Signal transduction signal transduction system composes: receptor, enzyme,pathway
Perturbation of Signal Transduction
description
Transcript of Perturbation of Signal Transduction
Perturbation of Signal TransductionPathways for Growth Factors and Growth Inhibitors in
Malignant Transformation
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Different isoforms of PDGF form three types of receptor dimers
PDGF-AB PDGF-BB PDGF-DDPDGF-AAPDGF-CC
Ligandisoform
Extra-cellular
TM
Intra-cellular
Receptortype
In vivo function of PDGF
Embryonal development• kidneys (mesangial cells)• blood vessels (smooth muscle cells, pericytes)• lungs (alveolar smooth muscle cells)• CNS (oligodendrocytes)
Stimulation of wound healing
Regulation of interstitial fluid pressure
Ligand-induced activation of PDGFR causesa conformation switch in the receptor C-terminus
0 10 20 30 40 50 60 700
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5000
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10000
Wt
ct29
ct46
ct75
Time (min)
Kin
as
e a
ctv
ity
0 10 20 30 40 50 60 700
2500
5000
7500
10000
PDGFR autoinhibition by a C-terminal motif
No PDGF
+ PDGF
Time (min)
Kin
as
e a
ctv
ity
PDGFR
ct75-
ct46-
ct29-
- Y
Control
PDGF
Wt ct46 ct46-D850N
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10
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vect
orW
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D850N
ct46
ct46
-D85
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M-R
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71K
Nu
mb
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PDGFR activation by mutating the activation loop and the C-terminus
Binding of SH2 domain molecules to autophosphorylatedtyrosines in the PDGF -receptor
P
P
P
P
PP
P
P
P
P
PP
P
PP
P
P
P
P
P
P
P
PP
PLC- mitogenicity
562579581589
716740751763771775778
857
966 97010091021
SHP-2 Grb2/Sos1 Ras Erk MAPK mitogenicity
STAT
RasGAP
Nck
Src mitogenicity
Shc Grb2/Sos1 Ras Erk MAPK mitogenicity
PI3’-kinase Rho, Rac,JNK MAPK actin reorganization and chemotaxis
Grb2/Sos1 Ras Erk MAPK mitogenicity
SS S
S
Akt survival
200
116
97
66
50
37
25
1 2 3
PDGFRPDGFR
RasGAP / FAK
Hsp84
Hsp A5SHP2Stam1
Erk1
Grb2
Alix
Identification of tyrosine phosphorylated proteins after PDGF stimulation
1. Serum-starved cells
2. PDGFR expressing cellsstimulated with PDGF-BB
3. PDGFR expressing cellsstimulated with PDGF-BB
Alix overexpression decreases the rate of PDGFR removal from the cell surface
PAE/PDGFR
PAE/PDGFR-Alix
0
50
100
0 10 30 60PDGF-BB stimulation (min)
Cel
l su
rfa
ce P
DG
FR
(% o
f in
itia
l)
1.0 8.2
Alix
PAE/PDG
FR-
Alix
-actin
PAE/PG
FR
K Monoubiquitylation
Ub
KKKMultimono-ubiquitylation
UbUbUb
Ubiquitylation of proteins
KPolyubiquitylation(via K48 and K63)
UbUb
UbUb
UbUb
UbUb
K
0
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0 60 120Time (min)
Ub
iqu
itin
atio
n (
a.u
.)
PAE/PDGFR
PAE/PDGFR-Alix
PDGF-BB (min) 0 30 60 120 0 30 60 120
Ip: PDGFRIb: HA-Ub
Ip: PDGFRIb: PDGFR
Ub-PDGFR
PDGFR
PAE/PDG
FR
PAE/PDG
FR-
Alix
Alix overexpression reduces PDGFR ubiquitination
AlixAlix AlixAlixP
P
Cbl
Ub
Alix may stabilize PDGFR by inducing Cbl degradation
Downregulation of receptor tyrosine kinases
TC-PTP ko wt
PDGF-DD - + - +
IB: P-Tyr
IB: PDGFR
Ligand-activated PDGF -receptor is hyperphosphorylated in TC-PTP knockout cells
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10 wt
TC-PTP ko
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rec
epto
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hos
ph
oryl
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Decreased rate of PDGF -receptor degradation in TC-PTP ko MEFs
Direct detection of recycled PDGF -receptors in TC-PTP ko MEFs
The PDGF -receptor co-localizes with Rab4a after 10 min stimulation in TC-PTP ko MEFs
PDGF -receptor Rab4a-EGFP
No colocalization between Rab11EGFP and PDGFR inTC-PTP ko MEFs after 20 min of stimulation
degradation
TC-PTP?
PDGFRPDGFR
TC-PTPP
XP
ESCRT?P
YP
P
XP
P
P
P
PTP?
Trafficking of PDGF receptors
cc
c cc c
N-glycosylation
O-glycosylation
Glycosaminoglycan chain
c Cysteine residues
P
P
P Phosphorylationsite
Structure of the hyaluronan receptor CD44
Link domainHyaluronan-binding domain
Basic motif
Variant exon (v1-v10)Extracellular
Transmembrane
IntracellularERM
Actin
Co-immunoprecipitation of PDGF ß-receptor and CD44
IP:
IB: PDGFRCD-44 PY99
PDGF-BB: - - + - - + - - +
PDGF-BB: - - + - - + - - +
IP:
rabbit IgG
rabbit IgG
rabbit IgG
PDGFR PDGFRPDGFR
ratIgG
CD-44 CD-44CD-44 ratIgG
ratIgG
IB: CD44 PDGFRPY99
PDGFR
CD44
PDGFR
CD44
Hyaluronan - + +Monoclonal Hermes1, that block hyaluronan binding to CD44, restores PDGF ß-receptor activation
Hermes 1 - - +
PDGF-BB + + +
PY99
PDGFRß
Co-exposure of skin fibroblasts to PDGF-BB and hyaluronan inhibits PDGF ß-receptor activation
Hyaluronan (µg/ml) 0 10 25 50 100 0 10 25 50 100PDGF-BB - - - - - + + + + +
PY99
PDGFRß
PTPases account for the hyaluronan-induced PDGF ß-receptor dephosphorylation
Quantification
PervanadateHyaluronan
PDGFR
PDGF-BB
PY99
1 1 10 20 100 220 55 225
Cross-talk between CD44 and the PDGF ß-receptor
CD44-receptor
CD44
-receptor
PPP
PPTPinactive
PP
PP
PDGF-BBPDGF-BB
PTPactive
Signaling
Hyaluronan
Modulated signaling
PDGF in disease
Fibrotic conditions• lung fibrosis• glomerulonephritis• liver cirrhosis• myelofibrosis
Atherosclerosis
Malignancies• autocrine stimulation (glioblastoma, sarcoma)• paracrine stimulation (cancers)
Dermatofibro-sarcomaprotuberans
Chronic mono-myclocytic leukemia
Hyperoesinophilicsyndrome
Gastrointestinalstromal tumor Glioblastoma
PDGF antagonists
PDGF aptamer
-DNA molecule whichbinds PDGF B-chainwith high affinity
-specific
-expensive, cumbersometo administer
STI571 (Glivec, imatinib)
-LMW inhibitor of PDGFreceptors, c-Kit, Abl,and Arg
-not absolutely specific
-inexpensive, easy toadminister
Difference in STI571(Glivec/imatinib) sensitivitybetween different glioblastoma cultures
Clustering of mRNA profiles of glioblastoma cell cultures
PDGF1
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Effects of PDGF in tumors
bloodvessel
stromal fibroblast
lymphvessel
tumorcells
Effect of anti-PDGFR and anti-VEGFR treatment on B16 melanoma tumor growth
PDGF and the interstitial fluid pressure (IFP) of tumors
•PDGF -receptor activation increases IFP in normal connective tissue
•Many solid tumors display increased IFP
•Increased tumor IFP prevents drug uptake into tumors by decreasing transcapillary transport
•Many solid tumors express PDGF receptors on pericytes and stroma cells
Src
PLC-
Shc
PI3'- kinase
SHP-2
Grb2/Sos1
mitogenicity
mitogenicityRas MAP kinaseGrb2/Sos1
actin reorganization /chemotaxis/migration
Rho, RacAkt
mitogenicityRas MAP kinaseGrb2/Sos1
mitogenicityRas MAP kinase
/ mitogenicityactin reorganization
survival
Binding sites for signal transduction molecules
on the PDGFR-
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-1.5
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Regulation of Interstitial fluid homeostasis
by the PDGFR- via PI3'kinase
mice with wild type PDGFR-
subdermal injectionof PDGF-BB
injection of C48/80
interstitial fluid pressurepif [mmHg]
time[minutes]
mice with PI3´K binding site mutant PDGFR-
Forces that regulate transcapillarytransport in tissues
b Tumour tissuea Normal tissue
Blood vessel
Net outwardfiltration pressure1-3 mm Hg
Net inward oroutward pressure(-18-2 mm Hg)
COPIF
8 mm HgPIF
-1 to -3 mm HgCOPIF
20 mm HgPIF
10 -30 mm Hg
b Tumour tissuea Normal tissue
b Tumour tissue
Structural differences between normal and tumourtissues that affect interstitial fluid pressure
PDGF and tumor IFP
Tumor-derived PDGF contributes to increased tumor IFP•
- decreases tumor IFP
- synergizes with chemotherapy of tumors
Treatment with PDGF antagonist•
- increases drug uptake in tumors
AcknowledgementsLICR, Uppsala Uppsala University
Bengt WestermarkLena Claesson-WelshKristofer Rubin
Linköping UnivÅke WastesonKarolinska InstituteMonica NisterArne ÖstmanChrister BetsholtzLund Univ. in MalmöLars RönnstrandGothenburg Univ.Keiko FunaLICR, StockholmUlf Eriksson
Johan Lennartsson Piotr Wardega Lotti Rorsman Alexandra JurekCarina Hellberg Aga Koslowska-Wardega Susann Karlsson Christian SchmeesEvi Heldin Lingli LiRainer Heuchel Monica Krampert Shioto Suzuki Aive Åhgren
Ken AmagasakiGudrun BäckströmFederica ChiaraJean-Baptiste DemoulinSimon EkmanMasao FuruhashiYuko HasumiDaniel HägerstrandAnders KallinKaska KowanetzRonggui LiCamilla LoomanKristian PietrasAkira ShimizuTobias Sjöblom