WHAT DOES A EUKARYOTIC CELL NEED TO DIVIDE & TO SURVIVE?

What will you answer ?

Whenever we are asked with

this question definitely we will answer that yes, the growth factors are required. But, is it enough to permit a cell to divide &/or survive??????

What will you answer ?

Some facts in course of

time have been unveiled:-

What will you answer ?

[1] Fibroblasts, if suspended in an appropriate

liquid medium endowed with EGF, will not proliferate, as growth factors will fail to trigger Ras-Raf-MEK-ERK pathway. Not only that but also increased level of p27 would linger in G1 phase instead of expression of CyclinD. After failure cells undergo apoptosis. But in normal culture system, where cells are grown onto a substratum, this phenomenon have not been recorded yet.

[2] Epithelium & endothelium cells always fail to survive if they do not adhere to one another or to matrix. In this case they automatically undergo apoptosis. In this particular context apoptosis is called anoikis.

 [3] In accord with previous two evidences blood cells

shouldnt 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 exquisite mechanism 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 .

Paramount significance of focal adhesion sites in non-conventional function

Answer revealed
From those fore- mentioned facts it is likely that cell-matrix

adhesion indeed has a role in maintaining cell survival &/or proliferation. Perhaps a signaling may emanate from cell-matrix adhesion sites which might promote cell survival &/or proliferation. This is exactly what happens. Researchers have found that in the juxtamembrane region of Focal adhesion sites, an intracellular signaling complex assembles, 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.

Lets recapitulate the growth factor signaling

pathway once more

(for proliferation)

(Step:1 ) a growth factor binds and dimerizes its cognate

receptor. As for example, a PDGF binds to PDGFR. (Step:2) activation of intrinsic tyrosine kinase activity of receptor; 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 of Sevenless) , a GEF, destined to convert Ras-GDP to Ras-GTP. (Step:5) Ras then signals via a series of downstream effector molecules, which are in the following ordered sequence:-Raf( MAP3K) MEK( MAP2K)- ERK( MAPK). All these mediators should be in close juxtaposition to one another. ERK translocates into nucleus to activate

continued.
transcription factors like SRF, AP-1 etc. (Step:6) These factors in turn bind to promoter of early responsive genes( so named because they express within a minute of growth factor stimulation. (Step:7) Early responsive gene products now regulates expression of Late responsive genes maximum of which encoding cell-cycle machineries, replication- transcription machineries etc, i.e. preparing the cell to proliferate.

Growth factor signaling augments through activation of Ras.

..transmitting signal

.nuclear localization of ERK.

How do focal adhesions enforce growth factor signaling cascade?

To execute this non-conventional, yet

important function, focal adhesion sites recruit 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 of the cell. If crouched in terms of non-conventional functions FAK is the organizer of the Focal Adhesion Complexes.

Focal Adhesion signaling complex

FAK recruitment platform is the protein

talin( another important scaffolding component of Focal Adhesion sites) FAK is targeted to FERM domain of talin by mean of its Focal Adhesion Targeting (FAT) domain.
After recruitment FAK is autophosphorylated

at some specific Tyr residues.

Focal Adhesion signaling complex

Src, a cytoplasmic tyrosine kinase, binds to

FAK by virtue of its SH2 domain. Meanwhile, FAKs one of the two Proline-rich domain tethers CAS(Crk-associated substrate) with FAK.

 CAS acts as a docking protein as it possesses

multiple phosphorylating sites (Tyr residues) . This protein, indeed is phosphorylated by both FAK & Src kinase (a reminiscent of IRS1 of Insulin receptor) Mutation in CAS wont transmit any signal.

FAK signaling complex formation

Vinculin

Alphaactinin

FAK mediated signaling(direct)

The infancy of signaling cascade may be

different , originating differently but ultimately converge on activation of transcription factors like c-jun, c-fos, jun-D etc, which are regulators of cell-cycle progression. Grb2-SOS can be directly recruited to Tyrphosphorylated FAK activation of Ras
bRaf MEK1 ERK 2

CAS mediated pathway

Another trajectory proceeds through CAS.

Crk an adaptor protein docks onto p-Tyr residues of CAS. Then Crk by virtue of its SH3 domain tethers C3G(a GEF of Rap) and/or 180DOCK (a GEF of Rac). Rap1 & Rac1 both belong to monomeric GTPase family protein. Rap signals via Raf MEK1 ERK2 pathway to execute its function.

 Rac1 also passes on its signal through PAK1

JNK1 pathway.
All of these signals lead to activation of a set

of transcription factors such as AP1(c-fos+ cjun), SRF leads to expression of a set of immediate early genes. ,encoding cyclin-D, &eventual formation of active cyclin-D/cdk4 complex.

This complex drives the cell past the restriction point in G1 phase & cell gets committed

to complete the cell cycle.

 Products of few early genes are the activators

of a battery of secondary response genes.

their expression
leads to
Production of multiple components of

replication & transcription apparatus,

CAS mediated signaling

Growth factor signaling emanating from hemidesmosomes is slightly different

In this case ERbB transmembrane kinase is

recruited in close juxtaposition to focal adhesion sites. ERbB kinase enhances the growth factor signaling output( see the figure in next slide). to be continued.

Proliferation signaling from hemidesmosomes

 continuum
In this context Src phosphorylates cytosolic

tail of subunit of integrins, facilitating crosslinking of plectins with integrins (as we know at hemidesmosomes, integrins are connected to intermediate filaments). As depicted in the diagram Src also phosphorylates STAT3 at a Tyr residue. This signal is enforced by phosphorylation at a Ser/Thr residue. Two of this factor additively controls cell-cell contact, cell cycle progression.

FAK also controls the level of p27 CKI

The details of this pathway remains to be

understood but it has been observed that FAK 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 of SCF-E3 complex. Induced expression of skp2 results in E3 complex formation( see the figure in the next slide)

SCF-E3 complex [skp2 is the ligand binding

component; Cul1 CTD & Rbx1 together function as E2( conjugating enzyme)]

Showing p27 destruction

FAK contributes to cell survival

FAK recruits type1 PI3-kinase as it binds to

FAK by virtue of its p85 regulatory subunit Architecture of the two subunits of PI3-kinase is shown(SH3 domain is ascribed for binding FAK

Lets recapitulate what are the basic mechanisms employed by Akt ( 4 main pathways are there)

[1] Phosphorylation of Forkhead superfamily

transcription 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, otherwise they would induce apoptosis inducing proapoptotic genes( TRADD, Fas-ligand, TRAIL, BIM).

Mechanism of FOXO (though, here it is shown in context of growth factor signaling but the concept remains same

Continuum

[2] Phosphorylation of Bad shifts its

location from mitochondrial outer membrane to cytosol & then sequestered by 14-3-3 chaperons.( the details of Bad & FOXO mediated pathway are shown in next figure)

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.

[3] This pathway is much more prevalent in a

cell) that has recently undergone a genotoxic stress (DNA damage) . In such circumstances cell generate large amount of p53 for maintaining cell cycle arrest in order to repair the damage. But unfortunately if p53 lingers in nucleus , then it induces expression of BH3 only proteins e.g. PUMA & NOXA leading to cell death.

Genotoxic stress promoting apoptosis

p53

So how does Akt intervens?

So Akt phosphorylates Mdm2, a E3, which

specifically targets p53.

p53 is ubiquitinated & targeted for

destruction by Proteosome.
p Akt Mdm2
phosphorylates

Akt sustains glucose uptake

Sometimes cells become deprived of growth

factors, nutrients(glucose) for a variety of reasons. As a result they fail to sustain their normal metabolism, & to perform any physical or physiological function. The consequence is inevitable death as apoptosis ensues due to mitochondrial membrane permeability to cytochrome C.

a new role of Hexokinase unveiled

Glucose absence results in release of Hexokinase from mitochondrial outer membrane into

cytosol. Other than its non-conventional function Hexokinase 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 augments apoptosis inducing pathway.

so what is the role of Akt in this context

Akt tries to sustain glucose uptake by

promoting transport of GLUT4 containing vesicles to membrane.

Thereby preventing release of cytochrome C

as best as it can.

conclusion
So at the end of our discussion it seems that

the adhesion dependent Ras-Raf &/or PI3kinase-Akt signaling & growth factor mediated these two signaling pathway are apparently overlapping, but virtually they are in a mutually enforcing circuit , amplifying one another. Absence of any one of them brings cell-cycle into stall.

.conclusion
Every cells have intrinsic drive to self-

destruct. But they are prevented from doing so by several signals, radiating from different source. Such a signaling emanates from cell-matrix adhesion sites.

The end