Human Tumors Harboring Ras Mutations

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Imaging Signal Transduction in Living Cells with
GFP
Molecular Pharmacology of Receptors Fall 2003
Mark Philips, M.D. Depts. of Medicine, Cell
Biology and Pharmacology
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A Cellular Signaling Network
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Signaling Data
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The Jellyfish Aequorea victoria
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Structure of the Green Fluorescent Protein of
Aequorea victoria
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Fluorophore of GFP
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The Wonderful World of GFP
GFP-Actin
GFP Mouse
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Labeling Proteins with Tetracysteine Motifs and
FlAsH
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Nascent Connexin 43 is Added to the Periphery of
Gap Junctions Pulse labeling with FlAsH followed
by ReAsH
Gaietta et al., Sciene 296503, 2002
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GFP in Signaling I Protein Localization
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Posttranslational Processing of GTPases with a
CAAX Motif
Prenylation
Farnesyl or Geranylgeranyl Modification
Proteolysis
AdoMet
AdoHcy
AFC
Carboxyl Methylation
Methyl Esterase
MeOH
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Putative Topology of Mammalian pcCMT
GFP
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Targeting Ras to the Plasma Membrane
Direct
Cytosol
Ras
Indirect
ER/Golgi
Plasma Membrane
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Localization of GFP-N-Ras in Live Cells
MDCK
COS-1
CHO
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GFP in Signaling II
Analysis of Protein Diffusion by Fluorescence
Recovery After Photobleaching (FRAP)
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Limits to Diffusion of Transmembrane Proteins in
the Plane of the Membrane
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Principles of FRAP
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Principles of FRAP
VSVG-GFP at 40C Tunicamycin
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GFP in Signaling III Relocalization
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Gq Signaling Through PLCb
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PLC Signaling Monitored by Relocalization of
PLCd-PH-GFP Angiotensin II Stimulation of COS-7
Cells
Várnai Balla, J. Cell Biol. 143501, 1998
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B-Cell Antigen Receptor Signaling
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B-Cell Antigen Receptor Signaling Reported by
GFP-PKCg Translocation
Oancea Meyer, Cell 95307, 1998
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Downregulation of GPCRs via bArrestin-mediated
Endocytosis
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The Wnt Signaling Pathway
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Disheveled is Reguired for bArrestin-mediated
Internalization of Frizzled
Chenet al., Science 3011391, 2003
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T cell receptor signaling
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T cell receptor signaling
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H-Ras but not K-Ras Associates with Patched TCR
in Lipid Rafts of Jurkat T cells
NT
CD3CD28
TR-Anti-mouse IgG
GFP
Overlay
GFP
CD8-GFP
GFP-Hras
GFP-Kras
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GFP in Signaling IV
Fluorescence Resonance Energy Transfer (FRET)
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Principles of FRET
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The EGF Signaling Pathway
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Tyrosine Phosphorylation of Cytoplasmic Domain of
EGFR Monitored by FRET
Verveer et al., Science 2901567, 2000
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Tyrosine Phosphorylation of Cytoplasmic Domain of
EGFR Monitored by FRET
Verveer et al., Science 2901567, 2000
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Lateral Propogation of EGFR Activation
Verveer et al., Science 2901567, 2000
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Fas Signaling
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FRET Demonstrates Preassembly of Fas Trimer
Siegel et al. Science 2882354, 2000
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GFP in Signaling V FRET Biosensors
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Signaling by Second Messenger Cyclic AMP
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Intracellular FRET Probe for cAMP
Zaccolo Pozzan, Science 2951711 1 March 2002
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Phosphodiesterase-dependent Localization of
cAMP in Microdomains of Neonatal Cardiac Myocytes
Demonstrated by FRET
Zaccolo Pozzan, Science 2951711 1 March 2002
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The Ras Signaling Pathway
Growth Factor
Membrane
GTP
GRB
SOS
ras
ras
GDP
PTKR
raf
Phosphotyrosine
SOS
Phosphorylation
GRB
MEK
SH2
SH3
Phosphorylation
Erk
Cytoplasm
Phosphorylation
Nucleus
Elk
Transcription
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Membrane Targeting of Raichu-Ras FRET Probe
Mochizuki et al., Nature 4111065 2001
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Ras Activation in COS Cells Reported by Raichu-Ras
Mochizuki et al., Nature 4111065 2001
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Hypothesis Ras Localized on Intracellular
Membranes is Capable of Signaling
GFP-H-Ras
GFP-H-RasC181,184S
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In vitro Probe for RasGTP
Binds RasGTP with 103 Fold Higher Affinity than
RasGDP
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Raf-1 RBD is an in vivo Reporter of Activated
H-Ras
RBD(Raf-151-131)-GFP
RBD(Raf-151-131)-GFP H-Ras61L
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Activation of H-Ras on Internal Membranes
GFP-H-Ras
GFP-H-Ras61L
GFP-H-Ras17N
GFP-RBD
GFP-RBD H-Ras
GFP-RBD H-Ras61L
GFP-RBD H-Ras17N
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Activation of H-Ras on Plasma Membrane and
Golgi of EGF-stimulated COS-1 Cells
0 min
5 min
10 min
20 min
40 min
60 min
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Detection of RBD Recruitment to Membranes By
Bystander FRET
CD8
Membrane
YFP
Ras
508 nm
530 nm
458 nm
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Activation of Endogenous Ras on Plasma
Membrane and Golgi of Insulin-stimulated COS-1
Cells (Bystander FRET)
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EGF Rapidly Stimulates Activation of
Palmitoylation-deficient (C181,184S) H-Ras on
Internal Membranes of Serum-starved COS-1 Cells
0 min
1 min
2 min
3 min
5 min
10 min
20 min
30 min
60 min
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Fin