Spatiotemporal Regulation of ERK by Spatiotemporal Regulation of ERK - - PowerPoint PPT Presentation

Spatiotemporal Regulation of ERK by Spatiotemporal Regulation of ERK by Dual Dual-

• specificity Phosphatases

specificity Phosphatases

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 Stafford Lightman Lightman – – Transcriptional regulation by Transcriptional regulation by glucocorticoids glucocorticoids Christos Christos Paraskeva 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 Cell signalling -

• Mechanisms of Hormone Action

Mechanisms of Hormone Action

reproductive endocrinology reproductive endocrinology – – GnRH, gonadal steroids GnRH, gonadal steroids IN Cell 1000 Projects IN Cell 1000 Projects Context Context-

• dependence of steroid receptor signaling

dependence of steroid receptor signaling

• 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. Ctrl. E2 E2

Benit Benit Maru Maru

Cell Signalling Cell Signalling -

• Mechanisms of Hormone Action

Mechanisms of Hormone Action

reproductive endocrinology reproductive endocrinology – – GnRH, gonadal steroids GnRH, gonadal steroids 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 (HA-

• GnRHRs)

GnRHRs) Ann Finch & Kris Sedgley Ann Finch & Kris Sedgley IN3 ( IN3 (µ µ µ µ µ µ µ µg/ml) g/ml) 1 1 10 10 100 100 1000 1000

Cell signalling Cell signalling -

• Mechanisms of Hormone Action

Mechanisms of Hormone Action

reproductive endocrinology reproductive endocrinology – – GnRH, gonadal steroids GnRH, gonadal steroids IN Cell 1000 Projects IN Cell 1000 Projects ERK signalling: compartmentalisation and kinetics ERK signalling: compartmentalisation and kinetics

Jim Caunt Jim Caunt & & Steve Armstrong Steve Armstrong

DAPI DAPI nucleus nucleus ERK2 ERK2

• GFP

GFP ppERK1/2 ppERK1/2 DAPI/ DAPI/GFP GFP

c-Raf

MEK1/2 ERK1/2 MKK4/7 JNK1/2/3 MKK3/6

MAPKKK

MAPKK MAPK

A-Raf B-Raf ASK1 MLK3 MEKK1/4 MLK3 TAK DLK

p38α α α α/β β β β/γ γ γ γ

Stress, Cytokines, Stress, Cytokines, Growth Factors, Growth Factors, GPCRs GPCRs Growth Factors, Growth Factors, GPCRs GPCRs Stimulus Stimulus Inflammation, Inflammation, Apoptosis, Apoptosis, Growth, Growth, Differentiation Differentiation Growth, Growth, Differentiation, Differentiation, Development Development Response Response

Raf

MEK ERK ERK ERK

Nuclear targets: Ets family transcription factors, IEGs, nuclear phosphatases etc. Cytoplasmic targets: cytoskeletal regulators, kinases, cytoplasmic phosphatases etc. In resting cells, ERK is chiefly cytoplasmic and bound to its upstream regulator, MEK, either in a free heterodimer or in larger multiprotein complexes

MEK ERK ERK MEK ERK MEK ERK

Phosphorylation of ERK by MEK causes dissociation and nuclear translocation Dephosphorylation of ERK by nuclear phosphatases facilitates reassociation with MEK and nuclear export

Ebisuya, M. et al. J. Cell Ebisuya, M. et al. J. Cell Sci

• Sci. 2005;118:2997

. 2005;118:2997

Schematic Schematic Representation of Representation of Regulatory Regulatory Mechanisms for Mechanisms for ERK Activation ERK Activation and Cellular and Cellular Responses Responses

Paxillin RTK Integrins Arrestin Arrestin Src KSR Src Raf MORG-1 p14 MP-1 ERK MEK ERK MEK

FAK

PEA-15 MEK Sef ERK MEK ERK MEK Raf ERK MEK Src Raf ERK MEK DUSP5 DUSP4 DUSP6 ERK ERK ERK Nucleus Golgi Focal Adhesion Early Endosome Late Endosome Cytosol Plasma Membrane ERK 7TMR

Caunt & Caunt & McArdle McArdle; Trends ; Trends Endocrinol Endocrinol. . Metab Metab., 2006 ., 2006

DUSP MEK ERK MEK ERK ERK DUSP ERK DUSP ERK Signalling Phosphatase binding Dephosphorylation and anchoring Dephosphorylation and release

Dual Dual-

• specificity Phosphatases (

specificity Phosphatases (DUSPs DUSPs) Regulate ) Regulate MAPKs MAPKs by Removing Both Phosphate Groups by Removing Both Phosphate Groups Required for Activation Required for Activation

Nuclear integrity and definition: DAPI stain (blue) Total ERK2 localisation: ERK2-GFP (green) Localisation of phosphorylated (active) ERK2: anti phospho-ERK2 Ab (red) ppERK2 ERK2-GFP DAPI

Graded Activation and Trafficking of ERK2 Graded Activation and Trafficking of ERK2-

• GFP

GFP Revealed by Frequency Distribution of Single Cell Revealed by Frequency Distribution of Single Cell Data Data

0.5 1.5 2.5 3.5 80 160 240

0nM 0.01nM 1nM ERK2-GFP N:C Ratio Cell Number

150 300 450 600 750 100 200 300

0nM 0.01nM 1nM ppERK Intensity Cell Number

Transient Transient phosphorylation phosphorylation (peak 5 (peak 5-

• 15min)

15min) Transient nuclear Transient nuclear localisation localisation Prolonged Prolonged phosphorylation phosphorylation (peak 5 (peak 5-

• 30min)

30min) Prolonged nuclear Prolonged nuclear localisation localisation

Ras Raf MEK

ERK ERK

PKC Raf MEK

ERK ERK This model of Tyr kinase This model of Tyr kinase receptor versus PKC receptor versus PKC-

• mediated activation of ERK

mediated activation of ERK allows exploration of the allows exploration of the factors affecting transient factors affecting transient

• r sustained ERK
• r sustained ERK

responses, which in turn responses, which in turn influence cell fate. influence cell fate.

Targeted siRNA Screening of Dual Targeted siRNA Screening of Dual-

• specificity

specificity Phosphatases ( Phosphatases (DUSPs DUSPs) )

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).

60 120 180 240 1.4 2.1 2.8

EGF PDBu Time (mins) ERK2-GFP N:C Ratio

60 120 180 240 200 300 400

EGF PDBu Time (mins) ppERK2 Intensity

siRNA Screening Parameters siRNA Screening Parameters

A B C D E F G H I J K L M N O P Ctrl Basal 5’ EGF 120’ EGF 15’ PDBu 120’ PDBu Basal 5’ EGF 120’ EGF 15’ PDBu 120’ PDBu

ppERK2 Intensity ppERK2 Intensity N:C ERK2 N:C ERK2-

• GFP Ratio

GFP Ratio

• 112

+112

• 0.55

+0.4

1 46 10 Basal Ctrl siRNA ERKsiRNA WT ERK2-GFP D319N ERK2-GFP Basal Ctrl siRNA ERKsiRNA WT ERK2-GFP D319N ERK2-GFP

EGF EGF PDBu PDBu

A B C D E F G H I J K L M N O P

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 Craig McArdle McArdle Benit Benit Maru Maru Ann Finch Ann Finch Stephen Armstrong Stephen Armstrong