Martin Luther University Halle-Wittenberg
Lecture 6:Signal Transduction through
Integrins and Receptor TyrosineKinases
Prof. Thomas Groth
Biomedical Materials Group
Martin Luther University Halle-Wittenberg
Martin Luther University Halle-Wittenberg
Content
• Understanding mechanisms of signal transduction from fixed effectors (ECM molecules) into the cell.
• Understanding also mechanism by which soluble signaling molecules (growth factors) activate cells
• Learning that integrin signaling and growth factor receptors talk with each other
• Effect of material chemistry on integrin-mediated signaling and subsequent behavior of cells
• Role of integrins, cadherins, growth factor receptors and signal transduction in tumor development & progression
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Effectors of Signal Transduction
• Adhesive contact with extracellular matrix molecules (fixed, insoluble effector) – e.g. collagen, fibronectin, laminin
• Soluble effector –hormons, cytokines, & growth factors
• Adhesive contact with neighbouring cells – e.g. Cadherins
• Mechanical stress – e.g.. shear stress in blood vessel, mechanical load of bone, etc.
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Transduction of Signals– Immobilised Effector
target cellsignal emitting
cell
Signal transfer mediated by trans-membrane proteins (e.g.
cadherins in epithelia)
target cell
extracellular matrix ECM
Signal transfer from ECM molecules to specific cell receptors (e.g. integrins)
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Transduction of Signals – Soluble Effectorendocrine gland
blood vessel
target cells
endocrine signaling (e.g.
thyroid hormone)
paracrine signaling (e.g. growth factors)
secreting celltarget cell
autocrine signaling e.g. growth factors
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Intracellular signaling from integrinsand growth factor receptors –a detailed view
Note:- Focal adhesion (FA) turnover- Filipodia- Lamellipodia- Stressfibre formation- Cell cycle progressionmitosis(FERM – spezielle Bindungsdomäne)
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Phosphorylation Reactions
ATP or GTP (Guanosin triphospate) as phosphate donors
Transfer of one phosphate group to an organic acceptor (e.g. alcohol)
Phosphorylation of aminoacids with hydroxyl groups like serin, tyrosine, threonine
UC Davies ChemWiki
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Function of Phosphorylation in Signal Transduction
thermofisher.com
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ECM Synthesis
Integrin Functions
Motility Growth Differentiation
Apoptosis
Nucleus
Cytosol
Matrix adhesion
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Simplified Model of Focal Adhesion Plaque
Signalling:
FAK – Focal Adhesion Kinase
ILK – Integrin-linked Kinase
Src – Sarcoma-derived Kinase
Linker to cytoskeleton:
α-Act- actinin
Fil – Filamentin
Tal – Talin
Ten – Tensin,,
Pax – Paxillin, Vin - Vinculin
Plasma
membrana a a ab b b b
Extracellular Matrix
Aktin
Ten
a b
Fil Tal
a-Act Pax
ILKFAK
Pax
Vin
Vin
Src
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Integrin-Ligand-Binding – Opening of Intracellular Binding Sites by Separation of a & b Subunits
• Integrin transmit signals from outside into the cell Change of conformation upon ligand binding
• exposure of cryptic binding sites for cytoskeletal and signaling proteins
Gupta et al. Blood, 2007 15;109(8):3513-20.
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Integrin conformation-function relationships: a model.
Askari J A et al. J Cell Sci 2009;122:165-170
Ligation of Integrins Signal Transduction & Mechanical Tension
• Ligation of integrins to ECM induces signal transduction, cytoskeletal arrangement and mechanical tension
• Mechanical tension affects signal transduction & assembly and dissassembly of cytoskeleton
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Signal Transduction via Integrins -Phosphorylation of Focal Adhesion Kinase (FAK)
a b
FN
FAK
Paxillin
Talin
Vinculin
Actin
Src RTKP
Contact integrin -fibronectin
Binding of N-terminus of FAK tointegrin b subunit
conformational change of FAK followed by autophosphorylation
active conformation of FAK withformation of binding site SH2 domain for Src kinase.
Src – Sarcoma
RTK – receptor tyrosine kinase
SH – sarcoma kinase homologous bindingdomains
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Integrin Clustering is Prerequiste for Full Catalytic Activity of Kinases
ab
FN
FAKP
Paxillin
Talin
Vinculin
ab
FN
FAKP
Paxillin
Talin
Vinculin
P P
PP
srcsrc
PP
P
P
• Oligomerisation of FAK through clustering of integrins.
• Trans-phosphorylation of FAK.
• Involvment of Src-receptor tyrosine kinases phosphorylation of Tyr-576 and Tyr-577 residues and full catalytic activity of FAK.
• Phosphorylation of Tyr-925 – Formation of binding site for adaptor proteins (growth factor receptor-bound protein 2 (Grb2) und SOS (son of sevenless protein)).
• Paxillin and vinculin become phosphorylated
by Src as well.
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Colocalisation of FAK with Integrins in Focal Adhesion Plaques
a b
FN
FAK
FAK Alpha 5
Cell membrane
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Tyrosine Phosphorylation of FAK in Focal Adhesion Plaques
Colocalisation of Phosphotyrosine and FAK (orange)
a b
FN
FAK
P
Cell membrane
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Hydrophobic Biomaterials Reduce Clustering of Integrins and Signal Transduction
Hydrophobic material
Hydrophilic material
Tyrosin phosphorylationav Integrin
Groth, Altankov (1996) Biomaterials
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Tyrosine Phosphorylation Decreased on Hydrophobic Substrata
0
1
2
3
4
5
6
7
8
9
glass APS ODS SINo
rma
lize
d p
ho
sp
ho
tyro
sin
e
ac
tivit
y
Water contact angles
20° 60° 90° 110°Groth, Altankov (1996), Biomaterials
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0
20
40
60
80
100
0
0,5
1
1,5
2
2,5
3
CH3 PEG NH2
C11
NH2 C3 COOH OH
Growth index
Observation: Growth and Viability of Fibroblasts Depends on Type of Surfaces
Viability(%)
Cells are seeded and cultured under same conditions -only head groups on substrata are different
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Receptor Tyrosine Kinases (RTK) and Signal Transduction
• Ligand (e.g. epidermal growth factor - EGF) binds on RTK domain outside the cell.
• RTKs – transmembrane protein
• RTKs possess intracellular tyrosine kinase domain.
• Cytosolic part of RTK has several tyrosine residues.
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Function of Receptor Tyrosin Kinases
• Receptor becomes activated by ligand binding.
• Followed by dimerisation of receptors and trans-phosphorylation.
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Growth Factor-Dependent Signal Transduction –Role of Ras I
• EGF – Epidermal growth factor
• Ras – rat sarcoma protein
• Grb2 - growth factor receptor-bound protein 2
• SOS - son of sevenless protein
• SH 2/3 – Src homologous binding domains
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Growth Factor-Dependent Signal Transduction –Role of Ras II
• Ras (rat sarcoma) – GTPase switch protein
• Signal transducer for many receptor tyrosine kinases
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MAP Kinase Pathway I
• MAP – mitogen-activated proteins
• Active Ras binding + activating Raf
(Rapidly growing fibrosarcoma)
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MAP Kinase Pathway II
• MEK – mitogen activated protein kinase
• MAPK – mitogen activated protein kinase
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Growth Factor-Dependent Signal Transduction - Overview
P
P
P
P
EGF
Cytosol
Adapter
proteins
Ligand binding induces Dimerisation and trans phosphorylation of receptor tyrosine kinases (RTKs).
Ras
Membrane
MAPK
active
Cascade of kinases:
Raf-MEK-MAPK
Mitogen-Activated-Protein-
Kinase (MAPK) inactiv
Nucleus
P
P
Grb2SOS
MAPK
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Cooperation Between Growth Factor and Integrin Dependent Signal Transduction
P
P
P
P
EGF
Cytosol
Adapter-
protein
RasMembrane
MAPK
activ
MAPK
inactiv
Nucleus
P
P
b
FN
aCaveolin
Integrins connected by b subunit & p-FAK via same adaptor proteins like RTKs!
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Cooperation Between Growth Factor and Integrin-Dependent Signal Pathways: Caveolin and a-Subunit of Integrins
P
P
P
P
EGF
Cytosol
Adapter-
protein
Ras
Membrane
MAPK
aktiv MAPK
inaktiv
Nucleus
P
P
b
FN
aCaveolin
Fyn/
ShcGrb2/
SOS
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Induction of Gene Transcription
• TCF – ternary complex factor
• SRF – serum response factor
• SRE – serum response element of DNA
• RSK - ribosomal s6 kinase c-fos gene – (like c-jun) encoding proteins for progress through cell cycle Mitosis
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Survey on Signal Transduction Pathways induced by RTKs
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Regulation of RTK Activity
Inhibition of kinase activity by phosphorylation of domains near membrane by protein kinase C (PKC), or dephosphorylation of tyrosine resdiues by protein tyrosinephosphatases (PTP), by endocytosis or by ubiquitinylation and degradation in proteasome
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Contact Inhibition (Blocking Mitosis of Cells) by Ligation of Cadherins Related to RTKs
VE–cadherin plays a pivotal role in contact inhibition of cell proliferation
Vascular endothelium (VE)–cadherin (, through its interaction with Dep-1 or other tyrosine phosphatases inhibits vascular endothelial growth factor (VEGF) proliferation signal via mitogen-activated protein kinases (MAPK) activation in three ways:
(i) by reducing VEGFR2 phosphorylation,
(ii) (ii) by inhibiting VEGFR2-associated Shcphosphorylation and
(iii) (iii) by decreasing VEGFR2 internalization and signaling from endosomal compartments.
VE–cadherin complex interaction with activated VEGFR2 also enhances VEGF survival signaling by PI3kinase/Akt pathway.
Biochimica et Biophysica Acta (BBA) – Biomembranes Volume 1778, Issue 3, March 2008, Pages 794–809Apical Junctional Complexes Part I
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Contact Inhibition by Ligation of CadherinsRelated to RTKs
Okada T et al. J Cell Biol 2005;171:361-371© 2005 Rockefeller University Press
Cadherin-initiated adhesion prevents activation of Pak in contact-inhibited cells and thereby causes accumulation of dephosphorylated Merlin. This closed form of Merlin suppresses integrin-mediated recruitment of Rac, and hence mitogenic signaling. Upon release from contact inhibition, Pak phosphorylates and inactivates Merlin, allowing recruitment of Rac to the membrane and mitogenic signaling.
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Role of Receptors and Signal Transduction in Tumor Development and Progression
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The Metastatic Cascade
Changes in adhesion duringmetastatic journey
Loss of E-Cadherin mediatedcell-cell adhesions
Migratory phenotype penetratesbasal lamina & invadesinterstitial extracellular matrix
Tumor angiogenesis with entryinto blood stream
Aggregation of tumor cells withplatelets and leukocytes
Occlusion of capillaries in targettissue enables extravasation ofcells
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Focal adhesion kinase (FAK) and Src-family kinases (SFKs) integrate pro-migratory signals from integrins and RTKs (receptor tyrosine kinases)
SMAD/AP-1 –transcription factors involved in progression of cell cycle (c-Jun, c-fos genes)
Integrin-RTK-Signaling Induces Cell Migration and Invasion
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Integrin-RTKs-Signaling Disrupts Cell-Cell-Adhesion
• Signaling to FAK through integrins activates ILK & SFKs like v-Src
• v-Src phosphorylation & internalisation of E-cadherin
• Signaling through ILK and Src supression of E-Cadherin gen expression
• Activation of Hakai that ubiquitinylates E-Cadherin endocytosis
Decrease of expression & increased endocytosis of E-Cadherin
Loss of cell-cell adhesions
Snail & Slug – Transkription factors blocking E-Cadherin expression
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Integrins and Matrix Remodelling
• avb3 integrin recruits metallomatrixprotease – 2 (MMP-2) to cell surface local degradation of extracellular matrix during invasion of cancer cells
• Integrins like avb3 associate with urokinase plasminogen activator (uPA) receptor (uPAR)
• Plasminogen converted to plasmin activates other MMPs precursors increased degradation of ECM
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Integrins Promote Oncogenic Signals of Activated RTKs
• Constitutively activated RTKs (mutation or overexpression) induce phosphorylation of b4 subunit of a6b4 integrin (hemisdesmosomes) docking sites for SHCs und PI3K kinases
• Cytoplasmatic domain of b4 signaling adaptor amplifying mitogenic, survival and motogenic signals
• SHC – adaptor protein, PI3K –Phosphatidylinositol-3- kinase
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Receptor Cross-Talk in Metastatic Dissemination
• “Resting” (healthy) cell integrins are engaged, cadherin-mediated adhesion is intact and proteinase (MMPs) expression is low.
• “Activated” (cancer - for example, ligand or mutational activation of EGFR signaling, or other mechanisms to stimulate cadherin or integrin-mediated signal transduction Disruption of cadherin-based cell adhesion and/or integrin clustering initiate signaling cascades increased proteinase production and activation Secreted and membrane-associated proteinases disruption of cadherin function through cleavage protein domains and generation of the soluble E-cadherin ectodomain shed E-cadherin ectodomain potentiate junction dissolution through protein-protein interactions with endogenous E-cadherin.
Copyright 2009 Stack Scientific
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TK
EGFR Function in Normal Cells
TKP P
Cell Proliferation Antiapoptosis
Angiogenesis
Gene Transcription
Cell Cycle Progression
+
© Dr.M.Jayanthi
Growth factor
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Growth Factor-Dependent Signal Transduction – Role of Ras II
• Ras (rat sarcoma) – GTPase switch protein
• Signal transducer for many receptor tyrosine kinases
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Constitutively Active Ras/Raf Mutations in Cancer
Nat Rev Cancer 2007;7:295
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RTK
Gene transcription
Cell Cycle Progression
Cell Proliferation Metastasis
Anti Apoptosis
Cancer
ATP
Aberant Mutation of EGF Receptor Constitutively Active
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Difference between EGFR Variant from Wild Type
EGFR - Variant III EGFR Wild type
No extracellular domain Present
Ligand cannot bind Can bind
RTK constitutively active RTK activated by ligand binding
Cannot dimerize Can dimerize
Not found in normal cells Found in normal cells
More propensity for cancer Up regulation leads to cancer
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Normal Cell Cancer Cell
Up Regulation
Mutation
Consequence of Overexpression of EGFR Receptors (Mutants & Wild Type)
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Expression of Aberant EGF Receptors in Different Tumors
Tumor EGFR Expression Rate
Breast 14 % - 91 %
Colon 25 % - 77 %
Lung Cancer (Non small cell) 40 % - 80 %
Head & Neck 80 % - 95 %
Ovarian 35 % - 70 %
Pancreatic 30 % - 50 %
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Strategies to Inhibit EGFR Signaling
RTK RTK RTK RTK
- - - -
EGFR tyrosine
kinase inhibitorsAnti-EGFR mAbs
Anti-ligand mAbs
BispecificAbs
Imm
un
e effector
cell
ATP
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Biopharmaceutical for Treatment of EGF Receptor Malignancies
Biopharmaceutical Effect
Gefitinib Highly selective, potent & reversible EGFR Tyrosine Kinase inhibitor
Erlotinib (see Gefitinib)
Cetuximab Monoclonal Anti-EGF receptorantibody
H 447 Bispecific anti EGF receptro antibodylinked to anti CD 64
MDX 210 (see H 447)
CD 64 – Anti-Fc receptor present on monocytes & macrophages
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Take Home Messages
• Signal transduction processes crucial for normal life of cells controlling tissue structure, motility, growth & differentiation
• Cross-talk between receptors for fixed and soluble signals in a cooperative manner
• Cancer development linked to disturbance in signal transduction processes by mutations or overexpression of signaling molecules, loss of inter-cellular adhesions and uncontrolled growth, proteolytic activity and migratory phenotype
• Biopharmaceuticals try to block RTKs activity by inhibiting binding of extra and intracellular ligands
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Literature
• Molecular Cell Biology 4th Edition; Lodish, Berk, Zipursky, Matsudaira, Baltimore, Darnell (2002) Freeman & Company
• Giancotti & Ruoslahti Science 285, 1999
• Danen & Yamada Journal of Cellular Physiology 189, 2001
• Hood & Cheresh Nature Reviews Cancer 2, 2002
• Guo & Giancotti, Nature Reviews Molecular Cell Biology 5, 2004
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