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					                                  Lecture 08,02.13.01
                         Integrin signaling contd., JAK/STAT

Slide1: References
Yamada, K, & Danen, E. 2000. Integrin signaling, pp1-25. From Signaling networks and
cell cycle control, Ed. Gutkind, JS, Human Press.
Giancotti & Rusolahti, 1999. Integrin signaling. Science 285:1028-32.
Hynes, R. 1992. Integrins: Versatality, modulation, and signaling in cell adhesion. Cell
69:11-25.
Kumar, CC 1998. Signaling by integrin receptors. Oncogene 17:1365-1373.
Schlapfer, et al., 1999. Signaling through focal adhesion kinase Progress in Biophysics
Mol. Biol. 71:435-478.
Holly et al., 2000. Multiple roles of integrins in cell motility. Exp. Cell Res. 261:69-74.

Slide 2: Integrin in focal adhesion
Trasnmembrane glycoproteins
Alphas, 1-10; E, IIb, v, L, M, X. Alphas are about 120-180kd.
Betas: 90-110kd. 1-8.
Beta1 associates with a1-9, av
Beta 2, aL, aM, aX
Beta3, av, aIIb
Beta4, a6
Beta5, av
Beta6, av
Beta7, a4, aE
Beta8, av

Cytochalasin D can disrupt integrin signaling without affecting growth factor signaling.
Most subunits have short cytoplasmic tail.
Beta4 is exception, associates with keratin filaments in hemidesmosomes.
17 alpha subunits and 8 beta subunits.
But only 22 combinations. Therefore, restricted diversity.
Alpha chain maybe involved in ligand specificity. Many splice variants.
B1, b3,b4,a3,a6,a11b can be alternatively spliced.
Integrins bind their ligands with a low affinity when sparsely distributed.
The integrins can bind their ligands only when they exceed a certain minimal no. of
integrins in certain places called focal adhesions and hemidesmosomes.
The weak binding to ligands make it poss for cells to move and explore ECM.

Variety of mols including talin, a-actinin, FAK binds integrin.
Binding of cytoplasmic mols to integrin may modulate integrin activity by
phosphorylation of integrin tails. Tyrosine phosphorylation of b4 has been shown to be
necessary for hemidesmosome formation and for triggering of binding of Shc/Grb2 to
initiate signaling.
Cytoplasmic tail of beta connects to focal adhesion inside and actin csk as well as FAK.




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Integrins lack intrinsic kinase activity.
Therefore their cytoplasmic tails associate with adaptor proteins that connect integrins to
csk, kinases and RTKs.
They regulate cell proliferation, differentiation, motility, and survival.

Integrins and cell proliferation
Slide3: FAK and Src/Grb2/SOS signaling
Non receptor tyrosine kinase with no SH2 and SH3 domians.
One of the first mols activated by integrin.
Talin may recruit FAK to integrin, but mechansim not completely understood.
Upon recruitment FAK may undergo confirmational change and interact with beta tail
thru N-terminus.
The N-terminus of FAK is autoinhibitory, therefore this interaction may initiate FAK
catalytic activity.
Integrin mediated adhesion with concommitant FAK autophosphorylation due to
oligomerization, enhances its activity.
FAK phosphorylation by integrin is not dependent on ligand occupancy, only clustering.
You can make them cluster by binding to non- functioning antibody.
Autophosphorylation of FAK at Y397 results in binding of SH2 domain of Src and Fyn.
Src can phosphorylate Y925 of FAK and create a binding site for Grb2-SOS.
In vitro FAK substrates= Tensin, Paxillin (biological significance not determined.)
These are two cytoskeletal proteins with signaling function.
(PAK and Paxillin paper)

Slide 4:
FAK structure
Catalytic kinase domain in center.
Nterminal domain shares sequence homologywith Band 4.1 proteins and directs
interactions with integrins and growth factor receptors.
Y397 Src binding
C-term has multiple protein-protein interaction sites.
Catalytic domain has Y576, Y577 regulatory site
Next 2 proline rich sites. Site I and II.
Site 1 binds Cas
Site II binds GRAF/ASAP SH3 domains
GRAF, a GAP for Rho and ASAP1 a GAP for Arfs 1 and 6.
But role unclear. Mutations in this site does not affect FAK signaling.
4serines 722, 843, 846, 910, role unclear.
FAT focal adhesion targeting
Paxillin and Talin binding
Grb2 biniding
FRNK, truncated version, negative regulator of full lebgth. found in chicken fibroblasts.
Generated by promoter located within intron downstream of catalytic domain.

Slide 4A
Fun Facts on Fak



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FAK homologs in human, chicken, frog .
FAK homolog, Pyk-2
Clearest biological effect of FAK=cell migration (Yamada)
FAK activates Ras MAPK signaling

Slide 5: Integrin associated FAK signaling (Giancotti and Rusolahti, 1999. fig 2)
Ability of FAK to enhance cell migration requires autophosphorylation at Y397.
Autophosphorylation of FAK at Y397 can activate SH2 domains of Src or Fyn as well as
PI3K.
Src can phosphorylate a no. of focal adhesion components. Major targets Paxillin and
Tensin.
Src also phosphaorylates p130CAS, FAK binds p130CAS (Crk associated substrate), a
docking protein thatr ecruts adaptor proteins Crk and Nck.
FAK binds: c-Src, CSK, PI3K, p130Cas, Grb2, paxillin, talin, vinculin, and a actinin.
Y397 binds Src
Src and Fyn phosphorylates paxillin, tensin, p130Cas.
Y925 (phosphorylated by Src) binds Grb-2
Binding of FAK to src and Grb2 is dependent on phosphoY but the other proteins will
bind to non phosphorylated FAK.
FAK is phoaphorylated on tyr by adhesion, but during mitosis, FAK becomes
phosphorylated on serine and dissociates from Src and p130CAS.

Shc can signal to Ras/ERK pathway.

FAK also signals to PI3K

FAK knockouts: die with defects in gastrulation and show defective migration.
Embryonic fibroblasts formed from FAK nulls have numerous small focal contacts, but
fail to form large peripheral focal contacts and do not spread well.
Dominant negative FAK can disrupt cell motility and proliferation.
Overexpression enhances cell migration.

Both FAK and Shc are negatively regulated by phosphatases
Cytoplasmic tyrosine phosphatases:
PTP-PEST and PTP-1B
Targets of p130Cas may specifically inhibit signaling downstream of FAK.
PTP-PEST has a C-terminal extension that anchors it to cytosolic face of ER. Integrin
mediated adhesion activates calpain, which cleaves this extension and releases
phosphatase so relocate to the focal adhesions.

PTEN
Lack of PTEN in certain cancers increase phosphorylated FAK, Shc and PIP3.

ILK, Integrin linked kinase:
Serine-threonine kinase.
In contrast to FAK, ILK is inhibited in response to integrin ligation.



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Binds b integrin and colocalizes to focal adhesions.
implicated in anchorage dependent growth because overexpression of ILK induces
anchorage- but not serum-independent growth.
ILK overexpression also induces cyclin D1 expression, cdk4 expression and Cyclin
E/Cdk2 activity. Overexpression of ILK therefore leads to transformation.
However, signaling pathway from integrin cytoplasmic tails to these activities not known.

ILK promotes translocation of b catenin to the nucleus, and b catenin can regulate
cyclinD1 promoter thru TCF/LEF transcription factor.

Slide 6: Integrin associated caveolin signaling
Novel signaling apthway involving integrin alpha subunits, oligomeric membrane protein
caveolin complexes, Fyn, and Shc phosphorylation.
Caveolin-1 may help iintegrins to cluster on the pm. And function as an adaptor.
This function of caveolin is consistent with its ability to bind cholesterol and
glycosphingolipids and organize specialized pm “rafts”, which are enriched in Src family
kinases that carry both a myristoyl and a palmitoyl lipid group.
Eg. of such Src family members: Fyn, Yes, Lck.
Upon integrin binding Fyn is activatedand its SH3 grp interacts with proline rich domain
on Shc.
This pathway may be cell type specific and and also specific to alpha.

Slide 7: X-talk
Integrins can modulate growth factor receptros:
Integrin clustering and occupancy can produce a transient local accumulation of EGFR,
PDGFR, FGFR.
Integrins are necessary for optimal activation of growth factor receptors.
Results in receptor phosphorylation.
IRS-1 become bound to avb3 integrin after vitronectin adhesion. AlphavBeta3 can be
immpttd with IGFR, PDGFR, and VEGFR.
A5b1 associates with EGFR.


Slide 8: Integrin signaling
Different functions of integrin.
How are they coordinated?
Show connections to Rho family members

Slide 9: Rho family and integrin signaling
Cell motility
Via MAPK activity by directly phosphorylating MLCK (Klemke, et al., 1997).

1. Cell Spreading
Rac and CDc42
A5b1 activate Rac and Cdc42
(syk and vav in hemopoietic cells are activated by integrins).



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In fibroblasts, plating on fibronectin first stimulates Rac and Cdc42 activation which
activates PAK.
Both PAK and ROCK can phosphorylate and activate LIM kinase, which inactivates
cofilin.
Focal complexes first form at the ends of filopodia. Then cells spread by lamellipodia.
Rho activity increases at the end of spreading in response to ECM proteins. Reaching a
maximum as focal adhesions and stress fibers are assembled.
2. Contractility
Rho (A-E)
A6b4 activate RhoA.
1. Rho activation results in focal adhesion and stress fiber assembly.
Serum or LPA lead to Rho activation in 2-5min.
Other growth factors (PDGF, EGF, insulin) activated stress and FCs but with longer time
course via Rac.
2. Integrin can also activate Rho.
Integrin activated Rho stimulates PIP5-kinase producing elevated levels of PIP2.
PIP2 can modulate actin binding of profiling, gelsolin, etc.
PIP2 is enriched in focal adhesion plaques.
AntiPIP2 antibodies can inhibit LPA Rho induced stress fibers and focal adhesions.

Burridge, 1996 reported that association of PIP2 with vinculin induces a confirmational
change in vinculin allowing it to interact with talin.

Dominant negative Rho also partially suppresses ERK activation. Therefore, Rho
regulated pathways are necessary for full activation of ERK thru integrin mediated cell
adhesion.

Constitutive activation of p190RhoGAP disrupts integrin clustering and Fas.

Rhokinase
   A. phoosphorylates the myosin binding subunit of myosin light chain phosphatase,
      and increase myosin light chain phosphorylation.
   B. Phosphorylate MLC at same site as MLCkinase

Slide 10: filopodia and lamellipodia
Slide 11: lamellipodia and focal adhesions
Slide 12: moving cell
Cells use focal adhesions to move.
Alpha6beta4 has been implicated in chemotaxis and invasion of cancer cells.
A6b4 integrin stimulates PI3K, cAMP phosphodiesterase, and Rho. In these cells Rac
and Rho are in lamellipods.

Integrins and cell survival
Slide 13:
Anchorage-dependent cell cycle progression:




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Cell attachment and spreading are imp for cell proliferation and that loss of this
requirement leads to tumorigenecity.
Control of growth by cell anchorage is due to regulation of G1.
G1 progression: cyclin D/Cdk4/6 and cyclin E/cdk2 activation leading to phosphorylation
of Rb proteins p107 and p130 and release of E2F family members, and induction of
cyclin A.
This is thru integrin control of :
    1. MAPK and PI3-K activation of ets-2 and elF4E transcription factors which turn
        on cyclin D1 transcription.
    2. Downregulation of p21 and p27 inhibitors of cyclinE-Cdk2 activity.
    3. Integrins activate PI3K independent of RTKs. Akt will turn on p70S6K which
        degrades and inactivates p27, inhibit Bad, etc.
    4. By an unknown mechansim p53 is inhibited so Bax is not synthesized.
    Inhibition of p53 prevents FAK deficient cells from undergoing apoptosis, therefore,
    FAK may inhibit p53.
    A5b3 can suppress p53 and activate NFkB
    5. a5b1 induces expression of Bcl-2.
    6. Loss of matrix adhesion accumulate hyper phosphorylated Rb, apparentky because
    the Rb target E2F transcription factor protects cell from apoptosis, suggesting that
    hyperphosphorylated Rb can under some conditions provide an apoptotic signal.
Alpha5Beta1 expressing CHO cells survived in serum free cultures by increasing Bcl-2
expression. Integrin mediated adhesion prevents anoikis by stimulating a high ratio of the
levels of Bcl2/Bax ratio.

Therefore, integrin-mediated cell adhesion increases cell proliferation and cell survival,
in a cell type and integrin type manner.

Slide 14: stretching is good for a cell
Chen et al., 1997 Geometric control of cell life and death Science 276:1425.
Showed that cells forced to spread over large surface had better chances of survival and
proliferated faster than smaller rounded cells.
They used microfabricated surface with ECM dots. They allowed cells to attach to a
single circular dot or several small dots that were the same size collectively as one large
dot. Therefore, chemical signals from integrins is not the predominant factor but cell
spreading over large area.
Integrin mediated adhesion rescues many cell types from apoptosis.
Anoikis: Induction of apoptosis by loss of integrin-mediated adhesion.

 Anoikis involves a positive feedback loop that includes activation of caspases that cleave
and activate MEKK-1, which in turn activates more caspases (Cardone et al., 1997 Cell
90:15-323.)

Apoptosis-induced cell rounding has been associated with caspase cleavage of FAK.
PTEN transfection into tumor cell lines can restore this form of apoptosis, termed
anoikis, which is characteristic of many normal cells, via dephosphorylation of PIP3.
And may be also via its action on FAK



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Integrins in cell transformation
Cell detachment induced by Ras and Src oncogenes don’t induce anoikis Tumor cell
proliferation is generally independent of both adhesion and serum through anchorage
independence. Anchorage dependence and serum-dependence of g separated.
Martin Shwartz constitutively activated the oncogenes dbl and ibc (RhoGEFs) and
showed that they induced anchorage independent growth but not serum independent
growth.
Constitutive activation of both integrin-mediated and growth factor receptor activated
pathways are imp for cell transformation.

Slide 15: ”Inside out signaling”
1. Activation of aIIbbeta3 in platelets
2. Activation of b2 integrins in monocytes, neutrophils, lymphocytes
3. Activation of b1 integrins in lymphocytes
4. Inactivation of a5b1 in keratinocytes, teratocarcinomas, mitosis
5. Inactivation of a6b1 in retinal ganglion axons
Imp in hemapoietic cells.
Allows attachment of platelets to soluble ligands and of lymphocytes to antigen
presenting cells and the phagocytosis of opsonized targets by granulocytes and
macrophages.

Rho GTPases regulate Integrin activation
Botulinum C3 exoenzyme which ADP ribosylates and inactivates Rho was shown to
block integrin mediated functions such as platelet aggregation (mediated by integrin
IIb3), lymphocyte aggregation (aLb2) or adhesion (aLb2, a4b1, a5b1), and fibronectin
matrix assembly by fibroblasts (a5b1).

Platelet aggregation:
Most intensely studies integrin inside out signaling is in platelet aggregation.
External signals like thrombin activates integrin to bind fibrinogen and mediate platelt-
platelet adhesion. (Shattil, 1998. Blood 91:2645-2657.)

Regulation of leukocyte adhesion and migration
Neutrophils and monocytes need to attach to endothelial layers in order to leave the
bloodstream at sites of inflammation. These extravasation sites involve several different
adhesive proteins, central b2 integrins. In the gentic disease leukocyte adhesion
deficiency (LAD) b2 integrins are abs.

Slide 16
Leukocyte extravasation can be divided into:
1. Rolling: Leukocytes loosely attach to the surfaces of endothelial cells, thru
    interactions between selectins an their ligands, and roll on the endothelium.
In certain situations integrins (a4) are imp for rolling. CAMs are locted on leukocyte
microvilli that bind to integrins. Selectin expression on the endothelium is induced by
inflammation.



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Adhesion requires integrin activation. Beta2 Integrin activation can be accomplished by
phorbol esters, or more physiologically by various inflammatory mediators (TNF, C5a,
platelet activating factor, fMet-Leu-Phe) produced by the endothelium or underlying
tissue.
2. Firm adhesion requires adhesion of b2 integrins to counterreceptors on endothelial
cells. Integrin binds ICAM1.
3. transendothelial migration: Integrins essential for this step.

ICAMs Intracellular cell adhesio molecule)
Integrins recognize integral membrane proteins of the immunoglobulin superfamily
(ICAM1, ICAM2, VCAM1) and mediate direct cell-cell adhesion.
Intracellular cell adhesion mol and vascular cell adhesion mol.
B2aL binds ICAM1 and ICAM2.
ICAMs are structurally related members of the IgG supergene family and act as ligands
for b2 integrins on leukocytes.
ICAMs are expressed constitutively at a low levels in endothelial cells and on some
lymphocytes and monocytes, its expresson is increased by cytokines like TNFa, IL-1 and
IFNa, and ROS.

An analagous situation exists for T lymphocytes. Specificity of their adhesion to antigen-
presenting cells comes from the T-cell receptor, which recognizes antigenic peptides
bound to major histocompatibility mols. However, adhesion also relies on aLb2 integrin
(lymphocyte function antigen-1, LFA-1).

Alpha subunit of integrins bind to Cytohesin-1 Cytoplasmic protein serves as mediator of
PI3K activation of inside out signaling and affects b2 activity in lymphocytes.

Slide 17: Modulation of inegrin function by uPAR
An imp serine protease involved in ECM degradation is urokinase plasminogen
activator
Immediate target of u-PA is plasminogen. Plasminogen is inactive until uPA cleaves a
single bond in plasminogen to yield plasmin.
Plasmin has a wide substrate range and cleaves fibrin (blood clots), fibronectin, and
laminin.
UPA system and plasmin generation (protease) facilitates cell migration.
Following injury to the vascular wall, vascular smooth muscle cells migrate to create a
new inner lining.

Urokinas-type plasminogen activator R (GPI anchored p) associates with avb3 and
modulates cell migration. Also b1 and b2. Colocalizes with integrin and leading edge of
migrating cells.
UPAR is thought to signal to integrin

(What other proteases are imp? MMPs that are expressed by some cells when they bind
matrix)




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UPAR knockout mice has severley impaired b2 integrin-dependent leukocyte
extravasation.
UPAR inhibitor Plasminogen activator inhibitor-1 (PAI-1) . PAI can modulate cell
migration thru interacting with vitronectin at the same site where uPAR binds, and
compete for ECM binding.
PAI-1 can interfere with ECM binding to both integrins and UPAR.

In addition to leukocytes, uPAR-bound uPA is also required for avb5-mediated tumor
cell migration.

Agurre et al., 1999. Showed that uPAR can noactivate ERK/MAPK with integrins in
tumor cells. If they interfered with upAR/integrin (a5b1) they were able to mimic in vivo
tumor dormancy (G0/G1).

MMPs have also been implicated in integrin activation. But not well established.

GPI anchored proteins on surface of leukocytes other than uPAR, such as Fcgamma
receptor IIIB (CD16b) and LPS/LPS binding proteins receptor (CD14) may also bind
integrins.


Integrin based therapeutics:
anti b3 antibody available in market for prevention of arterial restenosis.
RGD peptides and antibodies to specifically block integrins in developmental stages to
treat thrombosis (aIIb beta3 in platelets), Osteoperosis (avb3 in osteoclasts), and tumor
induced angiogenesis (avb3 in neovascular endothelial cells).
Avb3 mediated endothelial cell migration during angiogenesis can be blocked by
antibodies.

Slide 20: receptors and signaling
c. Tyrosine kinase associated receptors JAK/STAT
d. receptor tyrosine phosphatases
usually negative regulators.
CD45 a single pass glycoprotein with tyrosine phosphatase domain on cytoplasmic
surface palys a positie role in activation of T and B lymphocytes.

Slide 21: JAK/STAT pathway
    1. STATs (signal transducers and activators of transcription)
    2. SH2 domain containing transcription factors required for activation of immediate
       early genes by interferons, other cytokines, growth factors
    3. JAKs are activated by ligand induced receptors.
    Confirmational change in receptor associated JAKs make them transphosphorylate
    each other and get activated.
    JAKS then tyrosine phosphorylate cytokine receptors.
    4. STATs bind p-tyro residues on receptors.
    5. STATS are phosphorylated by JAK family non-receptor tyrosine kinases



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   6. This leads to dimerization of phosphorylated STATs and their localization to the
      nucleus to control expression of target genes.

JAK family: JAK1, JAK2, JAK3, and Tyk2
6 STATs

Slide 22: Interferon action on JAK/STAT
Interferon receptor: IFNAR1 and IFNAR2
1. Upon ligand binding these receptors heterodimerize.
2. Tyk2 is constitutively associated with IFNAR1,
        JAK1, STAT1, and STAT2 bind IFNAR2
    3. Activated JAK1 and Tyk2 can phosphorylate both subunits of the receptor.
    4. Tyrosine phosphorylated receptors provide docking sites for SH2 domains on
        STATs.
    5. STATs become phosphorylated in response to ligand binding.
    Y701 in STAT1
    Y690 in STAT2
    6. Phosphorylated Stats homo or heterodimerize via SH2 domains and bind DNA.
    7. Both stat1 and stat 2 bind to general transcriptional coactivators p300 and CBP
        (chromatin binding protein).
    These proteins appear to open chromatin near transcriptional start sites and link
    enhancer binding factors to basal transcriptional machinery.

   Glucocorticoid receptor can act as a transcriptionl coactivator of STAT5.

   Multiple cytokine and growth factor receptors activate JAK/STATS.
   See table in p399.
   Usually JAKS are involved in STAT activation. Exceptions are EGF and PDGF
   which can stimulat tyrosine phosphorylation of stats in Jak deficient cells.
   Expression of common oncoproteins activate JAKS,

   MAP kinases:
   Raf1, ERK2, PKA,PI3K can also be recruited to JAK/STAT complex.
   Serine 727 of STATs is a site for proline-directed serine kinases such as MAP
   kinases.
   IFNs rapidly stimulate MAPK activity and association with IFNAR1 receptor and
   STAT.
   Activation of Raf1 by IFN requires both STAT and JAK.

   Role of tyrosine phosphatases in initiation and dowwnregulation of stat signaling
   1. SHP1 and SHP2, SH2 domain containing tyrosine phosphatases can also interact
      with receptor. SHP1 is a positive regulator and SHP2 is a negative regulator of
      interferon signaling.
   2. A nuclear tyrosine phosphatase that dephosphorylate stats.
   3. Ubiquitin-proteosome pathway that degrade activated stats.




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   4. CIS (cytokine inducible proteins) with SH2 domains. CIS proteins can bind to
      tyrosine phopshorylated receptors, activated JAKS and STATS and inactivate
      them.

Slide 23:
            (a) brizzi et al., 1999. Mol Biol Cell
            Integrin mediated adhesion of endothelial cells induces JAK and STAT.
            JAK/STAT pathway plays adominant role in integrin-mediated c-fos
            gene expression.

   In addition to the SRE sequences in promoters that are regulated by MAPkinase
   phosphorylating Elk1 and Sap1,
   c-fos promoter contains a Serum inducible element (SIE) which is a target for STATs.
   They showed that adhesion to fibronectin increased JAK and STAT phosphorylation,
   SIE binding activity,.
   Stable expression of dominant negative STAT5A in NIH3T3 cells reduced
   fibronectin activated c-fos mRNA expression.

   (b)
   In mammary epithelial cells interaction with laminin is required for prolactin to
   trigger JAK/STAT pathway to start milk protein production.




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