Characterization of the interaction between the adaptor protein Nck and the protein kinase PKR
By: Afnan Abu-Thuraia
Department of Medicine Division of Experimental Medicine
McGill University Montreal, Quebec, Canada
A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Master of Science
© Afnan Abu-Thuraia, October 2010
II
Abstract
Tight regulation of the double-stranded RNA (dsRNA)-activated
protein kinase (PKR) is critical for the maintenance of cellular homeostasis
due to its potent inhibitory role on general translation. Previously, we have
identified the adaptor protein Nck-1 as a novel cellular regulator of PKR
activation through its interaction with PKR. In this study, we further
confirmed that Nck-1 limits PKR activation under normal conditions.
However, we demonstrate that the control of PKR activation by Nck-1 is
reversible, since significant levels of dsRNA override Nck-1’s negative
control of PKR activation and induce dissociation of Nck-1 from PKR. Our
data show that Nck-1 needs to be in full length to interact and modulate
PKR. In addition, we observed that the interaction of Nck-1 with PKR is
independent of any functional Src-homology domains of Nck-1, but our
findings showing that Nck-1 interacts with both the N- and C-terminus of
PKR challenge this concept. Nonetheless, we uncovered that upon
significant levels of dsRNA, dissociation of Nck-1 from PKR is due to the
activation of the catalytic activity of PKR rather than to competition by
dsRNA binding or change of PKR conformation during its activation.
Finally, we provided further evidence supporting the occurrence of Nck-1
phosphorylation by PKR in vivo. Hence, Nck-1 not only buffers PKR
activation but appears to be a substrate of PKR. Therefore, we propose
that PKR-mediated phosphorylation is part of the mechanism that
promotes Nck-1 dissociation from activated PKR. Taken together, our data
confirm Nck-1 as a novel cellular modulator of PKR that limits PKR
activation under physiological conditions.
III
Résumé
La protéine kinase PKR, activée par l’ARN double brins (ARNdb), est
connue pour jouer un rôle inhibiteur de la traduction des protéines. La
régulation de PKR est donc critique pour le maintien de l’homéostasie
cellulaire. Nous avons précédemment identifié la protéine adaptatrice
Nck-1 comme étant un potentiel régulateur de l’activation de PKR, suivant
son interaction avec PKR. Dans la présente étude, nous avons été en
mesure de confirmer que, dans des conditions physiologiques, Nck-1 peut
limiter l’activation de PKR par l’ARNdb. Cependant, le contrôle qu’exerce
Nck-1 sur PKR est réversible puisque, lorsque la quantité d’ARNdb
dépasse une certaine concentration, PKR est activée et alors Nck-1 se
dissocie de PKR, l’empêchant ainsi de limiter son activation. Nos données
démontrent également que Nck-1 doit être dans sa forme native pour
interagir et moduler l’activation de PKR. De plus, il semble que l’interaction
entre Nck-1 et PKR ne nécessite pas que les différents domaines
homologues de Src (SH2 et SH3) présents chez Nck-1 soient
fonctionnels. De plus, nous avons observé que Nck-1 interagit à la fois
avec les domaines N- et C-terminaux de PKR. Nous démontrons
également que lorsque les niveaux d’ARNdb atteignent un niveau seuil,
Nck-1 se dissocie de PKR non pas à cause d’une compétition avec
l’ARNdb, ni à cause d’un changement de conformation de PKR ou son
autophosphorylation, mais est plutôt dû à l’activation du domaine
catalytique de PKR. De plus, il semble que Nck-1 puisse être phosphorylé
par PKR in vivo. Nck-1 est donc non seulement un modulateur de
l’activation de PKR mais peut également servir de substrat pour PKR.
Ceci nous amène donc à proposer que la phosphorylation de Nck-1 par
PKR activée soit responsable du mécanisme de dissociation entre Nck-1
et PKR. En conclusion, nos résultats confirment Nck-1 comme étant un
nouveau modulateur cellulaire de PKR, en limitant son activation dans des
conditions physiologiques.
IV
CONTRIBUTION OF AUTHORS
This thesis was entirely written by me with editorial comments and
corrections by my supervisor, Dr. Louise Larose and the French
translation of my abstract by our Post-Doc, Dr. Julie Dusseault.
V
TABLE OF CONTENTS Page
Abstract ..................................................................................................................II Résumé................................................................................................................ III CONTRIBUTION OF AUTHORS ..................................................................... IV TABLE OF CONTENTS Page.................................................................................................................................V LIST OF FIGURES Page .......................................................... VI ACKNOWLEDGMENTS..................................................................................VIII CHAPTER I ........................................................................................................... 1 INTRODUCTION AND LITERATURE REVIEW ............................................. 1 1.1 Cellular Stress Response .................................................................... 2
1.1.1 Overview ................................................................................................. 2 1.1.2 Stress Induced-Cell Death .................................................................... 3
1.1.2.1 Apoptosis........................................................................................ 3 1.1.2.2 Autophagic Cell Death................................................................. 4 1.1.2.3 Necrosis .......................................................................................... 5
1.1.3 Stress and Survival Pathways .......................................................... 6 1.1.3.1 The Heat Shock Response ......................................................... 6 1.1.3.2 DNA Damage Response.............................................................. 9 1.1.3.4 The Unfolded Protein Response............................................. 12
1.2 eIF2α Kinases ............................................................................................. 15 1.2.1 Heme-Regulated Inhibitor (HRI) ..................................................... 18 1.2.2 General Control Non-derepressible-2 (GCN2)............................ 21 1.2.3 PKR (RNA-dependent protein kinase)-like ER kinase (PERK)23 1.2.4 RNA-dependent Protein Kinase (PKR) ......................................... 26
1.3 Nck Adaptor Proteins ............................................................................... 32 1.3.1 Nck gene and proteins...................................................................... 32 1.3.2 Nck interaction partners and functions ....................................... 33 1.3.3 Role of Nck in regulating eIF2α phosphorylation and cell response to stress ....................................................................................... 35
CHAPTER II ........................................................................................................ 38 HYPOTHESIS AND PROJECT OUTLINE .................................................... 38 CHAPTER III....................................................................................................... 41 EXPERIMENTAL PROCEDURES .................................................................. 41 CHAPTER IV ...................................................................................................... 47 RESULTS ............................................................................................................ 47 CHAPTER V ....................................................................................................... 65 DISCUSSION...................................................................................................... 65 REFERENCES ................................................................................................... 74
VI
LIST OF FIGURES Page
CHAPTER I: INTRODUCTION AND LITERATURE REVIEW Figure 1: Induction of heat shock proteins inhibits apoptosis and promotes
cell survival ................................................................................................ 8
Figure 2: DNA Damage Response ......................................................... 11
Figure 3: ER stress and the Unfolded Protein Response ...................... 14
Figure 4: Regulation of eIF2 ................................................................... 16
Figure 5 a, b: a) Protein kinases, PKR, HRI (heme-regulated inhibitor),
PERK and GCN2 are activated by different stress conditions; b) Domain
structure of protein kinases, PKR, PERK, HRI and GCN2....................... 17
Figure 6: HRI balances heme and globin synthesis by sensing
intracellular heme concentrations ............................................................ 19
Figure 7: Domain Structure of HRI ......................................................... 19
Figure 8: GCN2 pathway of activation .................................................... 22
Figure 9: Model for activation of PERK in response to ER stress........... 24
Figure 10: Signalling pathways involving PKR........................................ 27
Figure 11: Schematic representation of PKR activation ......................... 30
Figure 12: Modular composition of Nck adapter proteins........................ 33
Figure 13: Schematic model of the regulation of translation by Nck-1
during ER stress ...................................................................................... 36
CHAPTER II: HYPOTHESIS AND PROJECT OUTLINE Figure 1: Nck-1 reduces PKR activation induced by dsRNA……………..39
CHAPTER IV: RESULTS
Figure 1: Endogenous PKR co-immunoprecipitates with HA-Nck-1…….48
VII
Figure 2: Nck-1-PKR interaction is independent of Nck-1 functional SH
domains…………………………………………………………………………49
Figure 3: SH3 mutated Nck-1 reduces (A) PKR activation and (B)
phosphorylation of eIF2α induced by dsRNA……………………………….51
Figure 4: SH2 mutated Nck-1 reduces (A) PKR activation and (B)
phosphorylation of eIF2α induced by dsRNA……………………………….52
Figure 5: Nck-1 full length binds PKR………………………………………53
Figure 6: Loss of Nck-1 binding upon PKR activation by dsRNA………..54
Figure 7: Nck-1 binds to Dominant negative-PKR…………………………56
Figure 8: Nck-1 binds inactive PKR independently of dsRNA……………57
Figure 9: Nck-1 binds PKR N-terminus and inactive C-terminus
domains…………………………………………………………………………59
Figure 10: Nck-1 binds the N-terminus domain of PKR independently of
dsRNA…………………………………………………………………………..60
Figure 11: Nck-2 binds PKR………………………………………………....61
Figure 12: Nck-1 is phosphorylated in MEFs PKR+/+ and in HEK 293 cells
over-expressing wild type PKR……………………………………………….63
CHAPTER V: DISCUSSION Figure 1: Model of mechanism of PKR’s regulation by Nck-1……………67
VIII
ACKNOWLEDGMENTS
Many individuals have been instrumental through out the course of
this project. Many thanks go to my supervisor Dr. Louise Larose, to whom
I am indebted and grateful for the endless support, guidance and
encouragement she has provided me with for the last two years. I do
appreciate the great opportunity she has given me to work in her
laboratory which made these two years an unforgettable experience.
Special thanks also go to all present and past members of Dr. Larose
laboratory such as Eric Cardin, Geneviève Bourret, Lama Yamani, Hui Li
and Julie Dusseault. I would also like to thank everyone else at the
Polypeptide Laboratory (PPL) for their help whenever it was needed and
their friendship. My thanks also go to my thesis committee meeting for
their great advice and support: Dr. Stephane Laporte, Dr. Arnold Kristof
and my academic advisor, Dr. Jun-Li Hui.
Words are not sufficient to express my feelings of gratitude towards
my family. I want to thank my parents, Mr. Nabil Abu-Thuraia and Mrs.
Awatef Al-Khatib for being there for me all the time. I extend a heartfelt
thank you to my family and friends and specially my very good friend,
Kathy Malas, for the endless stream of love and care that nourished my
success and allowed me to excel in my studies. Of course, my greatest
gratefulness goes to Allah, the most merciful and most generous, for
everything that I am blessed with in my life.
1
CHAPTER I INTRODUCTION AND LITERATURE REVIEW
2
1.1 Cellular Stress Response
1.1.1 Overview
Cellular stress can be defined as the threat of damage to
macromolecules. Hence, the main essence behind cellular stress
response, once a cell is exposed to environmental conditions that
significantly perturb its’ homeostasis, is the protection of macromolecules.
Cells respond to cellular stress in many ways ranging from activation of
survival pathways to eliciting programmed cell death which eliminates
damaged cells. The initial response a cell takes upon stress is geared
toward fighting the stress stimuli and recovering the cell from this insult. If
the cell is unable to recover from this stress, then the death signaling
pathways are activated in order to abolish damaged cells. The stressed
cells’ fate is determined upon the interplay the stressed cell has between
these two responses. Hence, a cells’ survival depends on the ability to
initiate an appropriate response towards environmental or intracellular
stress stimuli. A rapid and transient stress response is required for the cell
to reestablish cellular homeostasis (1). Moreover, this reaction is highly
conserved throughout evolution due to its’ extraordinary significance for
many areas in biology and medicine. For example, antioxidant defense
mechanisms against oxidative injury and stress proteins such as heat-
shock proteins exist in lower organisms, as well as in mammals (2).
There are many different types of stress a cell can encounter.
Depending on the type and level of the stress, the cell mounts different
responses to deal with these conditions. For example, protective
responses such as the unfolded protein response against accumulation of
unfolded proteins in the endoplasmic reticulum, to enhance protein folding
by increasing chaperones protein activity, counteracts the stress and
promotes cell survival. Hence, the cells mount different responses to
promote survival; if stress is not resolved, then cell death programs are
activated.
3
1.1.2 Stress Induced-Cell Death
1.1.2.1 Apoptosis
Cell death comes in many forms and types. Cell death research has
attracted much attention in the last two decades, mainly due to its
relevance to several diseases and cancer. The term programmed cell
death refers to controlled or regulated form of death associated with a
series of biochemical and morphological changes (3). Programmed cell
death, nowadays, is synonymous with apoptosis. The term apoptosis was
first used to describe a particular morphology of cell death common to the
vast majority of physiological cell deaths (4). During the 1980s, apoptosis
became the focus of attention in the field of cell death. The discoveries of
Bcl-2 family of proteins (5-7), death receptors (8), caspases (9),
mitochondrial cytochrome-c release (10), and a role for the endoplasmic
reticulum (11) in apoptosis were just a few major milestones in the history
of the cell death field. Several types of cellular stress stimuli are known to
trigger apoptosis, such as chemotherapeutic agents, irradiation, oxidative
stress and ER stress. Cysteine proteases, known as caspases, are
common death effector molecules in apoptosis. During apoptosis,
caspases are activated by different mechanisms. Death receptors of the
tumor necrosis factor (TNF) receptor family or TNF-related apoptosis
inducing ligand (TRAIL) receptors are stimulated with their respective
ligands and agonistic antibodies, leading to receptor aggregation and
recruitment of the Fas-associated death domain (FADD) and procaspase-
8, forming death inducing signaling complex (DISC) (12). Upon
recruitment, caspase-8 is cleaved and activated to further cleave
downstream caspases (12). The release of cytochrome-c and other
apoptogenic factors into cytosol from mitochondrial intermembrane space
leads to activation of caspase-3 through the formation of cytochrome-
c/Apaf-1/caspase-9-containing apoptosome complex (13). Caspases
activation must be tightly controlled due to their potential detrimental
effects on cell survival if they are inappropriately activated. Inhibitors of
4
apoptosis proteins (IAPs) represent a group of endogenous inhibitors of
caspases (14). Among the IAP family, XIAP is the most potent inhibitor of
caspases and blocks apoptosis by binding to active caspase-3 and
caspase-7 and by blocking activation of caspase-9 (14). In addition to
IAPs, apoptosis is regulated by anti-apoptotic proteins, such as Bcl-XL,
Bcl-2 and Mcl-1 and pro-apoptotic proteins such as Bax, Bak and BH3
domain only molecules (15). Moreover, apoptosis sensitivity is controlled
through the regulation of additional signaling cascades, for example, NF-
κB, JNK, TNFR, and the ubiquitin/proteosome pathway (14, 17).
1.1.2.2 Autophagic Cell Death
Autophagy, or self eating, is a catabolic process where a cell eats
its own components through the lysosomal machinery. It is characterized
by the vesicular sequestration and degradation of long-lived cytoplasmic
proteins and organelles (18). When cells are exposed to metabolic and
therapeutic stresses, such as growth factor deprivation, inhibition of the
receptor tyrosine kinase/ Akt/ mammalian target of rapamycin (mTOR)
signaling, shortage of nutrients, ischemia/reperfusion, inhibition of
proteasomal degradation, the accumulation of intracellular calcium and
endoplasmic reticulum (ER) stress, autophagy is typically observed (19-
22). Under most cellular conditions, autophagy functions as a stress
adaptation that prevents cell death, and in some circumstances, it
constitutes an alternative route to cell death, hence the functional
relationship between autophagy and cell death is complex. In addition to
that, this complex interrelationship implies these responses are linked at a
molecular level.
Although it is not clear if autophagy plays a protective or toxic role
in the cell, there is evidence that it plays a beneficial role in the heart
under physiological and pathological conditions (23). Constitutive
autophagy in the heart under baseline conditions is a homeostatic
mechanism for maintaining cardiomyocyte size and global cardiac
5
structure and function, and that upregulation of autophagy in failing hearts
is an adaptive response for protecting cells from hemodynamic stress (23).
Consistent with this, rapamycin, which induces autophagy by inhibiting
mTOR, protects myocardium against ischemia/reperfusion injury (24).
However, it has been shown that down-regulation of transcription factors
such as activating transcription factor 5 or 7 (ATF5 or ATF7) using siRNA
prevents stress-induced cell death (25, 26). Hence, what is critical for
deciding the fate of the cell is the timing and level of autophagy. Moreover,
there is some evidence of crosstalk between apoptosis and autophagy at
the molecular level, and accumulating evidence have shown that inhibition
of apoptosis induces cell death that is dependent on or associated with
autophagy. The anti-apoptotic proteins from Bcl-2 family have also been
shown to inhibit autophagy and autophagic cell death (27-29). Thus, Bcl-2
not only functions as an anti-apoptotic protein, but also as an anti-
autophagy protein via its inhibitory interaction with Beclin 1, an autophagy
protein (28). This anti-autophagy function of Bcl-2 may help to maintain
autophagy at levels that are compatible with cell survival, rather than cell
death.
1.1.2.3 Necrosis
Necrosis is the term used for accidental cell death, implying it is an
unregulated process within a multicellular organism. It is known to be
associated with inflammation and is said to occur in severe forms of injury
(30). Necrosis results from the additive effect of a number of independent
of biochemical events that are activated by depletion of cell energy stores
(30). Moreover, the inflammation observed in necrosis, is mostly evidence
of the phagocytosis of cell debris produced by the necrosis process.
Morphologically, necrosis is characterized by an increase in cell volume,
swelling of organelles, and plasma membrane rupture (2). In the
propagation of necrotic cell death, several signaling cascades have been
shown to be involved. The serine/threonine kinase RIP1 has been shown
6
to be one of the key mediators of necrotic cell death in the case of death
receptors or Toll-like receptors (31, 32). Studies have revealed the
requirement of RIP1 for death receptor-induced necrosis (33, 34) and
lipopolysaccharide-induced cell death of macrophages (35). In addition,
molecules known as inhibitors of RIP1 kinase were reported to protect
against ischaemic brain injury in an in vivo model of necrosis (36-38).
Another serine/ threonine kinase RIP3 has shown a critical role in necrotic
cell death in response to TNF stimulation and during virus infection (39-
41).
Other mediators are known to be involved in the propagation of
necrotic cell death, such as ROS and calcium (42, 43). Mitochondria are
known to promote ROS generation from oxidative phosphorylation
stimulated by mitochondrial calcium (42, 43). Hence, both ROS and
calcium lead to the damage of macromolecules and organelles
contributing to the loss of cell integrity. Furthermore, the stimulus that
drives necrosis has shown to inhibit apoptotic machinery. Caspases
inactivation and cleavage are mediated by calcium-dependent activation of
calpain (44) and ROS rendering caspases inactive (45).
1.1.3 Stress and Survival Pathways
1.1.3.1 The Heat Shock Response
Upon exposure to cellular stress, depending on the type and level
of stress, a cells’ response can be manifold. If the stress level is not very
severe, then the cell can cope with it by promoting one of its survival
pathways to ensure its survival. One of the main prosurvival pathways a
cell can mount upon stress is the heat shock response (46). The main
stress this response was known originally to be activated upon was mild
heat stress; however other stresses like oxidative stress and heavy metals
are now known to activate this response (46). One of the main
consequences of these stresses is protein damage leading to the
accumulation and aggregation of unfolded proteins (46). During the initial
7
phase of heat shock response, gene transcription and protein translation
are halted in order to lessen the aggregates of unfolded proteins in the cell
(46). However, in these conditions, some transcription factors known as
heat shock factors (HSFs) are selectively activated or expressed (56).
Many forms of HSFs exist in veterbrates, the most famous being HSF1
which is essential for heat shock response (56). Studies have shown that
mice lacking HSF1 are more sensitive to stress and unable to induce heat
responsive genes upon heat shock (57-59). HSF1 is maintained inactive in
the cytoplasm by binding to Hsp90 and co-chaperones (60, 61). When
cells are exposed to stress, unfolded proteins are accumulated and
compete with HSF1 for Hsp90 binding. HSF1 monomer is released and
forms a homotrimer, which is translocated to the nucleus to activate heat
responsive genes (Figure1). HSF1 binds to upstream sequences (heat
shock elements) of its’ target genes leading to the expression of heat
shock proteins (Hsps) (Figure 1).
The genes encoding Hsps are highly conserved and are grouped in
families on the basis of sequence homology and molecular weights:
Hsp110, Hsp100, Hsp90, Hsp70, Hsp60, Hsp40 and small Hsp families
(46). Hsps are known to function as molecular chaperones (46). Usually
Hsps work as oligomers, if not as complexes of several different
chaperones, co-chaperones and/or nucleotide exchange factors (46).
Their interaction with chaperones is responsible mainly for (a) maintaining
Hsps’ partner proteins in a folding-competent, folded, or unfolded state; (b)
organellar localization, import, and/or export; (c) minimizing the
aggregation of non-native proteins; and (d ) targeting non-native or
aggregated proteins for degradation and elimination from the cell (46).
Some Hsps are known to be stress-inducible, such as Hsp27 and Hsp70
(62). Hsp27 is regulated by phosphorylation and dynamic
association/dissociation into multimers (63). However, Hsp70 is regulated
by DnaJ cochaperones. DnaJ stimulates Hsp70 to hydrolyze ATP, a key
8
step that closes its substrate-binding cavity and thus allows stable binding
of substrate (268).
Figure 1: Induction of heat shock proteins inhibits apoptosis and promotes cell survival (2)
Copyright © 2010 Simone Fulda et al.
Hsp27 and Hsp70 are known to protect the cell against stress-
induced cell death including apoptosis (64) and necrosis (65-67). These
effects are achieved by promoting prosurvival activities and inhibiting cell
death pathways. They promote prosurvival pathways by binding to the
unfolded proteins to aid in their refolding, thereby preventing protein
aggregation (68). In specific, Hsp70 is also known to interact with actin to
maintain the integrity of the cytoskeleton (55). In addition, they inhibit
apoptosis by modulating the extrinsic and intrinsic pathways and
interfering with caspase activation (49-51). In specific, Hsp27 and Hsp70
have been reported to block the release of pro-apoptotic factors, such as
9
cytochrome c from mitochondria (52-54). Hsps in the cytosol are also
known to block the activation of caspases and the formation of
apoptosome. Hsp70 can interact with and inhibit apoptosis-inducing factor
(AIF), therefore inhibiting apoptotic nuclear changes (47, 48). Overall,
Hsps can be activated or induced by a number of stresses to protect the
cell by influencing a variety of cellular processes which determine the
cells’ fate. Hsps are, in general, prosurvival and anti-apoptotic molecules.
1.1.3.2 DNA Damage Response
Several stressors such as chemotherapeutic agents, irradiation,
and environmental genotoxic agents such as ultraviolet light are known to
cause DNA damage (69, 70). Upon sensing key lesions such as double-
stranded breaks (DSBs) and single strand breaks (SSBs) in the DNA (72),
several processes are initiated. Cell cycle checkpoints are activated to
arrest cell cycle progression to allow time for repair before the damage is
passed on to daughter cells. In addition to checkpoint activation, the DNA
damage response leads to induction of transcriptional programs and
enhancement of DNA repair pathways. If the level of damage is too severe
and irreversible, the stressor is transmitted by the cellular stress response
to the activation of effector systems to mediate cell death and hence
apoptosis (71). All of these processes are carefully coordinated so that the
genetic material is faithfully maintained, duplicated, and segregated within
the cell.
Moreover, once DSBs are generated directly or indirectly by many
anticancer drugs, ataxia telangiectasia mutated (ATM) kinase is recruited
by the MRE-11-Rad50-NBS1 (MRN) complex to sites of broken DNA to
phosphorylate downstream substrates such as checkpoint kinase 2 (Chk2)
and p53 (73). p53 can then transcriptionally activate cell cycle regulatory
protein p21. The accumulation of p21, a cyclin-dependent kinase inhibitor,
suppresses Cyclin E/Cdk2 kinase activity thereby resulting in G1 arrest
(73). On the other hand, upon SSBs, ataxia telangiectasia and Rad3
10
related (ATR) kinase is the kinase that gets activated to phosphorylate
Chk1. Chk1 in turn phosphorylates and inhibits Cdc25c, a
tyrosine/threonine phosphatase which is a mitotic inducer, to mediate
G2/M arrest or alternatively phosphorylates and inhibits Cdc25a, required
for the progression from G1 to the S phase, to promote S-phase arrest
(73, 74).
Several DNA repair pathways are reported to be initiated once a
cell is experiencing any DNA damage. Direct reversal is the simplest
pathway where direct reversal of the highly mutagenic alkylation lesion O6-
methylguanine (O6-mG) by the product of the MGMT gene (O6-
methylguanine DNA methyltransferase) takes place (76). The alkyl group
is transferred from a guanine to a cysteine residue since the O6-mG is
extremely detrimental to the cell. Another pathway for DNA repair is the
base excision repair (BER) which corrects non-bulky damage to bases
resulting from oxidation, methylation, deamination or spontaneous loss of
DNA base itself (77). Nucleotide excision repair (NER) is another method
known to be the most flexible DNA repair pathway (75). The most
significant lesion it acts upon is pyrimidine dimmers caused by the UV
component of sunlight. Other NER substrates include bulky chemical
adducts, DNA intrastrand crosslinks and some forms of oxidative damage
(75). All these lesions share a common feature in which they all cause a
helical distortion of the DNA duplex and a modification of the DNA
chemistry (75). The DNA mismatch repair (MMR) pathway is another
method which plays a role in the correction of replication errors. Mispairs
generated by the spontaneous deamination of 5-methylcytosine and
heteroduplexes formed following genetic recombination are also corrected
via MMR. Lastly, DSB repair is the last method of DNA repair. DSBs are
the most serious form of DNA damage because they pose many problems
for transcription, replication, etc. This damage is caused by a variety of
sources including exogenous agents such as ionizing radiation and certain
genotoxic chemicals. Overall, coordination between the highly complex
11
methods of DNA repair pathways and the repair surveillance mechanisms
linked to cell cycle checkpoints as well as cell death pathways is required
to have an accurate genome transmission. Failure in DNA repair will lead
to the activation of cell death pathways.
Figure 2: DNA Damage Response
Pathway diagram reproduced courtesy of Paterson Institute of Cancer Research, Inc.
(http://www.paterson.man.ac.uk/dnadamage/)
1.1.3.3 Response to Oxidative Stress
At cellular homeostasis, pro-oxidant species and antioxidants
defense mechanisms are at equilibrium. The disturbance of this
equilibrium by the generation of reactive oxygen species (ROS) such as
superoxide anion (O2•-), hydrogen peroxide (H2O2), singlet oxygen,
hydroxyl radical (OH•), peroxy radical, as well as the second messenger
nitric oxide (NO•) which can react with O2•- to form peroxynitrite (ONOO−),
leads to oxidative stress (2). Antioxidant defense mechanisms constitute
of ROS-metabolizing enzymes including catalase, glutathione peroxidase,
and superoxide dismutases (SODs) and other antioxidant proteins such as
glutathione (GSH). ROS are usually generated intracellularly from the
mitochondrial electron transport chain and are dealt with SODs, enzymes
of defense against oxygen toxicity (2). In addition to the physiological
sources of ROS, exogenous agents can contribute to the intracellular
production of free radicals. Excess ROS can cause damage to major
12
biological macromolecules such as nucleic acids, proteins, carbohydrates,
and lipids (2). Furthermore, when the cell antioxidants are overwhelmed
and are unable to clear the ROS, ROS can induce cell death. Cytotoxic
agents induce ROS such as peroxide and O2•-, which can lead to
apoptosis (78). Peroxide lead to the release of cytochrome c from
mitochondrial intermembrane space into cytosol and activate nuclear
transcription factors like, NF-κB, AP-1 and p53 (79), which play a role in
up-regulating death proteins or down-regulating survival proteins. Different
proposed models have been reported of how ROS induce cell death.
Firstly, peroxide induction of apoptosis could be through up-regulation of
Fas-FasL system leading to activation of caspase-8 and downstream
caspases (80, 81). Another model could be that NO• induces apoptosis by
inactivating antioxidant enzymes and by the generation of ceramide which
leads to caspase activation, induction of mitochondrial permeability and
activation of Fas system (82-84). Several studies have reported that anti-
apoptotic proteins have antioxidant roles and that Bcl-2 reduces the
generation of reactive oxygen species by preventing the loss of
cytochrome c from the mitochondria (85). In addition to that, separate
studies have shown that Bcl-2 over-expressing cells have higher levels of
GSH (86).
Moreover, ROS can also interfere with apoptosis and adopt the
cells to use an alternative mode of cell death, necrosis. This is achieved
by inactivation of caspases (87) and reduction in levels of ATP (88, 89).
Studies have also illustrated that ROS can provide a link between cellular
stress and the initiation of autophagy, a strategy to ensure the cells’
survival (90).
1.1.3.4 The Unfolded Protein Response
The endoplasmic reticulum (ER) is the cellular organelle where
secretory and membrane proteins are post-translationally processed by
glycosylation, disulfide bond formation, correct folding and oligomerization
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(2). For all these processes to take place effectively, the ER environment
has to be monitored. If the influx of nascent, unfolded polypeptides
exceeds the folding and/or processing capacity of the ER, the
physiological function of the ER is perturbed (91). ER perturbation leads to
more accumulation of unfolded proteins in the ER, creating ER stress.
This results in the activation of signaling pathways that form the unfolded
protein response (UPR) (91, 92) (Figure 3). UPR involves the activation of
ER resident transmembrane proteins: Inositol-requiring enzyme 1 (IRE1),
double-stranded RNA activated protein kinase (PKR)-like ER kinase
(PERK), and activating transcription factor 6 (ATF6). Once they are
activated, their signaling pathways lead to the transcriptional regulation of
specific UPR target genes including, molecular chaperones, folding
catalysts, ER-associated degradation molecules (ERAD) and antioxidant
genes (91).
ATF6 is a type II transmembrane protein containing a basic leucine
zipper (bZIP) transcription factor in its cytosolic domain (93). Upon ER
stress, ATF6 is translocated to the Golgi complex. Site-1 protease (S1P)
cleaves ATF6 in the luminal domain and the N-terminal membrane
anchored half is cleaved by Site-2 protease (S2P). These proteolytic
reactions release the cytosolic (bZIP) domain, which translocates to the
nucleus to activate transcription of genes (93). Furthermore, IRE1 and
PERK are type I transmembrane proteins that dimerize to promote auto-
phosphorylation and activation upon ER stress (2). IRE1 is a
transmembrane protein with a cytosolic kinase and endoribonuclease
activities, and an ER luminal dimerization domain (94). Once IRE1 is
dimerized and auto-phosphorylated, its RNase domain is activated (95).
IRE1 cleaves mRNA that encodes a transcription factor X-binding protein-
1 (XBP-1) to produce XBP-1s (96, 97), which in turn activates the
transcription of UPR target genes. On the other hand, PERK, a ser/thr
protein kinase, once activated leads to the phosphorylation of the alpha
subunit of the eukaryotic translation initiation factor 2 (eIF2α), hence
14
translational attenuation (98). PERK-induced signaling pathway also leads
to the transcriptional activation of UPR target genes through up-regulation
of the CAP-independent translation of a transcription factor ATF4 (99).
PERK activation also leads to the phosphorylation of bZIP cap’ n ‘collar
transcription factor NF-E2 related factor (Nrf2), which contributes to
cellular redox homeostasis by inducing the expression of antioxidant
genes (100, 101).
Figure 3: ER stress and the Unfolded Protein Response (2)
Copyright © 2010 Simone Fulda et al.
Upon failure of UPR signaling to overcome ER stress, cells die
through ER stress-induced apoptosis. Three different pathways have been
found to be involved. One of which is caspase-4/caspase-12 pathway
where caspase-12 (103) is expressed in mice and caspase-4 (104) in
15
humans. Caspase-12 has been shown to cleave caspase-9 without
activation of the Apaf-1/cytochrome c pathway (102). Another pathway
involved is the C/EBP homologous protein (CHOP) pathway, where CHOP
transcription factor induced downstream of PERK and ATF6 leads to the
suppression of anti-apoptotic Bcl-2 expression (105) and induction of Bim
expression (106). The last mode involved in ER stress-induced apoptosis
is the activation of JNK through IRE1 by binding to Traf2 and ASK1 (107,
108). In addition, glucose-regulated proteins (GRPs) induced by glucose
starvation are also transcriptionally induced by ER stress (109, 110).
GRPs include molecular chaperones of the ER such as GRP78/Bip,
GRP94, etc. GRPs promote cell survival to various stresses such as
ischemia (111), glutamate excitotoxicity (112) and neurodegeneration
(113). GRPs also play important roles in survival during early mammalian
development (112). Interestingly, recent studies have revealed the
existence of small compounds that mimic the function of GRPs and those
that induce endogenous GRPs to prevent protein aggregation and protect
cells against stress-inducing conditions such as ischemia or
neurodegeneration.
1.2 eIF2α Kinases
Eukaryotic cells recognize and process diverse stress signals to
cope with the cellular damage or alternatively, induce cell death. An
important contributor to the alleviation of cellular injury is the family of
protein kinases that phosphorylate the α subunit of eIF2 on Ser51 residue.
In normal conditions, eIF2 is known to bind to initiator Met-
tRNAiMet (aminoacylated initiator methionyl-tRNA) and GTP, and
participate in ribosomal scanning for the start codon (114). After joining of
the small and large ribosomal subunits, GTP complexed with eIF2 is
hydrolysed to GDP, and eIF2-GDP is released from the translation
machinery. The GDP-bound eIF2 is recycled to active GTP-bound eIF2 by
a reaction catalyzed by the guanine nucleotide exchange factor eIF2B
16
(114). When the cells are exposed to different stress conditions, eIF2α
kinases phosphorylate the α-subunit of eIF2 at Ser51 (Figure 4). This
phosphorylation changes the translation factor, eIF2, from a substrate to
an inhibitor of eIF2B by increasing its affinity to and sequestering eIF2B.
By lowering the levels of eIF2-GTP, general translation is attenuated
(114). Hence, this allows cells to have sufficient time to correct the stress
damage and selectively enhance gene-specific translation, such as the
transcription factor ATF4 (122-125), which plays an important role in
stress adaptation.
Figure 4: Regulation of eIF2 (126)
Pathway diagram reproduced courtesy of Eunice Kennedy Shriver National Institute of Child Health and Human
Development (http://spb.nichd.nih.gov/index.htm)
In this family, four kinases have been identified in mammals and
each contains its own regulatory regions to recognize different sets of
stress conditions (Figure 5). Heme-regulated inhibitor (HRI) or EIF2AK1 is
one of the eIF2α kinases which is activated by heme deprivation and
oxidative and heat stresses in erythroid tissues (119, 120). General control
non-derepressible-2 (GCN2) or EIF2AK4 is activated upon amino-acid
17
deprivation (115, 116). EIF2AK3 or PERK ((RNA-dependent protein
kinase)-like ER kinase) is activated in response to misfolded proteins in
the ER as described above (117). Lastly, RNA-dependent protein kinase
(PKR) or EIF2AK2 is induced by interferons and by viral dsRNA following
viral infections (118).
A)
B)
Figure 5 : A) Protein kinases, PKR, HRI (heme-regulated inhibitor), PERK and GCN2
are activated by different stress conditions; B) Domain structure of protein kinases, PKR, PERK, HRI and GCN2
Adapted from reference (127), Generated by Afnan Abu-Thuraia ©
18
1.2.1 Heme-Regulated Inhibitor (HRI)
Iron and heme play a very important role in hemoglobin synthesis
and erythroid differentiation. The majority of iron in a human body is
present in heme iron. In addition, the most abundant hemoprotein,
hemoglobin, contains as much as 70% of total iron content in a healthy
body. Heme is not only a prosthetic group of proteins required for oxygen
transport and storage, respiration, and biosynthetic pathways, it is also
required for the regulation of gene expression by virtue of its ability to bind
Bach1, a transcription factor that functions in association with Maf proteins
(128, 129). Moreover, heme is also required for the control of translation in
erythroid precursors by modulating the eIF2α kinase activity of Heme-
regulated inhibitor (HRI) kinase (130). This regulation by heme of HRI
kinase is essential to repress translation of globin proteins in anemias due
to iron deficiency (131), erythropoietic protoporphyria, and β-thalassemia
(132). HRI has been discovered in reticulocytes under conditions of iron
and heme deficiencies. Later on, HRI was shown to be a heme-regulated
kinase that phosphorylates the α-subunit of eIF2 at Ser51. Under iron and
heme deficiencies, protein translation is halted at initiation with
disaggregation of polysomes. HRI, which senses intracellular heme
concentration, gets activated by multiple autophosphorylation upon heme
deficiencies (134). The first autophosphorylation reaction, happening once
HRI is newly synthesized, stabilizes HRI and prevents its aggregation.
This makes HRI an autokinase, but not yet an eIF2α kinase. The second
autophosphorylation reaction is required to form a stable HRI that can
dimerize and is heme-regulated (133). Under heme abundance, heme is
bound to HRI and keeps it inactive (135) (Figure 6). Upon heme
deficiency, the third stage of autophosphorylation takes place on Thr485
leading to its’ eIF2α kinase activation and hence phosphorylation of
eIF2αSer51 and attenuation of protein synthesis (Figure 6). HRI is
19
inhibited following heme repletion and HRI that is no longer heme-
regulated is then degraded (136).
Figure 6: HRI balances heme and globin synthesis by sensing intracellular heme
concentrations Adapted from reference (137)
Previous studies have revealed the importance of HRI in vivo in
regulation of protein synthesis in erythroid precursors. Using hri-/- mice,
they demonstrated that hri-/- reticulocytes had higher rate of globin protein
synthesis compared to hri+/+ reticulocytes (131), hence its essence in
ensuring balanced synthesis of globins and heme. Due to its important
role in differentiation of erythroids, the regulation of HRI is tightly
controlled. The autophosphorylation and eIF2α kinase activities of HRI are
inhibited by hemin with an apparent inhibition constant Ki of 0.2µM (131,
140). Once hemin is bound to HRI, it blocks the binding of ATP to HRI in a
concentration dependent manner (141).
Figure 7: Domain Structure of HRI
P P
20
Adapted from reference (137) HRI contains two regions where heme molecules bind, one in the N-
terminus and the other in the kinase insert (Figure 7). Heme-binding
region in the N-terminus is nearly saturated with stably bound heme and is
co-purified with HRI, while the other binding region is available for
exogenous heme and is reversible (140). The second site is responsible
for the down-regulation of HRI kinase activity. In addition to that, the N-
terminus is required for achieving higher eIF2α kinase activity, although it
is not essential for the kinase activity of HRI. N-terminus is also required
for the highly sensitive heme regulation of HRI. Histidine residues 75 and
120 in the N-terminus (Figure 7) were shown to be respectively proximal
and distal heme ligands (138, 139). Their mutation to Alanine further
underscored the significant importance of heme in the N-terminus for the
down-regulation of HRI activity.
Other HRI activators have also been identified. These include
arsenite-induced oxidative stress, osmotic shock and heat shock (134).
Arsenite-induced activation of HRI involves reactive oxygen species and
requires molecular chaperones such as hsp70 and hsp90 (134). NO was
also shown to activate HRI whereas CO was shown to prevent its
activation (142). NO activation of HRI requires HRI N-terminus domain to
be loaded with heme (139).
Additional studies have shown that in iron-deficiency anemia, HRI is
critical in determining red blood cell size, cell number and hemoglobin
content per cell. In addition, HRI is also responsible for the adaptation of
microcytic hypochromic anemia in iron deficiency (137). In the case of
erythropoietic protoporphyria (EPP) and β-thalassemia (132), studies have
indicated that HRI may be a significant modifier gene contributing to EPP
disease severity, particularly in the development of hepatic pathology
(143, 144).
21
1.2.2 General Control Non-derepressible-2 (GCN2)
During times of energy deprivation, the mammal goes through
complex metabolic responses to promote survival. Some of these
responses include the usage of triglycerides storage from adipose tissue
to provide energy in the form of fatty acids and ketone bodies (145). In
addition to that, protein degradation rates are increased in order to
increase the levels of amino acid precursors (145). These will provide
continued availability of essential amino acids that can be used to
synthesize essential proteins that are required to cope with the stress of
energy deprivation. In liver, the pathways that regulate lipid balance upon
energy deprivation have resulted in enhanced glycogenolysis,
gluconeogenesis, fatty-acid oxidation and ketone body formation (145).
Many responses of organisms and cells to amino acid deprivation have
also been characterized. Several studies have shown that GCN2 protein is
the main regulator of cellular responses to amino acid deprivation (154).
GCN2 was first identified in yeast, as a ser/thr protein kinase that
phosphorylates the α-subunit of the translation initiation factor 2 (eIF2) at
Ser51 (155-157). GCN2 eIF2α kinase is activated by uncharged tRNAs
which accumulate following amino acid deprivation (152, 155). Upon
phosphorylation of eIF2α on Ser51, global translation is attenuated to
ensure that sufficient amount of amino acids are available to support cell
growth and function (Figure 8). However, transcription and translation of
stress-inducible genes is up-regulated by the modification of eIF2α (148).
Upon GCN2 activation, translation of GCN4 is up-regulated in yeast.
GCN4 is a transcription activator of genes involved in amino acid
biosynthesis to ensure that cells have an adequate supply of amino acids
for protein synthesis during times of stress (152).
22
Figure 8: GCN2 pathway of activation (145)
Reprinted from reference 145 with permission Copyright © Elsevier
Mechanisms of GCN2 activation upon amino acid deprivation were
studied. GCN2 contains a domain homologous to histidyl-tRNA synthetase
(HisRS), an enzyme responsible for charging histidyl-tRNA with histidine
(150). This domain in GCN2 lacks the synthetase activity and some
residues that are critical for histidine-specific binding (150). It has been
proposed that in yeast, uncharged tRNA that are accumulated upon amino
acid limitations, bind to this HisRS-related domain in GCN2 and lead to the
activation of its catalytic domain (152, 153) (Figure 8).
A GCN2 ortholog in mammals is also found to be activated upon
amino acid deprivation (146, 147). In addition, the mechanism of
transcriptional activation of GCN4 gene expression seems to be
conserved through out eukaryotic evolution. In mammals, upon amino acid
deprivation, GCN2 is activated and the translation of the transcription
23
factor ATF4 is up-regulated. ATF4 activates the transcription of CHOP,
another transcription factor that regulates the expression of stress-induced
target genes (147). Although, GCN2 knockout mice under normal
conditions had no apparent phenotype (148), under amino acid deprived
states, they showed poor adaptation (148). Other studies have revealed
additional conditions activating GCN2 such as serum deprivation (146)
and UV irradiation (149), implicating GCN2 in other stress-induced
pathways. In addition, methylglyoxal, ubiquitous 2-oxoaldehyde derived
from glycolysis, has been shown to attenuate protein synthesis in yeast
through eIF2α phosphorylation mediated by GCN2 (159). Furthermore,
GCN2 eIF2α kinase activity is regulated by a complex formed of
GCN1/GCN20 (158, 161). This complex shows structural similarity to
eEF3, a factor important for the binding of tRNAs to ribosomes (158, 161).
The GCN1/GCN20 complex interacts with GCN2 by binding to its N-
terminus. This binding is proposed to facilitate the transfer of tRNAs from
ribosomal A site to the HisRS-related domain on GCN2 to result in its
activation (158, 161).
1.2.3 PKR (RNA-dependent protein kinase)-like ER kinase (PERK)
Perturbation of the ER homeostasis resulting in accumulation of
unfolded proteins modulates translation initiation by activating the ER
transmembrane ser/thr protein kinase PERK (163-165). Activated PERK,
as member of the eIF2α kinases family, is well known to phosphorylate
eIF2α on Ser51 (162). Concurrently, PERK is also known to induce
specific translation of stress-induced genes, such as the ATF4
transcription factor (166).
PERK has a luminal domain, similar to the ER-stress-sensing
luminal domain of IRE1, which acts as its regulatory domain, and a
cytoplasmic portion that contains a protein kinase domain most similar to
that of the known eIF2α kinases, PKR and HRI (163). Several studies
have shown that PERK oligomerization is critical for its activation by ER
24
stress. This is followed by PERK autophosphorylation at different sites,
one being Thr980 in the activation loop, and this results in facilitating the
binding of ATP and eIF2α (167, 168). Moreover, as for IRE1 in unstressed
conditions, PERK is bound to the ER chaperone BiP/GRP78 (169). After
the onset of ER stress, unfolded proteins compete with PERK for BiP
binding which release free PERK allowing its’ dimerization and
autophosphorylation (Figure 9). Once ER stress is over, BiP re-associates
with PERK and results in PERK dephosphorylation/inactivation. Hence,
PERK binding to BiP is dynamic and reversible (169).
Figure 9: Model for activation of PERK in response to ER stress (166)
Reprinted from reference 166 with permission Copyright © Mary Ann Liebert, Inc.
A central regulator for eIF2α kinases stress response is the
transcriptional activator ATF4 (173). Translation repression by PERK
leads to increased expression of ATF4 in response to ER stress (173). In
addition to ATF4, other genes regulated by PERK activation are known to
be involved in an array of diverse cellular functions, including folding and
processing of secretory proteins, clearance of misfolded secretory proteins
by the ERAD pathway, glutathione biosynthesis and control of the cellular
redox status, mitochondrial function, amino acid synthesis and import, and
25
regulation of signaling, apoptosis and transcription (166). PERK then
directs the UPR transcriptional program by mechanisms independent of
ATF4. An example of this is the protein p58IPK encoded by ER stressed
cells. P58IPK, transcriptionally induced by the UPR under the control of an
ER stress-response element (ERSE) in its promoter (172), triggers a
negative feedback mechanism by binding and inhibiting PERK (172). This
will lead to translational recovery and enhancing the expression of genes
to increase the capacity of the ER to process client proteins. Furthermore,
NF-κB activation is induced by PERK-induced phosphorylation of eIF2α in
response to ER stress (170, 174-178). Due to the reduction in global
protein synthesis, the translation of IκBα, an inhibitor of NF-κB, is
significantly reduced and consequently, this leads to an increase in free
and active NF-κB (170, 174-178).
In addition to eIF2α, PERK phosphorylates the transcription factor
Nrf2 (171). In unstressed conditions, Nrf2 is present in the cytoplasm in
complex with Keap1 (171, 179). PERK-dependent phosphorylation of Nrf2
is both sufficient and necessary for Nrf2/Keap1 complex dissociation and
subsequent nuclear import of Nrf2 to activate gene transcription. Nrf2
phosphorylation inhibits its re-association with Keap1 in vitro (171). Hence,
Nrf2 is believed to be an important effector of PERK-mediated cell survival
in response to ER stress.
Finally, PERK is widely expressed with higher expression levels in
pancreas (162). Interestingly, genetic studies have revealed that PERK
deficiency caused by the loss of PERK catalytic activity is the underlying
cause behind the pancreatic insufficiency in Wolcott-Rallison Syndrome,
an autosomal recessive disorder characterized by neonatal or early
infancy type 1 diabetes, epiphyseal dysplasia, and growth retardation
(180). A role for PERK in pancreatic endocrine function has been further
supported by PERK KO mice, in which atrophy of the exocrine pancreas,
as well as loss of the insulin-secreting β-cells of the endocrine pancreas
were observed in the PERK-deficient animals (269).
26
1.2.4 RNA-dependent Protein Kinase (PKR)
PKR is a 551 amino acid protein consisting of two RNA binding
motifs (RBMs) at its N-terminus and a ser/thr protein kinase domain at its
C-terminus. In absence of stress, PKR is hypothesized to be kept in a
monomeric latent state due to the auto-inhibitory function of its RBMs,
which occlude the kinase domain and in this manner regulate PKR
activation (211) (Figure 11). As a consequence of viral infection and
accumulation of viral dsRNA (at least 30 bps in length), dsRNA binds to
RBMs leading to PKR dimerization. Dimerized PKR undergoes
conformational changes that relieve the auto-inhibition, induce PKR
activation, and allow substrate recognition and phosphorylation. Upon
dsRNA binding, PKR phosphorylates eIF2α at Ser51 (190, 191), resulting
in attenuation of translation (189). The most characterized role of PKR is
to inhibit translation initiation of viral mRNA through the phosphorylation of
eIF2α at Ser51 (188), a reaction conserved from yeast to mammals.
PKR is induced by interferons (IFNs) and plays a role in the antiviral
defense mechanism as a translation inhibitor (184). IFNs of type I (IFNα/β)
induce the expression of PKR through the activation of a highly conserved
13-bp sequence of the IFN stimulated responsive element (ISRE) on the
PKR promoter (207). In addition to mediating a critical role in response to
dsRNA, thus acting as a sensor of viral infections, PKR is switched on by
a set of other activators, such as proinflammatory stimuli, growth factors,
cytokines, and oxidative stress (192). In agreement with multiple
conditions activating PKR, it integrates and transmits signals to various
factors such as STAT, interferon regulatory factor 1 (IRF-1), p53, Jun N-
terminal protein kinase (JNK), and p38, as well as engaging the NF-κB
pathway (Figure 10). NF-κB is a transcription factor that is retained in the
cytosol by binding to IκBs, inhibitory proteins of NF-κB. Upon activation of
PKR by dsRNA, large IκB kinase (IKK) is activated by phosphorylation,
resulting in phosphorylation of IκB and IκB preferential degradation (202),
releasing NF-κB which is translocated to the nucleus to activate the
27
transcription of genes involved in the immune response, inflammatory
response, cell adhesion, cell growth and apoptosis (202).
Figure 10: Signalling pathways involving PKR (227)
Reprinted from reference 227 with permission Copyright © Nature Publishing Group
In addition to eIF2α, PKR phosphorylates the human protein
phosphatase 2A (PP2A) regulatory subunit B56α (215). It was found that
28
B56α interacts with PKR in a kinase dependent manner. Upon
phosphorylation of B56α, the activity of PP2A trimeric holoenzyme is
increased (215) and mediates PKR biological effects besides translation
control, such as transcription and apoptosis (215).
PKR has been also implicated in cell growth regulation and
tumorigenesis that rely on control of apoptosis in vivo (204). In fact, over-
expression of PKR in murine cells induces apoptosis in response to
dsRNA, lipopolysaccharide (LPS), serum deprivation or TNF-α treatment
(203, 205). In contrast, cells over-expressing the catalytically inactive PKR
∆6 were completely resistant to dsRNA and TNF-α-induced apoptosis
(206), demonstrating that PKR kinase activity is required. Moreover, PKR
induces expression of tumor necrosis factor receptor (TNFR) family and of
pro-apoptotic proteins such as Bax (206). Hence, PKR mediates
expression of pro-apoptotic genes regulated by dsRNA and probably
functions in interferon-mediated host defense to trigger cell death in
response to virus infection. However, a variety of human malignancies
have developed ways to inhibit PKR activity by expressing or activating
cellular PKR inhibitors, one of which is nucleophosmin (NPM), a
multifunctional nucleolar protein usually over-expressed in a several
malignancies (214) that binds and inhibits PKR (214). In addition, PKR is a
p53 target gene, regardless of viral infection or type I IFN stimulation, and
plays an important role in the tumor suppressor function of p53, in part
through inhibition of translation and induction of cell apoptosis. PKR could
interact directly with the C-terminal part of p53 and phosphorylate p53 on
the Ser392 residue, to induce its activity (228). The ability of p53 to cause
cell cycle arrest and regulate transcription of target genes is impaired in
PKR−/− MEFs. Several other studies have reported that PKR promotes
proteosomal degradation of p53 in association with glycogen synthase
kinase 3 (GSK-3β) and Mdm-2 independently of translational control (208).
These results indicate that p53-induced PKR expression conversely plays
29
a role in the feedback down-regulation of p53. This could be considered as
a homeostatic control of p53-mediated PKR enhancements (210).
PKR modulates STAT proteins (signal transducers and activators of
transcription) function. PKR and STAT1 form a complex that is not
dependent on PKR catalytic activity but requires RBMs of PKR (229). It
has been suggested that this interaction leads to the inhibition of STAT1
DNA binding activity. PKR does not phosphorylate STAT1 directly but
seems to control a kinase cascade in which ERK2 is the kinase
phosphorylating STAT1 (230). In addition to that, PKR is shown to
associate with STAT3, leading to full STAT3 activation in response to
PDGF stimulation (231). STAT3 phosphorylation on Tyr and Ser residues,
which is necessary for full activation, is PKR dependent. As proposed for
STAT1, PKR regulates ERK activation ultimately involved in STAT3
phosphorylation (231). Moreover, PKR is an activator of signaling
cascades involved in stress-activated protein kinases and is shown to
mediate JNK and p38 activation in response to specific stimuli (232) where
their full activation is dependent on PKR. As p38 MAPK is a master
regulator that controls several transcription factors, such as NF-κB, ATF-2,
and STAT1, PKR might play an important role in these transcriptional
pathways (233).
30
Figure 11: Schematic representation of PKR activation (211)
Reprinted from reference 211 with permission Copyright © American Society for Microbiology
PKR dimerization may be mediated in part through the RBMs,
either through direct protein-protein interactions in this region (196, 197),
or through dsRNA bridging the protein subunits (198, 199). After
homodimerization, PKR undergoes rapid autophosphorylation at Thr446
and Thr451 in the activation loop, stabilizing the dimerization, in turn
increasing PKR catalytic activity (210). Given important roles played by
PKR in various cellular processes, the activity of this kinase must be tightly
regulated. Several viruses have developed mechanisms in order to
repress PKR activity (Table 1). Examples include the adenovirus-encoded
VAI RNA, which forms an inhibitory complex with PKR by functioning as a
competitive inhibitor of dsRNA binding (219). Both retrovirus and rotavirus
encode virus-specific dsRNA binding proteins to sequester dsRNA from
PKR. In addition, the 88-amino acid K3L gene product of vaccinia virus,
K3L, and the tat gene product of human immunodeficiency virus type-1
(HIV-1), physically interact with PKR, resulting in inhibition of PKR kinase
activity during viral infection (212, 220). In fact, K3L was shown to interact
31
with PKR catalytic domain to form a complex that interferes with active-site
function and/or substrate association (212). Another gene product of
vaccinia virus, E3L (221), is also known to inhibit PKR by binding to and
sequestering dsRNA. E3L also inhibits PKR by directly interacting with
PKR leading to heterodimer formation (221). Moreover, influenza virus
encoded protein NS1, which has dsRNA binding domains is known to
sequester dsRNA away from PKR to prevent its activation (222). Also,
NS1 interacts with PKR to form an inhibitory complex (223). Recently, it
was uncovered that influenza virus uses a novel mechanism to repress
PKR activity by activating the cellular inhibitor of PKR, p58IPK (224-226).
On the other hand, non-dsRNA molecules including heparin and
polyanions such as, dextran sulfate, chondroitin sulfate and poly (L-
glutamine) have been shown to activate PKR (200). Recently, a protein
named RAX that activates PKR has been identified in mice where its
human orthologue is called PACT (201). It contains three dsRNA binding
motifs (RBMs) where the first two RBMs of PACT heterodimerize with
PKR via its RBMs, leading to PKR activation in absence of dsRNA (201).
The third RBM of PACT binds weakly to the kinase domain of PKR and
promotes its activation. In contrary, the activity of PKR kinase is regulated
in part by, association with specific inhibitory proteins such as p58IPK, a
cellular protein of the tetratricopeptide repeat (TPR) family (212). P58IPK is
known to repress the activation and activity of PKR by interacting with the
ATP-binding region of the catalytic domain. Hence, by forming a complex
with PKR, p58IPK interferes with nucleotide binding and autoregulation
(212). Another mechanism negatively regulating PKR involves the catalytic
subunit of protein phosphatase 1α (PP1C) (213). PP1C reduced PKR
autophosphorylation activity by directly interacting with PKR N-terminal
regulatory region regardless of dsRNA binding (213). PP1C reduced PKR
enzymatic activity by promoting PKR dephosphorylation and subsequent
disruption of PKR dimers. Overall, regulation of PKR must be an important
32
process due to PKR’s significant role in many cellular processes that could
be detrimental to a cell. Table 1: Viral products that inhibit PKR activation and/or eIF2α phosphorylation
(211) Reprinted from reference 211 with permission Copyright © American Society for Microbiology
1.3 Nck Adaptor Proteins
1.3.1 Nck gene and proteins
A cDNA encoding the non-catalytic region of tyrosine kinase protein
(Nck-1) has been first randomly isolated from a human melanoma library
(234). This cDNA coded for a 377 amino acid protein composed of three
Src-homology 3 (SH3) domains at its N-terminus and one C-terminus Src-
homology 2 (SH2) domain (234). More recently, a second Nck related
cDNA encoding Nck-2 has been identified from a mouse embryonic cDNA
expression library (237). Nck proteins are the products of different genes:
human Nck-1 is localized to the locus q21 of chromosome 3 while Nck-2 to
chromosome 2 at locus q12. Nck-1 and Nck-2 display 68% identity at the
amino acid level (235) (Figure 12) and the amino acid variations fall largely
into the interval sequences between the SH domains. Moreover, Nck-1
and Nck-2 are to some extent functionally redundant and neither Nck-1
nor Nck-2 knock-out mice exhibit an apparent phenotype whereas double
33
knock-out mice die in utero (270). Moreover, only a single Nck-like gene
has been identified in several invertebrate species, including Xenopus
(Nck), Drosophila (Dock) (236) and C. elegans (235), suggesting that the
function of Nck might be evolutionary conserved. Mutations in Dock gene
in Drosophila photoreceptor cells (R cells) disrupt axon guidance and
targeting in the developing nervous system (236).
Figure 12: Modular composition of Nck adapter proteins (238)
Copyright © 2009 Lettau et al; licensee BioMed Central Ltd.
1.3.2 Nck interaction partners and functions
Studies over the last few years in mammals and invertebrates have
indicated that the main cellular function of Nck is to link activated cell
surface receptors to actin cytoskeleton reorganization involved in various
biological responses such as axon pathfinding, migration, chemotaxis and
endocytosis. Nck was detected in complexes with PDGF receptor
(PDGFR), VEGF receptor (VEGFR), Hepatocyte growth factor receptor
(HGFR), EGF receptor (EGFR) and Ephrin receptor (EphB1) where the
interaction between Nck-1/Nck-2 and the activated receptor tyrosine
kinases is mediated through the SH2 domain of Nck and a
phosphotyrosine residue in the receptors (239-241). Nck-1 has also been
shown to bind tyrosine phopshorylated proteins such as insulin receptor
substrate-1 (IRS-1) (271), p130-Cas (242) and the GTPase-activating
protein (GAP)-associated protein p62dok (243). All together, these
observations suggest that Nck participates in signal transduction at the
34
level of receptors or at the level of proteins recruited downstream in
specific signaling pathways.
Nck also contributes to signal transduction by interacting with
proteins through its SH3 domains, which bind to proline and hydrophobic
rich motifs (246). The Abl tyrosine kinase was the first shown to bind to the
SH3 domains of Nck-1 and this lead to Abl activation (244). A stable
complex between Nck-1 (SH3) and the Ras guanine nucleotide exchange
factor Sos was demonstrated in cells. This suggests a role for Nck-1 in
linking receptor tyrosine kinases to Ras activation (245) for Nck to
participate in the control of gene expression and proliferation. Moreover,
two Nck-SH3-binding proteins, PAK1 (middle SH3) and neuronal Wiskott -
Aldrich syndrome protein (N-WASP) (third SH3), have been implicated in
signal transduction by Rho GTPases such as Rac and Cdc42, in
regulating actin cytoskeleton (247). N-WASP is known to interact with
GTP-bound Cdc42 and cluster in polymerized actin structures at the
membrane level (248). Evidence supports that Nck plays a role in
translocating N-WASP from the cytoplasm to the plasma membrane, via
its interaction with receptor tyrosine kinases, and then would facilitate
N-WASP interaction with Cdc42 (248). Similarly, PAK1-induced actin
reorganization requires Nck binding (249). Activated PDGFR recruits
PAK1 to the cell membrane via Nck. As described for N-WASP, the role of
Nck is to target PAK1 to the plasma membrane, where PAK1 interacts
with the GTP-bound Rac1/Cdc42 (250), resulting in further increase in
PAK1 kinase activity (251).
Upon PDGF and EFG stimulation, Nck has been reported to be
phosphorylated on serine, threonine and tyrosine residues (240, 261)
mapped in the intervening sequence between the first and second SH3
domains (240). In addition, protein kinase A (PKA) and C (PKC) appear to
phosphorylate Nck on serine residues (240) however, the functional
relevance of Nck phosphorylation has still to be established.
35
In T-cells, Nck plays a pivotal role in T-cell receptor (TCR)-
mediated re-organization of the actin cytoskeleton as well as in the
formation of immunological synapses. Upon activation of T-cells, Nck is
recruited to a membrane proximal site via interaction with the tyrosine-
phosphorylated adaptor SLP76, providing a scaffold for the WASP-
dependent actin remodeling machinery (252-254). Nck is also directly
recruited to the CD3ε component of the TCR in an activation-dependent
manner (255), where the interaction between Nck and CD3ε is mediated
by the first SH3 domain of Nck. However, the role of the CD3ε-Nck
interaction is disputed (255-258).
1.3.3 Role of Nck in regulating eIF2α phosphorylation and cell
response to stress
In the past years, our laboratory has uncovered a novel role for
Nck-1 in modulating protein translation through its direct interaction with
an intrinsic component of the translational machinery (263). In fact, we
have shown that Nck-1 directly interacts with the C-terminal region of the
β-subunit of the eukaryotic initiation factor (eIF2β) via its first and third
SH3 domains (263). We detected the complex Nck-1-eIF2β in enriched
ribosomal fractions and shown that the levels of Nck associated with
ribosomes is dynamically regulated by insulin (263). Furthermore, we
demonstrated that over-expression of Nck-1 increases protein translation
through the same domains that it uses to bind eIF2β (263). This suggests
that Nck-1 modulates translation through interaction with eIF2β. In
addition, our work has revealed that the PERK arm of the unfolded protein
response (UPR) induced by ER stress pharmacological drugs like
tunicamycin or thapsigargin treatment that result in phosphorylation of
eIF2α at Ser51 and inhibition of protein translation, is modulated by Nck1
(264) (Figure 13). In addition, we found that Nck-1 over-expression
decreased basal and ER stress-induced eIF2α phosphorylation and
36
further induction of ATF4 and CHOP. We identified Nck-1 in a complex
with the serine/threonine protein phosphatase 1c (PP1c) (264). Therefore,
we proposed that Nck-1 contributes to maintain eIF2α dephosphorylated
and then indirectly promotes protein synthesis by recruiting PP1c in close
proximity to eIF2.
Figure 13: Schematic model of the regulation of translation by Nck-1 during ER
stress (260) Copyright © 2004, by the American Society for Biochemistry and Molecular Biology
In parallel, our group has provided strong evidence that Nck-1 also
regulates eIF2αSer51 phosphorylation by other eIF2α kinases, except
GCN2 (265). Given Nck-1 effects on eIF2αSer51 phosphorylation are not
universal to all eIF2α kinases, this suggests that more than one
mechanism could exist. In fact, we discovered that Nck-1 efficiently
prevents PKR activation, suggesting that Nck-1 acts at the PKR level,
37
rather or in addition to that of eIF2α (266). This was further supported by
showing that Nck-1 also impaired other PKR-induced signaling pathways
than eIF2αSer51 phosphorylation, where Nck-1 decreased PKR-mediated
p38 MAPK activation and greatly attenuated dsRNA-induced cell death
(266). We showed that the inhibition of PKR activation by Nck-1 was
reversible, since it could be neutralized by significant high levels of dsRNA
achieving a robust activation of PKR (266). Finally, we provided evidence
that upon PKR activation, Nck-1 is released based on the observation that
Nck-1 interacts only with inactive PKR. Combined to the observation that
Nck-1 was phosphorylated by PKR in an in vitro reaction, we proposed the
following model: In normal conditions, Nck-1 is bound to PKR to buffer
PKR activation in order to avoid inappropriate spontaneous PKR activation
and signaling that could be detrimental. In response to viral infection or
any condition that triggers significant PKR activation, Nck-1 is released
from PKR due to PKR change in conformation or alternatively following
PKR-mediated phosphorylation of Nck-1.
38
CHAPTER II HYPOTHESIS AND PROJECT OUTLINE
39
Previously our laboratory has reported that Nck-1 modulates eIF2α
phosphorylation by HRI, PERK and PKR, but not GCN2, demonstrating
that Nck-1’s regulation of eIF2α phosphorylation is specific to a subset of
eIF2α kinases. To delineate the mechanism underlying the effect of Nck-1,
we focused on Nck-1’s modulation of eIF2α phosphorylation by PKR. To
our surprise, we discovered that Nck-1 limits PKR activation induced by
dsRNA (Figure 1) and impairs other PKR downstream signaling events
than eIF2α phosphorylation and also protects cells against dsRNA-
induced cell death (266).
Figure 1: Nck-1 reduces PKR activation induced by dsRNA (266)
In addition, we provided evidence that Nck-1 binds the inactive form of
PKR, and is an in vitro substrate of PKR (266). From these results, we
proposed that Nck-1, in complex with inactive PKR, limits PKR activation
in absence of significant stress (dsRNA), but once PKR is activated by
high levels of dsRNA, Nck-1 dissociates from PKR, allowing full PKR
activation. My hypothesis is that during the process of PKR activation by
40
dsRNA, PKR phosphorylates Nck-1 and in this manner, promotes PKR-
Nck-1 dissociation. In this thesis, my research objectives were to a) define
the molecular determinants mediating the interaction between Nck-1 and
inactive PKR; and b) provide evidence that Nck-1 is a substrate of PKR in
vivo. My first objective included classical in vitro pull down and co-
immunoprecipitation assays involving Nck-1 and PKR proteins in their wild
type and/or truncated/mutated forms, with dsRNA stimulation when PKR
activation was required. To achieve my second objective, determining
whether Nck-1 is substrate of PKR in vivo, PKR+/+ and -/- mouse embryonic
fibroblasts (MEFs) challenged with or without dsRNA have been analyzed
for Nck-1 phosphorylation using a novel technology that allow separation
of phosphorylated from non phosphorylated proteins in SDS-PAGE and
western blotting. Using the same approach, I have confirmed Nck-1
phosphorylation in HEK 293 cells over-expressing wild type form of PKR.
41
CHAPTER III EXPERIMENTAL PROCEDURES
42
Cell culture and Transfection Cos-7 and Hela cells were grown in Dulbecco’s Modified Eagle’s Medium
(DMEM) (GIBCO™ invitrogen corporation, Grand Island, NY, USA)
supplemented with antibiotics, Antibiotic-Antimycotic (Anti-Anti) (GIBCO™
invitrogen corporation, Grand Island, NY, USA) and 10% heat-inactivated
fetal bovine serum (FBS) (GIBCO™ invitrogen corporation, Grand Island,
NY, USA) at 37ºC in 5% CO2/ 95% O2. 80% confluent cells grown in
60mm dishes were transfected using Lipofectamine-Plus reagent
(Invitrogen™, Carlsbad, CA, USA) according to the manufacturer’s
instructions.
PKR activation 24 hrs after transfection, PKR was activated by transfecting the cells again
for 2 hrs with synthetic double-stranded RNA (poly I:C, amount indicated
in the figures) (InvivoGen©, San Diego, CA, USA) using Lipofectamine-
Plus reagent according to the manufacturer’s instructions.
Cell lysate preparation and Immunoblot analysis Cells were washed once with cold 1x Phosphate Buffered Saline (PBS)
and lysed in ice-cold lysis buffer (50mM Hepes (pH 7.5), 150mM NaCl,
10% glycerol, 1% Triton X-100, 1.5mM MgCl2, 1mM EGTA, 10mM sodium
pyrophosphate, 10mM sodium fluoride) supplemented with protease
inhibitors (10µg/mL aprotinin, 10µg/mL leupeptin, 1mM PMSF, 200µM
activated sodium orthovanadate). Cell lysates were centrifuged at 13,000
rpm for 10 min at 4ºC. Supernatants were subjected to Bradford assay
(Bio-Rad® Laboratories, Inc, Hercules, CA, USA) for protein quantification.
Protein concentrations were normalized with lysis buffer and 6X Laemmli
buffer and samples were then heated at 90ºC for 5 min.
43
Western Blotting and Antibodies Equal amount of total cell lysate proteins (10-50µg) were resolved by
SDS-PAGE on 10% acrylamide gels and transferred onto polyvinylidene
difluoride (PVDF) membranes (Bio-Rad® Laboratories, Inc, Hercules, CA,
USA) to be immunoblotted with specific antibodies. PKR phosphorylation
was assessed by using the PKR pT446 antibody [E120] (Abcam, Inc®,
Cambridge, MA, USA) and eIF2αSer51 phosphorylation was detected by a
phosphospecific antibody directed against eIF2αSer51 (Invitrogen™,
Camarillo, CA, USA). Total eIF2α (FL-315), HA-probe (F-7)
(immunoprecipitation), HA-probe (Y-11) (western blotting), and PKR (K-
17) were purchased from Santa Cruz Biotechnology, Inc®, CA, USA. To
detect Flag-tagged PKR, an anti-FLAG antibody (M2, SIGMA®, St. Louis,
MO, USA) was used. Anti-Myc antibody (clone 9E10) was purchased from
Upstate® (Millipore™, CA, USA). Nck-1 and Nck-2 specific polyclonal
antibodies were generated in rabbits using a GST-fusion protein encoding
Nck-1 and Nck-2 specific amino acid sequence located between the third
SH3 and SH2 domains (QNNPLTSGLEPSPPQCDYIRPSLTGKFAGNP)
and (VVLSDGPALHPAHAPQISYTGPSSSGRFAGRE), respectively. Pan-
Nck antibody generation was described previously (267). Secondary
antibodies used were goat anti-rabbit, goat anti-mouse or Protein A
conjugated horseradish peroxydase (GAR-HRP, GAM-HRP and Prot A-
HRP) (Bio-Rad® Laboratories, Inc, Hercules, CA, USA). Signal detection
was performed using ECL Plus (Enhanced Chemiluminescence, GE
Healthcare©, Buckinghamshire, UK) according to the manufacturers’
instructions.
Phospho-affinity polyacrylamide gel electrophoresis Phos-tag™ acrylamide (AAL-107; NARD Institute, Amagasaki, Japan) was
prepared at 5 mM in water as stock solutions. The composition of the
resolving phospho-affinity SDS-polyacrylamide mini-gels containing
acrylamide-pendant Mn2+-Phos-tag ligand was the following: 8.25%
44
acrylamide, 0.375M Tris-HCl pH 8.8, 50µM Phos-tag™, 100µM MnCl2,
0.15% ammonium persulfate and 0.003% Tetramethylethylenediamine
(TEMED). Stacking gel was prepared according to the protocol used in
usual SDS-PAGE. The gels were run at 90V for 3 hrs and then soaked in
transfer buffer supplemented with 1mM of EDTA for 10min. Subsequently,
gels were washed with transfer buffer with no EDTA for 10min at room
temperature before proteins were transferred to polyvinylidene difluoride
(PVDF) membranes (Bio-Rad® Laboratories, Inc, Hercules, CA, USA) to
be immunoblotted with pan Nck antibody (267) as previously described.
Immunoprecipitation Cos-7 cells were treated with 2mM of the crosslinker agent
Dithiobis(succinimidyl)propionate (DSP) (Thermo Scientific®, Pierce
Biotechnology, Rockford, IL, USA) for 30 min at room temperature. Cells
were then washed with a Stop buffer (50mM Tris pH 7.4) and lysed in
RIPA buffer (50mM Hepes pH 7.4, 1% Triton X-100, 1% sodium
deoxycholate, 0.1% SDS, 150mM NaCl, 10% glycerol, 1.5mM MgCl2,
1mM EGTA, 10mM sodium pyrophosphate, 100mM sodium fluoride)
supplemented with protease inhibitors (1mM sodium orthovanadate,
10µg/mL leupeptin, 10µg/mL aprotinin, 10µg/mL Pefabloc SC, 10µg/mL
DTT). PKR was immunoprecipitated from 1.3mg of protein extracts using
lysates normalized at 1µg/µl with RIPA buffer and following overnight
incubation at 4ºC with 3 µg of anti-HA antibody. Protein A-agarose beads
(80 µL of 25% slurry solution) were added to the samples and further
incubated with agitation for 1 hr at 4ºC. Further on, beads were collected
by centrifugation (1 min, 13000 rpm at RT°), washed three times with
RIPA buffer and re-suspended in 60 µl of 2X Laemmli. Samples were
heated for 5 min at 90ºC and loaded on a 10% acrylamide gel for
SDS-PAGE and western blotting.
45
Pull-down Assay Cos-7 cell lysates were harvested in pull-down lysis buffer (10mM Tris-HCl
pH 7.5, 50mM KCl, 2mM MgCl2, 1% Triton X-100, 10mM sodium
pyrophosphate, 100mM sodium fluoride, 1mM DTT, 17.5mM β-
glycerophosphate, 4µg/mL aprotinin, 2µg/mL leupeptin, 100µg/mL PMSF,
1mM benzamidine). 4.5mg of proteins from lysate were incubated with
recombinant GST (20µg) or GST-Nck-1 proteins (20µg) previously
expressed in bacteria and immobilized on glutathione-agarose beads. This
incubation was done at 4ºC for 2 hrs. Proteins bound on beads were
collected, washed three times using pull-down buffer and re-suspended in
60 µL of 2X Laemmli buffer. Samples were heated for 5 min at 90ºC and
processed for western blotting with appropriate antibodies. Recombinant
protein expression was detected by Ponceau staining the membrane after
western blotting.
PKR plasmid construction Wild type C-terminal segment of PKR (amino acid 249 to 551) and the
mutant K296R C-terminal (amino acid 249 to 551) of PKR were amplified
using the forward primer containing a start codon and a HindIII restriction
site (5’ TTTTAAGCTTATGGCACCCAGATTTGACCTTC 3’) and reverse
primer that lacks a stop codon and has an XbaI restriction site (5’
TTTTCTCTAGACATGTGTGTCGTTCATTTTTCT 3’) from plasmids
pcDNA3.1 Flag-PKR wt and GST-PKR K296R mutant, respectively. Wild
type N-terminal (amino acid 1 to 248) of PKR was amplified using the
forward primer containing a start codon and a HindIII restriction site (5’
TTTTAAGCTTATGGCTGGTGATCTTTCAGC 3’) and reverse primer that
lacks a stop codon and has an XbaI restriction site (5’
TTTTCTCTAGAGATCTTTTTGCCTTCCTTTG 3’). PCR was carried for 24
cycles (94ºC 5 min, 94ºC 1 min, 55ºC 40 sec, 72ºC 40 sec) followed by a
final extension at 72ºC for 10 min. PCR products were separated on 1%
agarose gel containing ethidium bromide and PCR products purified using
46
QIAquick® PCR purification kit (50) (QIAGEN Sciences®, Maryland, USA).
Purified PCR products along with the vector pcDNA 3.1(+) Myc-His
version B (Invitrogen™, Carlsbad, CA, USA) were then double digested by
HindIII and XbaI at 37ºC for 2 hrs. Digested inserts were gel purified and
ligated with the vector at room temperature for 1 hr at 1:3 vector:insert
ratio. 2 µL of the ligation reaction were used to transform 40 µl of DH5α
electrocompotent cells using electroporation. Electroporation was carried
at 20 µF capacitance, 2.0 kV voltage and 200 ohms resistance. 1 mL of LB
media was added to the electroporated cells and incubated under shaking
at 37ºC for 1 hr. Cells were then plated on LB ampicillin agar plates and
incubated overnight at 37ºC. Colonies were collected and grown in LB-
ampicillin. QIAPrep® Spin Miniprep Kit (250) and QIAGEN® Plasmid Maxi
Kit (25) (QIAGEN Sciences®, Maryland, USA) were used to isolate the
plasmids DNA which were then sent for sequencing for confirmation.
Statistical analyses Statistical significance was determined using Student’s t-test with p values
≤ 0.05 considered as significant. In all tests, two groups with one changed
parameter were compared.
47
CHAPTER IV RESULTS
48
Nck-1 interacts with PKR independently of functional SH domains Due to the significant role of PKR in important processes such as
development, immune and inflammatory responses, cell adhesion, growth
and apoptosis, the activity of PKR must be tightly regulated. In our lab, we
have observed Nck-1-mediated attenuation of PKR activation by dsRNA.
The question addressed next was whether Nck-1 limits PKR activation by
interacting with PKR. Evidence from our laboratory has shown that Nck-1
binds to PKR in in vitro pull down assay (266). To confirm this in vivo, HA-
tagged wild type Nck-1 was transiently expressed in intact Cos-7 cells to
determine whether Nck-1 and endogenous PKR could be detected in a
common molecular complex. As shown in Figure 1, endogenous PKR was
co-immunoprecipitated with HA-Nck-1 in HA immunoprecipitates.
Figure 1. Endogenous PKR co-immunoprecipitates with HA-Nck-1. Cos-7 cells were
transfected with 1µg of wild type HA-Nck-1. Using an anti-HA antibody, HA
immunoprecipitates were prepared using lysates from Cos-7 cells previously exposed to
the cross linker agent DSP. PKR and HA-Nck-1 proteins in the immunoprecipitates were
detected by Western blotting using specific antibodies. This experiment was performed
three times.
In fact, Nck-1’s interaction with PKR was revealed to be independent of
any functional SH domain (266). Hence, to further confirm this, HA-tagged
wild type/mutants Nck-1 were transiently over-expressed in Cos-7 cells.
The HA-Nck-1 mutants contained point mutations which abrogate the
binding property of the three SH3 domains (3M) or the SH2 domain (2M)
(272, 273). As shown in Figure 2, equivalent amount of endogenous PKR
co-immunoprecipitated with HA-Nck-1 wild type and mutants. Western
blotting of the total cell lysates (TCL) with HA antibody revealed that
49
HA-Nck-1 mutated in its SH2 domain (2M) expressed at lower levels
compared to HA-Nck-1 wild type and the SH3 mutant (3M). Overall, Nck-1
interaction with PKR is independent of any functional SH domain.
Figure 2. Nck-1-PKR interaction is independent of Nck-1 functional SH domains.
Cos-7 cells were transfected with 1µg of wild type HA-Nck-1 (WT) or HA-Nck-1 mutants
containing point mutations which abrogate the binding property of all three SH3 domains
(3M) or the SH2 domain (2M). Using an anti-HA antibody, HA immunoprecipitates were
prepared using lysates from Cos-7 cells previously exposed to the cross linker agent
DSP. PKR and HA-Nck-1 proteins in the immunoprecipitated samples were detected by
Western blotting using specific antibodies. This experiment was performed three times.
dsRNA-induced PKR activation is reduced in cells over-expressing Nck-1 mutants Given that increased levels of Nck-1 significantly reduce
dsRNA-induced PKR activation (266) and that Nck-1 mutants still interact
with PKR, we predicted that Nck-1 mutants would also reduce PKR
activation induced by dsRNA. To test this, HA-Nck-1 with all SH3 domains
mutated (SH3M) was transiently expressed in Cos-7 cells and 24 hrs later,
cells were transfected with synthetic dsRNA (poly IC) to activate PKR. Low
concentration of poly IC (0.4 μg/ml) was used to activate PKR given that
robust PKR activation may overcome Nck-1’s effect. In fact, we have
shown previously that Nck-1 binds only to inactive form of PKR,
suggesting that upon PKR full activation, Nck-1 and PKR dissociate. Upon
poly IC stimulation, Nck-1 mutant SH3M significantly reduced PKR
activation and consequently phosphorylation of eIF2α on Ser51 compared
to the empty vector transfected cells (Figure 3). A similar experiment
carried with Nck-1 SH2M mutant revealed that poly IC-induced PKR
50
activation was also reduced by Nck-1 SH2M over-expression, as shown in
Figure 4. In agreement with lower levels of PKR activation, we found lower
levels of eIF2α phosphorylation on Ser51 in cells overexpressing Nck-1
SH2M.
A)
B)
51
Figure 3. SH3 mutated Nck-1 reduces (A) PKR activation and (B) phosphorylation of eIF2α induced by dsRNA. Cos-7 cells were transfected with 1µg of pRK5 empty
vector (vector) or HA-Nck-1 mutant containing point mutations which abrogate the binding
property of all three SH3 domains (SH3M) prior to be subjected 24 hrs later to a second
transfection with 0.4µg/mL of synthetic dsRNA (poly IC) for 2hrs. Indicated proteins were
detected by Western Blotting of cell lysates (adjusted for protein content) using indicated
specific antibodies (upper panel). Densitometry and statistical analyses (student t-test)
were performed on data obtained from three independent experiments. Results are
reported as the mean ± SEM of (A) the ratio phospho-PKR (p-PKR) over total PKR (lower
panel) and of (B) the ratio phospho-eIF2α (p-eIF2α) over total eIF2α (lower panel). *
p<0.05 relative to vector with poly IC, ‡ p≤0.05 relative to vector with poly IC.
A)
52
B)
Figure 4. SH2 mutated Nck-1 reduces (A) PKR activation and (B) phosphorylation of eIF2α induced by dsRNA. HEK-293 cells were transfected with 1µg of pRK5 empty vector (vector)
or HA-Nck-1 mutant containing a point mutation which abrogates the binding property of the SH2 domain
(SH2M) prior to be subjected 24 hrs later to a second transfection with 0.4µg/mL of synthetic dsRNA (poly IC)
for 2hrs. Indicated proteins were detected by Western Blotting of cell lysates (adjusted for protein content) using
specific antibodies (upper panel). Densitometry and statistical analyses (student t-test) were performed on data
obtained from three independent experiments. Results are reported as the mean ± SEM of (A) the ratio
phospho-PKR (p-PKR) over total PKR (lower panel) and of (B) the ratio phospho-eIF2α (p-eIF2α) over total
eIF2α (lower panel). * p<0.05 relative to vector with poly IC, ‡ p<0.05 relative to vector with poly IC.
Nck-1 full length is required to bind PKR Nck-1’s interaction with PKR was found to be independent of any
functional SH domain. Hence, we next asked if truncated Nck-1 molecules
composed of only the SH3 or SH2 domains interact with PKR. For this, we
used in vitro pull down assay, in which different constructs of GST-tagged
Nck-1 were expressed in bacteria and tested to pull down endogenous
PKR from Cos-7 lysates. As shown below in Figure 5, GST-tagged Nck-1
wild type (GST-Nck-1 FL) was the only construct that pulled down
endogenous PKR, whereas the truncated forms of Nck-1 (GST-Nck-1 SH3
53
and GST-Nck-1 SH2) did not. The level of expression of PKR in the cells
is shown in the total cell lysate detected by western blotting using PKR
specific antibody. Ponseau staining of the membrane was performed to
demonstrate that the same amount of GST-tagged proteins was used in
each pull down. Overall, Nck-1 needs to be full length to interact with PKR.
Figure 5. Nck-1 full length binds PKR. (A) Pull down of endogenous PKR with
GST-Nck-1 full length (FL) and GST- Nck-1 either SH2 or SH3 domains alone.
Cos-7 cell lysates (4.5mg of protein) were incubated with GST (20µg) or GST-
Nck-1 constructs (20µg) for pull down assays and endogenous PKR was
detected by Western Blotting (WB) using a PKR specific antibody as well as to
probe TCL, total cell lysate. (B) Levels of GST fusion proteins used revealed by
Ponseau staining (indicated by *).
54
Nck-1 binds only to inactive PKR Previously, our laboratory has shown that Nck-1 binds to inactive
PKR in vitro. To confirm this in vivo, a co-immunoprecipitation experiment
was carried out from cells transiently transfected with HA-Nck-1 wild type.
Cells were subsequently transfected with high amounts of poly IC (3.0
μg/ml) to induce robust full activation of PKR. Upon poly IC transfection,
interaction between Nck-1 and PKR was abolished, whereas endogenous
PKR was co-immunoprecipitated with HA-Nck-1 in absence of poly IC
(Figure 6). In addition, blotting for eIF2α phosphorylation showed that
upon poly IC treatment, high levels of eIF2α were phosphorylated,
confirming PKR activation. Phosphorylation of eIF2α under basal
conditions, however, decreased in Nck-1 over-expressing cells, confirming
our previous results showing that Nck-1 over-expression reduces the level
of PKR activation induced by dsRNA.
Figure 6. Loss of Nck-1 binding upon PKR activation by dsRNA. Cos-7 cells were
transfected with 1µg of pRK5 empty vector or wild type HA-Nck-1. Cells were subjected
24 hrs later to a second transfection with 3µg/Ml of synthetic dsRNA (poly IC) for 2hrs.
Using an anti-HA antibody, HA immunoprecipitates were prepared using lysates from
Cos-7 cells previously exposed to the cross linker agent DSP. PKR and HA-Nck-1
proteins in the immunoprecipitated samples and PKR, HA-Nck-1, Nck, p-eIF2α and total
55
eIF2α proteins in the total cell lysates (TCL) were detected by Western blotting using
indicated specific antibodies. This experiment was performed three times.
To determine whether activation of PKR activity is responsible for the loss
of HA-Nck-1 interaction, we assessed whether HA-Nck-1 binds to kinase
dead PKR molecule. For this we co-transfected Cos-7 cells with plasmids
encoding HA-Nck-1 and either Flag-tagged dominant negative PKR (Flag-
PKR∆6) or Flag-tagged wild type PKR (Flag-PKR WT). PKR∆6 is
dominant negative kinase dead due to the deletion of 6 amino acids in the
kinase domain that prevents PKR catalytic kinase activation, yet
interestingly PKR∆6 can still bind dsRNA, undergo dimerization and
change of conformation associated with the activation process (211). It
behaves as a dominant negative because it dimerizes with endogenous
PKR and prevents its activation. By over-expressing wild type form of
PKR, spontaneous PKR dimerization is induced due to the high levels of
PKR protein and hence is autophosphorylated and activated in the
absence of poly IC as shown by p-PKR (Figure 7). In HA-
immunoprecipitates prepared from cells expressing Flag-PKR WT or Flag-
PKR∆6, only Flag-PKR∆6 co-immunoprecipitated with HA-Nck-1 as
detected by anti-Flag specific antibody. Endogenous PKR was detected to
co-immunoprecipitate with HA-Nck-1 alone and to increase in binding with
Flag-PKR∆6 over-expression, since this dominant negative construct of
PKR dimerizes with and prevents endogenous PKR activation. HA-blotting
of the total cell lysate (TCL) showed that equal amounts of HA-Nck-1 were
over-expressed (Figure 7).
56
Figure 7. Nck-1 binds to Dominant negative-PKR. Cos-7 cells were transfected with
1µg of wild type HA-Nck-1 and 5µg of wild type Flag-PKR or kinase dead Flag-PKRΔ6.
Using an anti-HA antibody, HA immunoprecipitates were prepared using lysates from
Cos-7 cells previously exposed to the cross linker agent DSP. Flag-PKR, PKR and HA-
Nck-1 proteins in the immunoprecipitated samples and Flag-PKR, PKR, HA-Nck-1, Nck
and p-PKR in the total cell lysates (TCL) were detected by Western blotting using
indicated specific antibodies. This experiment was performed three times.
PKR activation is a multiple step process involving dsRNA binding,
dimerization and autophosphorylation. It was then interesting to determine
at which step Nck-1 dissociates from PKR. To test this, Flag-PKR∆6
construct was used due to its ability to bind dsRNA, dimerize and undergo
change of conformation without experiencing autophosphorylation and full
PKR catalytic activation. We showed that this dominant negative form of
PKR binds Nck-1 in the absence of poly IC (Figure 7), therefore the next
point addressed was whether HA-Nck-1 binds to PKR∆6 in cells treated
with dsRNA. For this, HA-Nck-1 and Flag-PKR∆6 were transiently over-
expressed in cells, followed by poly IC transfection 24 hrs later. As shown
in Figure 8, Flag-PKR∆6 interacts with HA-Nck-1 equally in the absence
and presence of poly IC treatment. Hence, we can conclude that loss of
interaction between Nck-1 and active PKR that is observed above (Figures
6 and 7) is due to PKR catalytic activity and not to dsRNA binding,
57
dimerization and change of conformation. Phosphorylation of PKR and
eIF2α was shown to increase with poly IC transfection in control cells yet
to decrease in Flag-PKR∆6 over-expressing cells treated with poly IC
since PKR∆6 dimerizes with endogenous PKR and prevents their
activation by poly IC. PKR blotting detected endogenous PKR binding to
and its dissociation from HA-Nck-1 in HA-immunoprecipitates in the
absence and presence of poly IC treatment, respectively. PKR binding to
HA-Nck-1 detected using anti-PKR antibody increased with Flag-PKR∆6
over-expression in the presence and absence of poly IC compared to
lysates without Flag-PKR∆6 over-expression.
Figure 8. Nck-1 binds inactive PKR in the absence and presence of dsRNA. Cos-7
cells were transfected with 1µg of wild type HA-Nck-1 and 5µg of kinase dead Flag-
PKRΔ6. Cells were subjected 24 hrs later to a second transfection with 3µg/mL of
synthetic dsRNA (poly IC) for 2hrs. Using an anti-HA antibody, HA immunoprecipitates
were prepared using lysates from Cos-7 cells previously exposed to the cross linker
agent DSP. Flag-PKR, PKR and HA-Nck-1 proteins in the immunoprecipitated samples
and Flag-PKR, PKR, HA-Nck-1, Nck, p-eIF2α, total eIF2α and p-PKR in the total cell
lysates (TCL) were detected by Western blotting using specific antibodies. This
experiment was performed three times.
58
Nck-1 interacts with truncated PKR N-terminal and inactive C-terminal region Thus far, Nck-1 interacts with inactive PKR and dissociates from
PKR once PKR is catalytically active. Also, Nck-1 full length is required to
interact with PKR; since truncated Nck-1, either N- or C-terminal segments
were not able to bind PKR (Figure 5). Therefore, to further understand
Nck-1 and PKR interaction, we investigated which domain of PKR
interacts with Nck-1. For this, several sections of PKR were generated as
Myc-tagged PKR constructs as shown in Figure 9A. Myc-PKR-N-terminus,
Myc-PKR-C-terminus WT or Myc-PKR-C-terminus K296R were transiently
co-over-expressed with HA-Nck-1 in Cos-7 cells to assess their binding
activity to Nck-1. We observed that HA-Nck-1 was able to co-
immunoprecipitate PKR-N-terminus and PKR-C-terminus K296R but not
PKR-C-terminus wild-type (Figure 9B). As shown in Figure 9B, over-
expression of PKR-C-terminus WT induces increased levels of
phosphorylated endogenous PKR. In addition, Nck-1 did not interact with
this construct, confirming that Nck-1 does not bind to catalytically active
PKR. As a control, we showed that transiently over-expressed Flag-
PKR∆6 full length was co-immunoprecipitated with HA-Nck-1 as detected
by anti-Flag antibody blotting. Overall, our data revealed that Nck-1
interacts with both N-terminal and inactive C-terminal domains of PKR.
59
A)
B)
Figure 9. Nck-1 binds PKR N-terminus and inactive C-terminus domains. A) Different
domains of PKR were amplified from PKR wild-type or kinase dead and cloned into
pcDNA 3.1(+) Myc-His version B vector. PKR-N-terminal expresses the N-terminus of
PKR (aa 1-248). PKR-C-terminal WT is the catalytically kinase domain of PKR (aa 249-
551) that is constitutively active. PKR-C-terminal K296R is the inactive C-terminal domain
(aa 249-551) of PKR with lysine 296 residue in the catalytic domain mutated to an
arginine. B) Cos-7 cells were transfected with 1µg of wild type HA-Nck-1 and with one of
the following plasmids: 5µg of mutant Flag-PKRΔ6, 5µg of PKR C-terminal wild-type
(Myc-PKR C-term), 5µg of PKR C-terminal mutated at K296R (Myc- PKR C-term K296R)
or 0.5µg of PKR N-terminal (Myc-PKR N-term). With an anti-HA antibody, HA
immunoprecipitates were prepared using lysates from Cos-7 cells previously exposed to
the cross linker agent DSP. Myc-PKR, Flag-PKR and HA-Nck-1 proteins in the
immunoprecipitated samples and Myc-PKR, Flag-PKR, HA-Nck-1, PKR, and p-PKR in
60
the total cell lysates (TCL) were detected by Western blotting using indicated specific
antibodies. This experiment was performed three times.
To confirm that Nck-1 dissociation is due to catalytic activation of
PKR and not to dsRNA binding, we investigated if PKR-N-terminus
interacts with Nck-1 in the presence of poly IC. Cos-7 cells were co-
transfected with HA-Nck-1 and PKR-N-terminus and treated with or
without poly IC. As shown in Figure 10, in HA-immunoprecipitates, we
observed that Nck-1 interacts with PKR-N-terminal domain and this
interaction is still detected upon poly IC treatment, suggesting that Nck-1
does not compete with dsRNA to bind PKR and does not dissociate from
PKR N-terminus upon dsRNA binding. Phosphorylation of PKR and
subsequently of eIF2α is shown to be induced upon poly IC treatment but
decreased in the presence of the N-terminus of PKR and poly IC due to
the dimerization of the N-terminus of PKR with endogenous PKR,
rendering them inactive. Total cell lysate (TCL) blotting shows the level of
Myc-tagged PKR construct to be equally expressed in Figure 9B and 10.
Figure 10. Nck-1 binds the N-terminus domain of PKR in the absence and presence of dsRNA. Cos-7 cells were transfected with 1µg of wild type HA-Nck-1 and 0.5µg of
Myc-PKR N-terminal domain. Cells were subjected 24 hrs later to a second transfection
61
with 3µg/mL of synthetic dsRNA (poly IC) for 2hrs. With an anti-HA antibody, HA
immunoprecipitates were prepared using lysates from Cos-7 cells previously exposed to
the cross linker agent DSP. Myc-PKR N-terminal and HA-Nck-1 proteins in the
immunoprecipitated samples and Myc-PKR N-terminal, PKR, HA-Nck-1, p-eIF2α, total
eIF2α and p-PKR in the total cell lysates (TCL) were detected by Western blotting using
specific antibodies.
Nck-2 interacts with PKR
Nck1 and Nck2 display 68% identity at the amino acid level (235)
and the amino acid variations fall largely into the interval sequences
between the SH domains. To determine whether Nck-2 also interacts with
PKR, HA-Nck-1 and HA-Nck-2 were transiently over expressed in parallel
in Cos-7 cells and immunoprecipitated with anti-HA antibody. As shown in
Figure 11, PKR co-immunoprecipitated with Nck-1 and Nck-2 in HA-
immunoprecipitates. Equal levels of HA-Nck-1 and HA-Nck-2 were over-
expressed as shown by anti-HA, anti-specific Nck-1 and anti-specific
Nck-2 immunoblotting (Figure 11).
Figure 11. Nck-2 binds PKR. Cos-7 cells were transfected with 1µg of wild type HA-Nck-
2 or wild type HA-Nck-1. With an anti-HA antibody, HA immunoprecipitates were
prepared using lysates from Cos-7 cells previously exposed to the cross linker agent
DSP. PKR and HA-Nck proteins in the immunoprecipitated samples were detected by
Western blotting using indicated specific antibodies. This experiment was performed
three times.
62
Nck-1 is phosphorylated by PKR in vivo
Earlier studies in our laboratory have shown that Nck-1 gets
phosphorylated in vitro by PKR (266). To assess whether this
phosphorylation event takes place in vivo, PKR+/+ and PKR-/- mouse
embryonic fibroblasts (MEFs) were treated with or without poly IC to
activate PKR and Nck-1 phosphorylation was addressed. Nck-1
phosphorylation was assessed using a novel technology called phos-tag
SDS-PAGE that allows the separation of phosphorylated from non
phosphorylated proteins in SDS-PAGE and western blotting. As shown in
Figure 12A, the amount of Nck-1 which showed gel retardation is
increased in PKR+/+ MEFs (indicated by an arrow) compared to PKR
deficient MEFs, suggesting that Nck is directly or indirectly phosphorylated
by PKR in vivo. As expected, PKR and eIF2α phosphorylation in the
absence of poly IC was higher in PKR+/+ in comparison to PKR-/- MEFs
and further enhanced by poly IC treatment. PKR blotting confirmed that
PKR is not expressed in PKR-/- MEFs.
63
Figure 12. Nck1 is phosphorylated in MEFs PKR+/+ and in HEK 293 cells over-expressing wild type PKR. (A) Mouse embryonic fibroblasts PKR+/+ and PKR-/- extracts
were transfected with 6µg/mL of synthetic dsRNA (poly IC) for 2hrs. Lysates were then
collected and analyzed by SDS-PAGE and phos-tag SDS-PAGE. Proteins were detected
by western blotting using indicated specific antibodies. (B) HEK 293 cells were
transfected with 5µg of wild type Flag-PKR or pcDNA3.1 empty vector and subjected
24hrs later to a second transfection with 6µg/mL of synthetic dsRNA (poly IC) for 2hrs.
Lysates were then collected and analyzed by SDS-PAGE and phos-tag SDS-PAGE.
Proteins were detected by western blotting using specific antibodies. This experiment
was performed three times.
To further support this, we used HEK293 cells transiently co-expressing
Flag-tagged PKR wild type (Flag-PKR) and HA-Nck-1 treated with or
without poly IC. Phosphorylation of Nck-1 was detected by phos-tag SDS-
PAGE and blotting for HA and Nck using anti-HA and anti-pan Nck
antibodies, respectively. As shown in Figure 12B, over-expression of wild
type PKR increased basal eIF2α phosphorylation, which is not further
augmented upon poly IC treatment, suggesting constitutive PKR activation
64
by over-expression. In parallel, the amount of HA-Nck-1 retarded in phos-
tag gels (indicated by the arrow) is increased in cells over-expressing wild-
type PKR and was not significantly further increased by poly IC treatment,
suggesting that PKR activation was maximally achieved by PKR over-
expression. Interestingly, when Nck western blotting is used to follow Nck
migration, we could observe that poly IC treatment induced further shift in
Nck-1 migration in both cells overexpressing or not overexpressing PKR.
These results provide evidence supporting Nck-1 as a novel substrate for
PKR in vivo.
65
CHAPTER V DISCUSSION
66
Regulation of eIF2α phosphorylation is essential to maintain global
cellular homeostasis because it controls an important step early in the
process of protein translation (280). This is consistent with the fact that
eIF2α kinases are subjected to multiple complex levels of control
governing their activation. Particularly, the eIF2α kinase PKR, which plays
a significant role in host antiviral defense, is known to be activated by
direct binding of viral dsRNA inducing PKR dimerization and
autophosphorylation. In these conditions, activation of PKR causes
transient inhibition of protein synthesis and apoptosis that altogether
contribute to limit viral production. On the other hand, it is well known that
PKR activity is simultaneously suppressed by inherent viral strategies
during infection. These involve the synthesis of viral proteins that directly
inhibit PKR (282) or recruit/activate a phosphatase that dephosphorylates
and inactivates PKR (285). However, independently of viral infection, only
two mammalian proteins so far, p58IPK and the glycoprotein p67, have
been reported to regulate PKR activation (212, 281).
In this study, we report that the adaptor protein Nck-1, which is
composed only of Src homology domains, restrains PKR activation by
dsRNA. We have demonstrated that Nck-1 functions like a reversible
inhibitor of PKR since its effects are overridden by significant intracellular
levels of dsRNA. To further support this concept, we demonstrated that
Nck-1 was only found in complex with inactive PKR and dissociates from
PKR during the process of PKR activation. We provide strong evidence
showing that the catalytic activation of PKR determines Nck-1 dissociation
from PKR. In fact, we demonstrated that Nck-1 interacts with the
dominant-negative kinase dead PKR (DN-PKR) in presence as well as in
absence of dsRNA, indicating that Nck-1 dissociation from PKR during
PKR activation by dsRNA does not result from dsRNA competition with
Nck-1 for PKR binding since DN-PKR still binds dsRNA (283). Overall, our
findings suggest that the interaction of Nck-1 with PKR under physiological
conditions limits PKR activation to prevent unwanted spontaneous
67
activation of PKR that could impair protein synthesis required for normal
cellular processes. Whether Nck-1 interaction with PKR is direct or
involves an additional player that acts as a PKR inhibitor has still to be
determined. Nonetheless, our study shows that the interaction between
Nck-1 and PKR requires full length Nck-1, while any of the functional SH
domains of Nck-1 appear to be dispensable. However, this hypothesis is
challenged by the fact that Nck-1 binds independently to the N-terminus
and inactive C-terminal region of PKR, suggesting that more than a unique
mechanism could support Nck-1 interaction and regulation of PKR
activation. Interaction of Nck-1 with the N- or C-terminal regions of PKR
could be driven by individual SH domains of Nck-1 and simultaneous
functional inactivation of all Nck-1 SH domains might be required to totally
prevent its interaction with inactive PKR. Further experiments aiming to
determine whether Nck-1 simultaneously inactivated in all SH domains still
binds to and regulates PKR activation need to be conducted to answer this
question. However, since Nck-1 mutated in its SH2 domain or in all SH3
domains interacts and prevents PKR activation by low concentrations of
dsRNA, this strongly suggests that the effects of Nck-1 on PKR activation
are direct and independent on Nck-1’s recruitment of an intermediary
protein in close proximity to PKR. Therefore, at this point our observations
demonstrating that Nck-1 binds only to inactive PKR and is a PKR
substrate allow us to propose that the interaction between Nck-1 and PKR
is a substrate-kinase interaction. We previously presented this hypothesis
supported by data revealing that Nck-1 was phosphorylated by PKR in
vitro (266). In this study, this hypothesis is further promoted by showing
that Nck-1 phosphorylation, revealed by Nck-1 migration shift in phostag-
containing acrylamide gels subjected to SDS-PAGE, was decreased in
mouse embryonic fibroblasts lacking PKR and increased following PKR
overexpression in human embryonic kidney cells (HEK293).
68
To delineate the exact mechanism(s) by which Nck-1 interacts with
and regulates PKR activation, we followed Nck-1 association with DN-PKR
in conditions activating PKR by dsRNA. PKR activation implies several
steps including dsRNA binding, PKR dimerization, conformational change
and finally, autophosphorylation (284). Any of these steps during the
process of PKR activation could induce dissociation of Nck-1 from PKR.
Here, we provide strong evidence in favor that Nck-1 dissociation from
PKR results from increased PKR catalytic activity independently of all
other steps occurring during the process of PKR activation. This comes
from our observation where Nck-1 still interacts with the DN-PKR, which is
kinase dead, in the presence of dsRNA. DN-PKR is known to undergo all
steps of wild-type PKR activation by dsRNA with the exception of the
catalytic activation due to the deletion of 6 amino acids in the kinase
domain that completely abolishes its activity (284). Taking this into
consideration with the fact that Nck-1 appears to be a substrate of PKR,
we proposed the following mechanism by which Nck-1 interacts with and
regulates PKR activation (Figure 1).
Figure 1. Model of Nck-1 and PKR interaction
69
In absence of dsRNA, PKR activation is buffered through its
interaction with Nck-1. Once intracellular levels of dsRNA increase over a
threshold level, PKR binds dsRNA, dimerizes, changes conformation, and
undergoes autophosphorylation leading to full activation of PKR catalytic
activity. During these processes, activated PKR phosphorylates Nck-1 and
promotes its dissociation to allow full PKR activation. We propose that
Nck-1 dissociation from PKR is mainly due to charge repulsion that lowers
the affinity of the interaction between Nck-1 and PKR. Interestingly, Nck-1
and PKR interaction could parallel the interaction of Nck-1 with the p21-
activated protein kinase PAK1 (274). In fact, through its direct interaction
with inactive PAK1 via its second SH3 domain, Nck-1 is believed to
translocate PAK1 from the cytosol to the plasma membrane where PAK1
will become activated. Plasma membrane activated PAK1 leads to PAK1-
mediated phosphorylation of Nck-1 and subsequent dissociation of Nck-1
from PAK1 (275). Therefore as for PAK1, Nck-1 only binds to the inactive
conformation of PKR and PKR-induced phosphorylation of Nck-1, during
dsRNA-mediated PKR activation, could promote Nck-1 dissociation from
activated PKR. To determine if the interaction between Nck-1 and PKR
plays a role in PKR sub-cellular localization and appropriate activation, like
it did with PAK1, this has to be further investigated.
Overall the exact mechanism by which Nck-1 regulates PKR activity
is still not completely elucidated. Our findings allow us to propose that
Nck-1 limits PKR activation directly, independent of any other protein.
However, we cannot rule out the possibility that Nck-1, as an adaptor
protein, may control PKR activation by recruiting PKR regulators in close
proximity of PKR. Candidates for such PKR regulators could include the
cellular chaperone p58IPK and the serine/threonine protein phosphatase
PP1c. It would be interesting to investigate whether Nck-1 interacts with
p58IPK and targets it to the catalytic domain of PKR to counteract PKR
activation, but up to now evidence of putative interaction between Nck-1
and p58IPK has never been reported. In contrast, previous work from our
70
laboratory has shown the participation of Nck-1 in assembling a molecular
complex including PP1c (264) and others have demonstrated that PP1c
down-regulates PKR activity through its interaction with PKR (213). PP1 is
regulated by its interaction with a variety of regulatory subunits that target
the catalytic subunit (PP1c) to specific subcellular localization or protein
substrates. Therefore, Nck-1 may play a role in targeting PP1c to PKR,
similar to other adaptor proteins such as CReP or GADD34 which target
PP1c to eIF2α (276, 277). Additional experiments need to be performed to
determine the molecular determinants which lead to the recruitment of
Nck-1 to a PKR-containing complex to maintain PKR inactive in absence
of significant levels of PKR activators. Moreover, Nck-1 has been shown to
be phosphorylated on serine, threonine and tyrosine residues upon the
activation of numerous growth factor receptors, yet the functional
relevance of its phosphorylation seems to be an unresolved issue. Our
results showing Nck-1 phosphorylation by PKR identifies Nck-1 as a novel
substrate of PKR and this can address the physiological meaning of Nck-1
post translational modification by phosphorylation in the perspective of
cellular response to stress. Hence it is of high interest to pursue the
significance of Nck-1 phosphorylation by PKR further and identify PKR
phosphorylation site(s) on Nck-1 to address their importance in interacting
with PKR and limiting its activation.
Due to the high percentage identity in amino acids (68%) between
Nck-1 and Nck-2, it was of importance to determine whether Nck-1
modulation of PKR activation was shared with Nck-2. In this study, Nck-2
was shown to interact with PKR, suggesting that PKR control is a common
function of both Nck. However, further investigation will determine if Nck-2
can also limit PKR activation and be phosphorylated by activated PKR, as
shown for Nck-1.
Nck-1 regulation of PKR activation could be of significance in many
processes involving PKR. For example, an important role for PKR in the
control of metabolic homeostasis and in specific, insulin signaling has
71
been recently uncovered (279). In addition, PKR has been shown to
negatively regulate IRS1, consequently inhibiting insulin signaling (278).
Considering this, it would be of interest to assess whether Nck-1 could
limit PKR activation to improve insulin signaling. By increasing the
threshold of PKR activation, Nck-1 could prevent PKR-mediated inhibitory
phosphorylation of IRS1 on Ser 307 and thus improve insulin signaling.
Furthermore, Nck-1 control of PKR activation could also be significant in
the pathogen sensing role of PKR. Under physiological conditions, Nck-1
bound to PKR limits PKR activation, even in the presence of low levels of
viral dsRNA. However, when a significant amount of viral dsRNA
accumulates upon a viral infection, this overrides Nck-1 control of PKR
and initiates PKR activation, Nck-1 phosphorylation and dissociation to
achieve full PKR activation and prevent virus from replicating. Some
viruses develop specific mechanisms to counteract by dsRNA. There are
viral proteins that interfere with PKR activation at different levels, by
inhibiting PKR activation, sequestering dsRNA, inhibiting PKR
dimerization, synthesizing PKR pseudosubstrates, activating antagonist
phosphatases, or degrading PKR. Therefore, it would be interesting to
assess whether a virus could use Nck adaptor proteins as a mean to
inhibit PKR activation and allow efficient viral replication.
Taken together, data presented in this thesis report that Nck-1
interacts with inactive PKR and limits its activation in normal conditions.
However, in conditions where PKR activators accumulate over a threshold
level, Nck-1 control of PKR is antagonized and PKR is activated. During
the process of PKR activation, Nck-1 dissociates from PKR and this can
be promoted by PKR phosphorylation of Nck-1. The interaction of Nck-1
with PKR can set the threshold at which PKR is activated and its re-
association with PKR determines the temporal of PKR de-activation.
Hence, Nck-1 can be considered as a modulator of PKR activation by
buffering the effects of small concentrations of dsRNA.
72
Conclusion and Future Perspectives Nck-1 limits PKR activation by interacting with inactive PKR
independently of its functional SH domains. Under significant levels of
stress, PKR is activated and Nck-1 is phosphorylated and dissociated from
active PKR to allow full PKR activation. Nck-1 dissociation from active
PKR appears to result in the catalytic activation of PKR, which we
propose, phosphorylates Nck-1 and promotes Nck-1 dissociation from
PKR. Full length Nck-1 interacts with the N-terminal and the inactive C-
terminal regions of PKR. Furthermore, Nck-2 also interacts with PKR.
Additional investigations are required to clearly understand the mechanism
by which Nck-1 regulates PKR. Nck-1 Null where all SH domains are
simultaneously functionally inactivated should be characterized in its ability
to interact with PKR and control its activation. This will be helpful to
determine whether the SH domains of Nck-1 could act in a cooperative
mode to interact with PKR and regulate its activation. Nck-1
phosphorylation could also be analyzed in P32 metabolically labelled
PKR+/+ and -/- mouse embryonic fibroblasts (MEFs) challenged with or
without dsRNA. By following incorporation of radioactive P32 into
immunoprecipitated Nck-1, it would be possible to further confirm in vivo
Nck-1 phosphorylation by PKR. Once in vivo PKR-dependent
phosphorylation of Nck-1 is established, Mass Spectrometry analysis of
immunopurified Nck-1 prepared from unlabelled PKR activated cells
should be performed to identify the exact PKR phosphorylated residue(s)
on Nck-1. Moreover, due to the evidence showing that Nck-2 interacts with
PKR, it is worthwhile to study its effect on PKR activation and to determine
whether it binds only to inactive PKR and is also phosphorylated by PKR.
Finally, the regulation of PKR activation by Nck-1 represents a novel
function for this SH domain-containing adaptor protein. Further
investigation addressing the role of other members of the SH domain-
containing adaptor family like Grb2 and Crk in PKR regulation is significant
to strengthen the specificity of Nck-1 in regulating PKR activation. Finally,
73
additional experiments are required to determine the biological relevance
of a role for Nck-1 in regulating PKR function in antiviral response, cell
apoptosis and insulin signaling.
74
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