The Experimental Design For Autophagy

Published Date: 02 Nov 2017

bovine brain homogenate and found on position 3.3 during electrophoresis and thus given the enigmatic name

14-3-3 based on column fractionation and electrophoretic mobility. These being a family of conserved

regulatory molecules, bind a wide array of functionally diverse regulatory molecules including transcription

factors, biosynthetic enzymes, cytoskeletal proteins, signaling molecules, apoptosis factors, and tumor

suppressors and are constitutively expresses in all eukaryotic organisms.(1-3) These acidic proteins thus play

critical role as a biochemical regulator in several cellular functions as signal transduction, apoptosis, neuronal

plasticity, cell adhesion, metabolic control and cell cycle regulation.(1, 3)

14-3-3 exists as a family of proteins encoded by distinct genes. These proteins are highly homologous with

seven isoforms found in mammals, named after their reverse phase HPLC elution profile as β ε γ η σ τ ζ. They

primarily exist as dimers (homodimers or heterodimers), having a monomolecular mass of ~30,000, with an

acid isoelectric point of 4-5. High degree of sequence conservation of these proteins among different species

indicates high degree of functional similarities. Being the most abundant in brain, 14-3-3 proteins are nearly

expressed in all tissues, (3) while on the cellular level they usually localize in the cytosolic compartment, but

may also be found within the nucleus, Golgi apparatus or plasma membrane. The isolation of 14-3-3 proteins

frequently from biochemical and genetic screens for different targets is an indicator of the physiological

relevance of these molecules. (1-3)

14-3-3 were the first phosphoserine/phosphothreonine (pS/T) -binding proteins to be discovered (1, 2) and are

thus integrated in phospho-regulatory pathways controlling multitude of cellular functions. Each member of the

14-3-3 dimer has ligand-binding capacity and thus a dimer can bind two pS/T sites on single or separate target

ligands. The consensus sequences on target proteins that are recognized by all isoforms of 14-3-3 are

RSx[pS/pT]xP and Rxxx[pS/pT]xP.(4) However, not all interactions of 14-3-3 interactome are phosphorylation

dependent.(2) The downstream effects of 14-3-3 dimer binding to target molecules mainly occurs through one

of the following modes of action: change of conformation of the binding protein, blocking accessibility to

enzymes or other proteins or masking specific sequences, acting as a scaffold to promote protein-protein

interactions of adjacent proteins. (2, 3)

Two of the most significant roles of 14-3-3 proteins is their involvement in the smooth regulation of cell cycle

progression and control of apoptotic events.(4)

Role in cell cycle progression (normal physiology)

In its role as controlling the transition from G2 into M phase, 14-3-3 acts as the key player that integrates

several kinases and phosphatases involved in the process of transition. Normally, the transition involves

activation of Cdk1/cyclin B. Cdk1 activation involves phosphorylation of a threonine residue, but is still

maintained in an inactive state by phosphorylation of a tyrosine residue. Cdc25 phosphatases play a significant

role in removing these inhibitory phoshorylations for allowing transition into mitotic phase. In turn, Cdc25 itself

are regulated through phosphorylations, activating or inhibitory. Specifically, the inhibitory phosphorylations

occur due to any endogenous or exogenous events causing DNA damage and the main mechanism that

underlies inhibitory Cdc25 phosphorylations involves phosphorylation dependent binding to 14-3-3. The

inhibition of Cdc25 function may be due to reduction in its phosphatase activity, or due to reduced access to its

nuclear substrate Cdk1/cyclin B. Besides inhibiting the phosphatase activity of Cdc25, 14-3-3 may activate

Cdk1 inhibiting Wee1 kinase by increasing Wee1 distribution throughout the nucleus. Thus 14-3-3 serves to

induce G2 checkpoint arrest to stop mitotic entry during abnormal conditions. Specifically, the 14-3-3σ isoform

plays a critical role in G2 checkpoint maintenance and has also been found to be up regulated under the

influence of p53 in certain carcinomas so that DNA damaged cells are prevented from moving into the M

phase. More importantly, the 14-3-3-binding sites on Cdc25, Wee-1, and other checkpoint modulators are Biomed 515, Cancer Biology

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generated through the action of DNA damage-responsive basophilic kinases, particularly the kinases Chk1,

Chk2, and MK2. Chk1 mainly responds to single strand breaks, lesions formed during replication fork damage

and DNA adducts induced by UV; Chk2 primarily responds to double strand breaks. MK2 is critical for G2

arrest especially in cells deficient in p53.

Following DNA repair during G2 arrest, re-entry into the mitotic phase involves Cdk1 and Plk1which would

inactivate the 14-3-3 dependent inactivation of the upstream kinase pathways, involving Chk1, Chk2, MK2 and

Wee1. Thus it involves blocking the binding of Cdc25 and Wee1 to 14-3-3 and permits re-entry into the cell

cycle. It is worth mentioning that G2/M checkpoint is not error free, since many normal and tumorous cells

enter the M phase despite of the presence of unrepaired DNA breaks. Thus pharmacological intervention

causing 14-3-3 function modulation to lead to a prolonged G2 arrest can be a potential mechanism to control

tumor proliferation.

Once cells have entered the mitotic phase, 14-3-3 may also contribute towards enhancing cell cycle

progression by stimulating cytokinesis. 14-3-3, by binding to protein kinase C epsilon (PKCϵ) activates it in a

lipid independent fashion, locking it in an open active conformation near the acto-myosin ring. This PKCϵ

activity is crucial during telophase for causing the downregulation of RhoA at midbody allowing cells to

complete abscission. Therefore, loss of 14-3-3 binding sites on PKCϵ, or loss of PKCϵ expression, or

expression of a catalytically inactive form of PKCϵ, or expression of a dominant negative form of 14-3-3 leads

to delayed or failed cytokinesis causing multinucleated cells. Importantly, the 14-3-3σ isoform has tumor

suppressor properties and is found absent in several carcinomas leading to the inability of such cells to

undergo cytokinesis and rather lead to the formation of tetraploid cells that may act as precursors to cancerous

cells.

Cdc25A isoform that has an important role in G1/S transition by regulating control over CyclinE and

CyclinA/Cdk2. 14-3-3-binding to Cdc25A may be important for short-term control of Cyclin/Cdk complexes

involved in both G1/S and G2/M progression.(4)

Role in cytoskeleton regulation

It has been found that14-3-3 interacts with actin. This data has been supported by the seeing the colocalization

of 14-3-3 gamma with actin filaments in astrocytes through fluorescent and confocal microscopy.

14-3-3 interacts with and regulates the activity of many protein kinases. It has been long known that 14-3-3

affects the activity of protein kinase C. One isoform of protein kinase C is involved in phosphorylation of heavy

chains of myosin, and in this way regulates formation of ordered myosin filaments. 14-3-3 forms a tight

complex with this isoform of protein kinase C and thus indirectly regulates the contractile activity.(5)

Role in Apoptosis

The critical role of 14-3-3 proteins in apoptosis emerged from the observation of interactions between 14-3-3

dimers and BH-3 domain containing proteins, especially BAD and BAX.

BAD was identified as a 14-3-3 target of an IL-3 dependent survival mechanism in the hematopoietic cell line.

The binding between BAD and 14-3-3 involves AKT-dependent phosphorylation of two sites on BAD. The

underlying mechanism of inactivation of BAD involves transient sequestration of BAD to prevent it from binding

to and causing inactivation of pro-survival protein Bcl-XL, along with locking BAD in a conformation that

enhances phosphorylation of an additional residue in the BH3 domain to cause a more fixed state of

inactivation.

Also, 14-3-3 proteins bind to and cause functional inactivation of the pro-apoptotic molecule BAX in the

unconventional phospho-independent manner. The binding of BAX to 14-3-3 renders it incapable of Biomed 515, Cancer Biology

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Prashant Dogra

transitioning into mitochondria and thus impeding its interaction with BAK to cause mitochondrial outer

membrane permeabilization to release cytochrome C for activating cell death causing caspases.

JNK plays a critical role in regulating the interaction of 14-3-3 with BAX. It directly phosphorylates 14-3-3 on

Ser-184 reside to block its binding with BAX and trigger its release. The activation of JNK occurs by MAP

kinase kinase ASK1 (an apoptosis stimulating kinase 1), which in turn is negatively regulated by 14-3-3

interaction. (6) Activation of ASK1 occurs downstream of TNFα receptor and FAS. TNFα mediated apoptosis is

negatively controlled by A20, a zinc finger protein, which in turn is also a target protein for 14-3-3.(7) The

signaling between TNF receptor family members and BAX involves formation of complex receptors and the

proteins MOAP-1 and RASSF1A. This process is controlled by the binding of RASSF1A to 14-3-3. (8, 9) Also,

as a response to deprivation of growth factors and presence of pro-apoptotic stimuli, certain transcription

factors belonging to FOXO family tend to drive the expression of pro-apoptotic genes. In turn, several FOXO

transcription factors have been shown to be under the control of 14-3-3 with their binding being regulated by

AKT-dependent phosphorylation.(10, 11)

14-3-3 proteins thus control apoptosis at several levels, ranging from upstream activators and transcription

factors to the effector BH3 domain-containing proteins.(4) As a direct evidence of this control and regulation, it

was shown that the blockade of 14-3-3 binding cleft with a peptide inhibitor enhanced apoptotic

responsiveness in several cell lines.(12)

Experimental design for Apoptosis

Since 14-3-3 β/α has been found to be an inhibitor of the pro-apoptotic Bax and thus possess antiapoptotic

potential, (13) it would be relevant to study the expression levels of this particular protein in cancer cell lines in

order to investigate its potential as an oncogene.

Hypothesis: 14-3-3 β/α has oncogenic potential due to its antiapoptotic activity.

Experiment: To start with, some cancer cell lines can be used and analyzed for the expression levels of 14-3-3

β/α through simple Western Blot analysis keeping normal cell lines as controls.

Further, we can study if rescuing the pro-apoptotic activity of Bax by deleting the expression of 14-3-3 β/α in

such cells can show anti-tumor effects.

In order to cause deletion of the 14-3-3 β/α, Cre-Lox system can be employed. Transgenic mice containing

Lox-P sites around 14-3-3 β/α are produced. They are then mated with mice that express Cre gene in a

particular or all cell types. This results in mice with both Cre gene and 14-3-3 β/α gene flanked by LoxP. Cells

expressing Cre will cause the deletion of LoxP flanked 14-3-3 β/α gene.

These mice cells can now be used in culture for further investigation. 14-3-3 β/α lacking cels are then

transformed through radiation or an established carcinogen. It would now be interesting to see if Bax can

counteract this transformation when 14-3-3 β/α is absent. As a control, transformed cells expressing normal

14-3-3 β/α can be used to see their effect on the apoptotic activity of Bax.

In order to avoid interference by other proapoptotic molecules, it would be necessary to silence their effects

through siRNA techniques.

Role in Autophagy

Although very little has been studied about 14-3-3 proteins from the context of autophagic cell death, but due

to the ubiquitous nature of 14-3-3 proteins and their crucial role in cellular processes like cell cycle regulation, Biomed 515, Cancer Biology

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14-3-3

Prashant Dogra

DNA damage check points, apoptosis, etc. prompts towards investigating the possibility of their involvement in

autophagy.

In a 2010 paper by Wang et al(14), the group investigates for the involvement of 14-3-3Ï„ in autophagy with the

hypothesis that 14-3-3Ï„ may be involved in the regulation of Becin1 probably through stabilization of E2F1.

This work is prompted from their previous study where they unravel the involvement of 14-3-3Ï„ in causing

stabilization of E2F1. In this study, using U2OS cell lines that were induced to express siRNAs against 14-3-3Ï„,

they observe a significant decrease (~seven fold) in Beclin-1 expression which returned to its basal level only

upon rescuing 14-3-3Ï„ depletion by using RNA-I resistant construct, thus supporting the evidence that 14-3-3Ï„

controls Beclin-1 expression.

Experimental design for Autophagy

As a probable extension of their work, it would be interesting to investigate if the induction of Beclin-1

expression by 14-3-3Ï„ is a direct or an indirect process.

Hypothesis: 14-3-3Ï„ interacts with promoter of Beclin-1 to induce its expression.

Experiment: In order to see if 14-3-3Ï„ binds to the promoter sequence of Beclin-1 to induce its expression, we

can perform chromatin immunoprecipitation and look for the co-immunoprecipitation of 14-3-3Ï„ with beclin-1

promoter sequence.

For this cross-linked ChIP may be performed. Protein cross linking to chromatin is done with the help of

formaldehyde. Shearing of the cross-linked chromatin is done by sonication. Bead conjugated antibodies

specific for 14-3-3Ï„ protein are then used to precipitate 14-3-3Ï„ along with its cross linked DNA sequence.

Cross-linked DNA is then purified and identified through PCR or microarray analysis.

This experiment would provide a good start in determining the exact mechanism for the induction of Beclin-1

by 14-3-3Ï„ proteins.