Spatiotemporal Regulation of ERK by Spatiotemporal Regulation of ERK by Dual- -specificity Phosphatases specificity Phosphatases Dual
University of Bristol IN Cell 1000 University of Bristol IN Cell 1000 WT Equipment Grant – WT Equipment Grant – “ “Use of a high content image analysis Use of a high content image analysis platform for biomedical research” platform for biomedical research ” Multi- Multi -user equipment user equipment Craig McArdle – Craig McArdle – Functional profiling of GnRHRs Functional profiling of GnRHRs Stafford Lightman Stafford Lightman – – Transcriptional regulation by Transcriptional regulation by glucocorticoids glucocorticoids Christos Paraskeva Christos Paraskeva – – proliferation, death and differentiation of proliferation, death and differentiation of colonic epithelial cells colonic epithelial cells Andrew Newby Andrew Newby – – migration, proliferation and survival of vascular migration, proliferation and survival of vascular smooth muscle cells smooth muscle cells Guy Guy Rutter Rutter – – roles of AMP roles of AMP- -activated protein kinase in hypothalamic activated protein kinase in hypothalamic neurone nutrient signalling neurone nutrient signalling
Cell signalling - - Mechanisms of Hormone Action Mechanisms of Hormone Action Cell signalling reproductive endocrinology – – GnRH, gonadal steroids GnRH, gonadal steroids reproductive endocrinology IN Cell 1000 Projects IN Cell 1000 Projects Context- -dependence of steroid receptor signaling dependence of steroid receptor signaling Context - translocation and transcription in breast and bone cells translocation and transcription in breast and bone cells - (ER/GFP, GR/GFP, ER/GR/GFP) (ER/GFP, GR/GFP, ER/GR/GFP) Ctrl. E2 Ctrl. E2 Benit Maru Maru Benit
Cell Signalling - - Mechanisms of Hormone Action Mechanisms of Hormone Action Cell Signalling reproductive endocrinology – – GnRH, gonadal steroids GnRH, gonadal steroids reproductive endocrinology IN Cell 1000 Projects IN Cell 1000 Projects GnRH receptor trafficking GnRH receptor trafficking Receptor internalisation and trafficking from ER to PM Receptor internalisation and trafficking from ER to PM (HA- -GnRHRs) GnRHRs) (HA 0 1 10 100 1000 0 1 10 100 1000 IN3 ( µ µ µ µ µ g/ml) g/ml) IN3 ( µ µ µ Ann Finch & Kris Sedgley Ann Finch & Kris Sedgley
Cell signalling - - Mechanisms of Hormone Action Mechanisms of Hormone Action Cell signalling reproductive endocrinology – – GnRH, gonadal steroids GnRH, gonadal steroids reproductive endocrinology IN Cell 1000 Projects IN Cell 1000 Projects ERK signalling: compartmentalisation and kinetics ERK signalling: compartmentalisation and kinetics DAPI DAPI nucleus nucleus Jim Caunt Jim Caunt & & ERK2 ERK2 -GFP GFP - Steve Armstrong Steve Armstrong ppERK1/2 ppERK1/2 DAPI/GFP GFP DAPI/
Stress, Cytokines, Stress, Cytokines, Growth Factors, Growth Factors, Growth Factors, Growth Factors, Stimulus Stimulus GPCRs GPCRs GPCRs GPCRs B-Raf MLK3 MLK3 MAPKKK A-Raf ASK1 TAK c-Raf MEKK1/4 DLK MAPKK MEK1/2 MKK3/6 MKK4/7 p38 α α α / β α β / γ β β γ γ γ MAPK ERK1/2 JNK1/2/3 Inflammation, Inflammation, Growth, Growth, Response Response Apoptosis, Apoptosis, Differentiation, Differentiation, Growth, Growth, Development Development Differentiation Differentiation
Raf In resting cells, ERK is chiefly cytoplasmic and bound to its upstream regulator, MEK, either MEK in a free heterodimer or in larger multiprotein complexes ERK Cytoplasmic targets: cytoskeletal regulators, ERK kinases, cytoplasmic phosphatases etc. ERK Nuclear targets: Ets family transcription factors, IEGs, nuclear phosphatases etc.
Phosphorylation of ERK by MEK causes dissociation and nuclear translocation MEK MEK ERK ERK MEK ERK ERK Dephosphorylation of ERK by nuclear phosphatases facilitates reassociation with MEK and nuclear export
Schematic Schematic Representation of Representation of Regulatory Regulatory Mechanisms for Mechanisms for ERK Activation ERK Activation and Cellular and Cellular Responses Responses Ebisuya, M. et al. J. Cell Sci Sci. 2005;118:2997 . 2005;118:2997 Ebisuya, M. et al. J. Cell
Integrins 7TMR RTK Plasma Membrane FAK Paxillin KSR MEK Arrestin ERK MEK ERK Src Raf ERK Src Raf MEK PEA-15 MEK Focal Adhesion ERK Early Endosome MORG-1 p14 MEK MEK Sef MP-1 ERK ERK Golgi Arrestin Late Endosome ERK Src Raf Cytosol MEK Nucleus DUSP6 ERK ERK ERK DUSP4 DUSP5 Caunt & McArdle McArdle; Trends ; Trends Endocrinol Endocrinol. . Metab Metab., 2006 ., 2006 Caunt &
Dual- -specificity Phosphatases ( specificity Phosphatases (DUSPs DUSPs) Regulate ) Regulate Dual MAPKs by Removing Both Phosphate Groups by Removing Both Phosphate Groups MAPKs Required for Activation Required for Activation Dephosphorylation and anchoring DUSP Signalling ERK DUSP MEK MEK ERK ERK ERK Phosphatase DUSP binding ERK Dephosphorylation and release
Nuclear integrity and definition: DAPI stain (blue) Total ERK2 localisation: ERK2-GFP (green) Localisation of phosphorylated (active) ERK2: anti phospho-ERK2 Ab (red) ERK2-GFP ppERK2 DAPI
Graded Activation and Trafficking of ERK2- -GFP GFP Graded Activation and Trafficking of ERK2 Revealed by Frequency Distribution of Single Cell Revealed by Frequency Distribution of Single Cell Data Data 240 300 0nM 0nM 0.01nM 0.01nM Cell Number Cell Number 1nM 1nM 160 200 80 100 0 0 0.5 1.5 2.5 3.5 150 300 450 600 750 ppERK Intensity ERK2-GFP N:C Ratio
Ras PKC This model of Tyr kinase This model of Tyr kinase receptor versus PKC- - receptor versus PKC Raf Raf mediated activation of ERK mediated activation of ERK MEK MEK allows exploration of the allows exploration of the factors affecting transient factors affecting transient or sustained ERK or sustained ERK ERK ERK responses, which in turn responses, which in turn influence cell fate. influence cell fate. ERK ERK Transient Transient Prolonged Prolonged phosphorylation phosphorylation phosphorylation phosphorylation (peak 5- -15min) 15min) (peak 5- -30min) 30min) (peak 5 (peak 5 Transient nuclear Prolonged nuclear Transient nuclear Prolonged nuclear localisation localisation localisation localisation
Targeted siRNA Screening of Dual- -specificity specificity Targeted siRNA Screening of Dual Phosphatases (DUSPs DUSPs) ) Phosphatases ( DUSPs comprise a diverse array of enzymes with a range of substrates, including non-protein targets. 10 genes in this group form a structurally similar subgroup of MAPK phosphatases (MKPs) which specifically dephosphorylate MAPKs and are characterised by MAPK docking motifs. Other DUSPs do not contain these motifs and are termed “Atypical”, but can also directly dephosphorylate MAPKs (e.g. DUSP3 acts on ERK: Kang et al. Nat. Cell Biol. 2006).
siRNA Screening Parameters siRNA Screening Parameters 400 2.8 ERK2-GFP N:C Ratio EGF ppERK2 Intensity PDBu 300 2.1 200 EGF 1.4 PDBu 0 60 120 180 240 0 60 120 180 240 Time (mins) Time (mins)
ppERK2 Intensity ppERK2 Intensity N:C ERK2- -GFP Ratio GFP Ratio N:C ERK2 Ctrl A B C D E F G H I J K L M N O P 15’ PDBu Basal 5’ EGF 120’ EGF 120’ PDBu Basal 5’ EGF 120’ EGF 15’ PDBu 120’ PDBu -112 0 +112 -0.55 0 +0.4
M O N K H G D C B A P E L J F I Basal Ctrl siRNA EGF EGF ERKsiRNA WT ERK2-GFP D319N ERK2-GFP 1 Basal 10 Ctrl siRNA PDBu PDBu ERKsiRNA 46 WT ERK2-GFP D319N ERK2-GFP
Summary Summary The knock-down add-back system is a highly informative and robust method for studying ERK signalling and offers information unobtainable with other methods. Observation of activation state and trafficking of ERK in response to different stimuli indicates varying signal termination mechanisms.
Craig McArdle McArdle Craig Benit Maru Maru Benit Ann Finch Ann Finch Stephen Armstrong Stephen Armstrong
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