dna2life_what does a cell need to divide &survive

7/29/2019 DNA2life_What Does a Cell Need to Divide &Survive

1/45

WHAT DOES A EUKARYOTIC CELLNEED TO DIVIDE & TO

SURVIVE?


2/45

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??????


3/45


Some facts in course oftime have been unveiled:-


4/45


[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.


5/45

[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.


6/45

[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 .


7/45

Paramount significance of

focal adhesion sites in

non-conventional function


8/45

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.


9/45

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


10/45

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.


11/45

Growth factor signaling augments

through activation of Ras.


12/45

..transmitting signal


13/45

.nuclear localization of ERK.


14/45

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.


15/45

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.


16/45

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.


17/45

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.


18/45

FAK signaling complex

formation

VinculinAlpha-

actinin


19/45

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


20/45

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.


21/45

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.


22/45

This complex drives the cell past therestriction point in G1 phase & cell

gets committed

to complete the cell cycle.


23/45

Products of few early genes are the activatorsof a battery of secondary response genes.

their expressionleads to

Production of multiple components ofreplication & transcriptionapparatus,


24/45

CAS mediated signaling


25/45

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.


26/45

Proliferation signaling from

hemidesmosomes


27/45

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.


28/45

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)


29/45

SCF-E3 complex [skp2 is the ligand bindingcomponent; Cul1 CTD & Rbx1 together function as E2(

conjugating enzyme)]


30/45

Showing p27 destruction


31/45

FAK contributes to cell survival


32/45

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


33/45

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).


34/45

Mechanism of FOXO (though, here it is shown in context of growth

factor signaling but the concept remains same


35/45

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)


36/45

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.


37/45

[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.


38/45

Genotoxic stress promoting

apoptosis

p53


39/45

So how does Akt intervens?

So Akt phosphorylates Mdm2, a E3, whichspecifically targets p53.

p53 is ubiquitinated & targeted fordestruction by Proteosome.

phosphorylatesAktMdm2

p


40/45

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.


41/45

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.


42/45

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.


43/45

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.


44/45

.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.


45/45

The end

dna2life_what does a cell need to divide &survive

Documents