The Cell Will Survive africangreyparrott to to tune of I Will Survive by Gloria Gaynor
DNA2life_What Does a Cell Need to Divide &Survive
Transcript of DNA2life_What Does a Cell Need to Divide &Survive
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WHAT DOES A EUKARYOTIC CELLNEED TO DIVIDE & TO
SURVIVE?
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What will you answer ?
Whenever we are asked withthis question definitely wewill answer that yes, thegrowth factors are required.
But, is it enough to permit acell to divide &/orsurvive??????
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What will you answer ?
Some facts in course oftime have been unveiled:-
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What will you answer ?
[1] Fibroblasts, if suspended in an appropriateliquid medium endowed with EGF, will notproliferate, as growth factors will fail to
trigger Ras-Raf-MEK-ERK pathway. Not onlythat but also increased level of p27 wouldlinger in G1 phase instead of expression ofCyclinD. After failure cells undergo apoptosis.
But in normal culture system, where cells aregrown onto a substratum, this phenomenonhave not been recorded yet.
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[2] Epithelium & endothelium cells always failto survive if they do not adhere to oneanother or to matrix. In this case theyautomatically undergo apoptosis. In thisparticular context apoptosis is calledanoikis.
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[3] In accord with previous two evidences blood cellsshouldnt survive as they neither are adhered to matrix
nor to other cells. Indeed, they have relatively short
lifespan. Maximum lifespan is of RBC(120 days).
Memory B or T cells, employ some exquisitemechanism in order to survive. They interact with
antigen-primed APCs. This evokes some signal, that is
transduced into nucleus & downregulate bax, bcl-Xs
and
others apoptosis promoting genes .
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Paramount significance of
focal adhesion sites in
non-conventional function
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Answer revealed
From those fore- mentioned facts it is likely that cell-matrixadhesion indeed has a role in maintaining cell survival &/orproliferation.
Perhaps a signaling may emanate from cell-matrix adhesion
sites which might promote cell survival &/or proliferation. Thisis exactly what happens.
Researchers have found that in the juxtamembrane region ofFocal adhesion sites, an intracellular signaling complexassembles, which passes on the signals via two major
pathways-[1] Ras-Raf-MEK-ERK pathway
[2] PI3-kinase pathway
in order to regulate cell survival &/or proliferation.
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Lets recapitulate the growth factor signaling
pathway once more (for proliferation)
(Step:1 ) a growth factor binds and dimerizes its cognatereceptor. As for example, a PDGF binds to PDGFR.
(Step:2) activation of intrinsic tyrosine kinase activity ofreceptor; autophosphorylation of receptor on specific Y
residues in cytosolic domain of receptor. (Step:3) adaptor protein with SH2 domain such as Grb2
now docks onto P-Tyr residues.
(Step:4) Grb2 now recruits Sos(Son ofSevenless) , aGEF, destined to convert Ras-GDP to Ras-GTP.
(Step:5) Ras then signals via a series of downstreameffector molecules, which are in the following orderedsequence:-Raf( MAP3K) MEK( MAP2K)- ERK( MAPK).All these mediators should be in close juxtaposition toone another. ERK translocates into nucleus to activate
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continued.
transcription factors like SRF, AP-1 etc.
(Step:6) These factors in turn bind to promoterof early responsive genes( so named because
they express within a minute of growth factorstimulation.
(Step:7) Early responsive gene products nowregulates expression of Late responsive genes
maximum of which encoding cell-cyclemachineries, replication- transcriptionmachineries etc, i.e. preparing the cell toproliferate.
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Growth factor signaling augments
through activation of Ras.
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..transmitting signal
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.nuclear localization of ERK.
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How do focal adhesions enforce growth
factor signaling cascade?
To execute this non-conventional, yetimportant function, focal adhesion sitesrecruit a cytoplasmic tyrosine kinase named
Focal Adhesion Kinase or FAK. FAK is the most crucial component for the
outside-in signal to transmit into interior ofthe cell.
If crouched in terms of non-conventionalfunctions FAK is the organizer of the FocalAdhesion Complexes.
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Focal Adhesion signaling
complex FAK recruitment platform is the protein
talin( another important scaffoldingcomponent of Focal Adhesion sites)
FAK is targeted to FERM domain of talin bymean of its Focal Adhesion Targeting (FAT)domain.
After recruitment FAK is autophosphorylatedat some specific Tyr residues.
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Focal Adhesion signaling
complex
Src, a cytoplasmic tyrosine kinase, binds toFAK by virtue of its SH2 domain.
Meanwhile, FAKs one of the two Proline-richdomain tethers CAS(Crk-associatedsubstrate) with FAK.
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CAS acts as a docking protein as it possessesmultiple phosphorylating sites (Tyr residues) .
This protein, indeed is phosphorylated byboth FAK & Src kinase (a reminiscent of IRS1
of Insulin receptor)
Mutation in CAS wont transmit any signal.
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FAK signaling complex
formation
VinculinAlpha-
actinin
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FAK mediated signaling(direct)
The infancy of signaling cascade may bedifferent , originating differently butultimately converge on activation of
transcription factors like c-jun, c-fos, jun-Detc, which are regulators of cell-cycleprogression.
Grb2-SOS can be directly recruited to Tyr-phosphorylated FAK activation of Ras
b-Raf MEK1
ERK2
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CAS mediated pathway
Another trajectory proceeds through CAS.Crk an adaptor protein docks onto p-Tyrresidues of CAS.
Then Crk by virtue of its SH3 domain tethersC3G(a GEF of Rap) and/or 180DOCK (a GEF ofRac).
Rap1 & Rac1 both belong to monomeric GTP-ase family protein.
Rap signals via Raf MEK1 ERK2pathway to execute its function.
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Rac1 also passes on its signal through PAK1JNK1 pathway.
All of these signals lead to activation of a setof transcription factors such as AP1(c-fos+ c-
jun), SRF leads to expression of a set of
immediate early genes. ,encoding cyclin-D,&eventual formation of active cyclin-D/cdk4complex.
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This complex drives the cell past therestriction point in G1 phase & cell
gets committed
to complete the cell cycle.
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Products of few early genes are the activatorsof a battery of secondary response genes.
their expressionleads to
Production of multiple components ofreplication & transcriptionapparatus,
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CAS mediated signaling
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Growth factor signaling
emanating from hemidesmosomes
is slightly different
In this case ERbB transmembrane kinase is
recruited in close juxtaposition to focaladhesion sites.
ERbB kinase enhances the growth factorsignaling output( see the figure in next slide).
to be continued.
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Proliferation signaling from
hemidesmosomes
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continuum
In this context Src phosphorylates cytosolictail of subunit of integrins, facilitatingcrosslinking of plectins with integrins (as weknow at hemidesmosomes, integrins are
connected to intermediate filaments). As depicted in the diagram Src also
phosphorylates STAT3 at a Tyr residue. Thissignal is enforced by phosphorylation at a
Ser/Thr residue. Two of this factor additively controls cell-cell
contact, cell cycle progression.
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FAK also controls the level of p27
CKI
The details of this pathway remains to beunderstood but it has been observed thatFAK sends a signal into nucleus in response to
inordinate level of p27. This signal induces expression of skp2 gene;
product is the ligand binding component ofSCF-E3 complex.
Induced expression of skp2 results in E3complex formation( see the figure in the nextslide)
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SCF-E3 complex [skp2 is the ligand bindingcomponent; Cul1 CTD & Rbx1 together function as E2(
conjugating enzyme)]
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Showing p27 destruction
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FAK contributes to cell survival
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FAK recruits type1 PI3-kinase as it binds toFAK by virtue of its p85 regulatory subunit
Architecture of the two subunits of PI3-kinaseis shown(SH3 domain is ascribed for binding FAK
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Lets recapitulate what are the basic
mechanisms employed by Akt ( 4 main
pathways are there)
[1] Phosphorylation of Forkhead superfamilytranscription factors such as FOXO1, FOXO3,
FOXO4. Phosphorylation causes their cytosolic retention
by 14-3-3 chaperons(see the next slide)
This renders FOXOs non-functional, otherwisethey would induce apoptosis inducing pro-
apoptotic genes( TRADD, Fas-ligand, TRAIL,
BIM).
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Mechanism of FOXO (though, here it is shown in context of growth
factor signaling but the concept remains same
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Continuum
[2] Phosphorylation ofBad shifts itslocation from mitochondrial outer membraneto cytosol & then sequestered by 14-3-3chaperons.( the details of Bad & FOXOmediated pathway are shown in next figure)
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The targets of Akt include the pro-apoptotic Bad and the FOXO transcription factor,
which stimulates transcription of another pro-apoptotic Bim. Phosphorylation by Akt
inhibits both Bad and FOXO. Thus production of BIM is prevented. Normally BAD & BIM
inhibit the anti-apoptotic protein Bcl-2. But as Bad & BIM are inactivated, also
functions promotes cell survival and inhibiting the release of cytochrome c from
mitochondria.
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[3]This pathway is much more prevalent in acell) that has recently undergone a genotoxic
stress (DNA damage) . In such circumstances cell generate large
amount of p53 for maintaining cell cycle arrest inorder to repair the damage.
But unfortunately if p53 lingers in nucleus , thenit induces expression of BH3 only proteins e.g.PUMA & NOXA leading to cell death.
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Genotoxic stress promoting
apoptosis
p53
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So how does Akt intervens?
So Akt phosphorylates Mdm2, a E3, whichspecifically targets p53.
p53 is ubiquitinated & targeted fordestruction by Proteosome.
phosphorylatesAktMdm2
p
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Akt sustains glucose uptake
Sometimes cells become deprived of growthfactors, nutrients(glucose) for a variety ofreasons.
As a result they fail to sustain their normalmetabolism, & to perform any physical orphysiological function.
The consequence is inevitable death asapoptosis ensues due to mitochondrialmembrane permeability to cytochrome C.
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a new role of Hexokinase
unveiled Glucose absence results in release of Hexokinase
from mitochondrial outer membrane into
cytosol.
Other than its non-conventional functionHexokinase maintains a structural integrity,
solute selectivity of Tim-Tom complex (channels
in mitochondrial inner & outer membrane). Release of Hexokinase results in release of
cytochrome C into cytosol, where it augmentsapoptosis inducing pathway.
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so what is the role of Akt
in this context Akt tries to sustain glucose uptake by
promoting transport of GLUT4 containingvesicles to membrane.
Thereby preventing release of cytochrome Cas best as it can.
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conclusion
So at the end of our discussion it seems thatthe adhesion dependent Ras-Raf &/or PI3-kinase-Akt signaling & growth factor
mediated these two signaling pathway areapparently overlapping, but virtually they arein a mutually enforcing circuit , amplifying
one another. Absence of any one of thembrings cell-cycle into stall.
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.conclusion
Every cells have intrinsic drive to self-destruct. But they are prevented from doingso by several signals, radiating from different
source.
Such a signaling emanates from cell-matrixadhesion sites.
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The end