP–Akt2 enabled monitoring of signalling outcomes in single cells at physiological doses of insulin with subcellular resolution and revealed two previously unreported features of Akt biology. In 3T3-L1 adipocytes, stimulation with insulin resulted in recruitment of Akt2 to the plasma membrane in a polarised fashion. Additionally, we observed oscillations in plasma membrane localised Akt2 in the presence of insulin with a consistent periodicity of 2 min. Our studies highlight the importance of fluorophore choice when generating reporter constructs and shed light on new Akt signalling responses that may encode complex signalling information. This article has an associated First Person interview with the first author of the paper.
- Published
- 2017
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