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A historical overview of protein kinase PKR…
PKR associated activator (PACT) (23, 24). PKR is also an
intermediary in TLR signalling (25). PKR is engaged in
dsRNA-activated TLR3 signalling, recruited by a TAK1-
containing complex in response to dsRNA binding to the
TLR3 receptor. In addition, PKR integrates and transmits
these signals not only to eIF-2a and the translational
machinery, but also to various factors such as STAT IRF1,
p53, JNK, and p38, as well as engages the NF-kB pathway
(1, 2, 26). In non-stressed cells, PKR is in a monomeric
latent state due to the autoinhibitory effect of its dsRBD,
which occludes the KD and regulates the activation of the
kinase. The different dsRNA molecules are recognised and
bound by PKR through the two N-terminal dsRBM,
resulting in PKR activation and autophosphorylation (27).
The structure of the PKR dsRNA binding domain was
determined by nuclear magnetic resonance (28) offering a
satisfactory explanation for the length required of dsRNA
molecules to be effective PKR activators. Most natural
dsRNA activators of PKR are synthesised in virus-infected
cells as by-products of viral replication or transcription.
For RNA viruses, dsRNA replicative forms are obligatory
intermediates for the synthesis of new genomic RNA
copies. Complex DNA viruses such as vaccinia virus
(VV), adenovirus, or herpes simplex virus (HSV) have
open reading frames in opposite orientation; they produce
overlapping mRNA transcripts that can fold to form
dsRNA stretches responsible for PKR activation in
infected cells (1, 2). After binding dsRNA, PKR undergoes
a number of conformational changes that relieve the
autoinhibitory interactions of the enzyme and allow
subsequent substrate recognition. Biochemical and genetic
data have underscored the importance of
homodimerisation in PKR activation (29). After
homodimerisation, PKR undergoes rapid
autophosphorylation in a stretch of amino acids termed the
activation segment. Among others, residues Thr446 and
Thr451 in this segment are consistently phosphorylated
during activation (29, 30). This further stabilises PKR
dimerisation, which in turn increases the catalytic activity
of the kinase. Whether of viral origin or pIC, dsRNA thus
not only induces effects on translation, but also influences
various signal transduction pathways that affect different
transcriptional activities. As such, PKR mediates the
dsRNA-induced transcription of many genes through
engagement of multiple transcription pathways (1, 2, 26).
2.2. Translation regulation by PKR
A number of reports have provided insights into the
mechanism of PKR activation and eIF2a phosphorylation,
which consists of a three-step pathway in which
dimerisation of the kinase domain triggers
autophosphorylation, in turn promoting specific
recognition of eIF2a. PKR activation-segment
phosphorylation on Thr446 promotes substrate recognition
and phosphorylation, although it has been reported that
phosphorylation at tyrosine residues in PKR also
contributes to the binding to dsRNA, autophosphorylation,
and eIF2a phosphorylation (31). To this day, a total of 14
phosphorylation sites have been identified in PKR, but
@Real Academia Nacional de Farmacia. Spain
only 12 are biochemically verified, and 8 have been
functionally characterised (32). Although phosphorylation
of most sites serves to augment kinase activity toward
eIF2a, only phosphorylation of T451 is required to
generate an active kinase (32). As predicted for a
translation regulator, PKR is associated to ribosomes,
mainly to 40S subunits (33). Ribosomal association of
PKR appears to be mediated by the dsRBD, strengthening
the role of these domains in the correct regulation of PKR
activity. PKR localisation in ribosomes offers a
satisfactory explanation for its local activation in response
to limited stimulus, as reported by several studies (1, 2,
33). Two models have recently suggested new evidence
for a sentinel model of ribotoxin-induced PKR activation
(34). One possibility is a sentinel model in which PKR
monomers basally associate with the ribosome and rRNA.
Upon interaction with a ribotoxin, one or more portions of
rRNA reposition and thereby promote dimerisation of the
PKR monomers followed by autophosphorylation and self-
activation. A second possibility is a sequential mode
whereby a ribotoxin first associates with rRNA and inflicts
damage and/or alters its structure, thereby exposing new
double-stranded (ds)rRNA regions. This could sequentially
elicit the binding of two or more PKR monomers in close
proximity to the damaged site, followed by the
dimerisation of these monomers and finally the
autophosphorylation and self-activation of the kinase (34).
2.3. NF-?B activation by PKR: identification of TRAF
family proteins linking PKR with NF-?B activation
The NF-?B family of transcription factors controls the
expression of genes involved in immune and inflammatory
responses, cell differentiation, and apoptosis, among others
(35). NF-?B activation is primarily regulated through its
interaction with the family of inhibitory proteins I?B
which retain NF-?B in the cytoplasm. Phosphorylation of
I?B on two conserved serine residues is mediated by the
I?B kinase complex (IKK complex) in response to a
variety of stimuli, leading to its subsequent ubiquitin-
dependent degradation by the 26S proteasome. This allows
NF-?B translocation to the nucleus, where it can activate
the transcription of a number of genes including those
encoding cytokines, chemokines, cell surface receptors,
and adhesion molecules (36). The IKK complex contains a
structural protein termed IKK? or NEMO and two kinase
subunits, IKKa and IKKß (37). The first clues suggesting a
role for PKR in NF-?B activation arose from observations
in 1989 that dsRNA could induce NF-?B activity in
different cell lines (38). Subsequent experiments using the
kinase inhibitor 2-aminopurine suggested a role for PKR in
this process. Additional evidence came from the analysis
of NF-?B activation following dsRNA treatment in cells
lacking PKR expression. When PKR expression was
downregulated using 2-5A antisense oligonucleotides,
diminished NF-?B activation was observed in response to
dsRNA, with no significant change in the response to
TNF-a (39). The design of mice deficient in PKR
expression in 1995 allowed carry out many critical
experiments for studying the mechanism of action of the
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