Global Control of Gene Expression via Translation

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Global Control of Gene Expression via Translation
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Translation Initiation Factors
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eIF-2 - composed of three subunits 36kDa (a),
38kDa (b), 52kDa (?)
GDP-bound eIF2 cannot bind Met-tRNAiMet
eIF2 binds the initiator Met-tRNAiMet to the P
site of the 40S ribosome subunit. GTP binding by
eIF2 is necessary for the formation of a stable
ternary complex. eIF2B is required to recycle
eIF2?GDP to eIF2?GTP eIF2? is a very important
target for the regulation of translation phosphor
ylation of eIF2? (on Ser51) converts eIF2 from
a substrate for eIF2B, to a competitive
inhibitor - a global modulator of gene
expression.
guanine nucleotide exchange factor (GEF)

initiator methionyl tRNA
ternary complex
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eIF2 phosphorylated by eIF2 kinases (GCN2,
PKR..) PKR is a double-stranded (ds) RNA
activated protein kinase whose expression is
induced by interferon. Component of the innate
immune response Activated PKR phosphorylates
the cellular substrate eIF2? - an essential
initiation factor of translation. PKR
autophosphorylation, and the phosphorylation of
its substrate eIF2?, is 7-40 fold higher in
lysates prepared from human breast carcinoma cell
lines than in those from non-transformed
epithelial cell lines. Correspondingly, a larger
proportion of eIF2? is present in a
phosphorylated state in carcinoma cell lines than
in non-transformed cell lines. Protein
synthesis is not inhibited by the high eIF2?
phosphorylation in carcinoma cells, probably
because they contain higher levels of eIF2B, the
initiation factor that is inhibited by eIF2?
phosphorylation PKR is proteolytically cleaved
in the early stages of apoptosis. The kinase
domain is cleaved from the N-terminal regulatory
domain. The released kinase domain efficiently
phosphorylates eIF2?.
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eIF2? PERK resident transmembrane eIF2?
kinase PERK is activated by an imbalance between
the load of client proteins translocated into the
ER lumen and the capacity of the ER to process
(transport) this load

- ER stress PERK
phosphorylates eIF2? thereby rapidly
down-regulating the translational activity of the
cell - reduces
load on the ER very important mechanism for
the cell to resist ER stress UPR ER unfolded
protein response also activates PERK
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eIF2? GADD34 (Growth Arrest and DNA
Damage-Inducible Protein)
regulatory subunit of protein
phosphatase 1 (PP1c) complex GADD34 is a
stress-induced protein implicated in the control
of protein synthesis and apoptosis. eIF2?
dephosphorylation is controlled by GADD34
stress recovery GADD34 protein levels are
elevated in human cancer cells in response to a
variety of stresses. GADD34 is rapidly degraded,
consistent with a temporal regulation of
stress-signalling. Herpes simplex virus encodes
a protein with GADD34-like activity
- dephosphorylates eIF2?,
- eIF2? phosphorylated by PKR in virus-infected
cells
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Model for heme-controlled protein synthesis in
reticulocytes.
In reticulocytes, the kinase that phosphorylates
eIF2? is haemin-controlled inhibitor (HCI). The
kinase activity of HCI is inhibited by binding of
haemin. Haeme is utilized in the formation of
haemoglobin by binding stoichiometrically to
globin polypeptides. Haemin is an oxidation
product of haeme. It accumulates in reticulocytes
when haeme production exceeds haeme utilization.
Hemin
Hemin

eIF2 remains active
HCI heme-controlled inhibitor - eIF2? kinase
HCI inactive as protein kinase
ATP ADP
P
eIF2 inactive
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eIF2B - composed of five subunits
guanine nucleotide exchange factor (GEF)
activity of eIF2B is also controlled
by phosphorylation / dephosphorylation eIF2B?
subunit phosphorylated at Ser540 by glycogen
synthase kinase-3 (GSK-3). phosphorylation
inhibits GDP/GTP exchange activity insulin
rapidly decreases eIF2B phosphorylation by
inactivating GSK-3 protein phosphatase PP1C (?
isoform) dephosphorylates and activates eIF2B

translation initiation
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Insulin / receptor
Glucose
Amino acids
Amino acid starvation
PI3-kinase
???
uncharged tRNA
???
mGCN2
PKB
eIF2? phosphorylation
GSK3 (off)
(inactivates)
inactivates (binds as competitive inhibitor)
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Interferon regulation of protein synthesis
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eIF4G A scaffolding protein
Binds poly(A) Tails
40S
Binds 7meG caps
eIF3
Binds eIF3
eIF4E
PABP
eIF4A
eIF4A
eIF4G
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Binds poly(A) Tails
40S
Binds 7meG caps
eIF3
Binds eIF3
eIF4E
PABP
eIF4A
eIF4A
eIF4G
eIF4G cleaved apart by caspase 3
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Picornaviruses e.g. Polio, FMDV shut-off host
cell capped mRNA translation
but virus RNA is translated
virus genome like a cellular mRNA, except cap is
different
An
single, long, ORF encodes a polyprotein
the polyprotein is processed by virus-encoded
proteinases
virus needs multifactor complex for its
translation..
multifactor complex (MFC)
cannot inhibit translation via eIF2? or eIF2B ..
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Binds poly(A) Tails
Binds 7meG mRNA caps
40S
eIF3
Binds eIF3
eIF4E
PABP
eIF4A
eIF4A
eIF4G
eIF4G cleaved apart by picornavirus proteinases
(Lpro, 2Apro)
uncouples the ability of the complex to bind to
both mRNA caps AND ribosomes
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eIF4E - a single 25kDa subunit
part of the cap-binding protein complex,
recognises mRNA caps eIF4E is phosphorylated on
Ser209 the activity of eIF4E is regulated by its
interaction with binding proteins. eIF4E-BPs th
e activity of eIF4E-BPs is regulated by
phosphorylation
PABP
PABP
eIF4G
eIF4E
eIF4A
m7GpppGAUUCGAUA..
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eIF4E-BPs a.k.a. PhosphorylatedHeat-and-Acid-Stab
le protein (PHAS)
4E-BPs bind eIF4E in a ratio of 11 there is an
excess of 4E-BPs in the cell binding of 4E-BP1
to eIF4E inhibits cap-dependent mRNA
translation 4E-BP binding to eIF-4E does not
inhibit eIF-4E binding to mRNA cap structures,
but inhibits the binding of eIF-4E to the
scaffolding protein eIF-4G and hence the
formation of eIF-4F the binding of eIF-4G and
4E-BP1 to eIF-4E is mutually exclusive.
4E- BP1
MAP kinase casein kinase 2
4E- BP1

P
P
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Insulin, Amino Acids
mTOR / FRAP pathway
P
P
P
P
P
P
PABP
PABP
eIF4G
eIF4A
eIF-4E
eIF3
4E-T
cap-binding protein complex
4E-T
4E-T shuttling protein which transports eIF-4E
to the nucleus
Nucleus
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eIF3 component of the of the 43S complex
binds scaffolding protein eIF4G (CBP)
Binds poly(A) Tails
40S
Binds 7meG mRNA caps
eIF3
eIF4E
PABP
eIF4A
eIF4A
Binds eIF3
eIF4G
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Model of inhibition of protein synthesis by mouse
P56 and human and mouse P54. Different P56 family
members block translation at different steps of
the initiation pathway. Human P54 and mouse P54
and P56 bind to the eIF3c subunit. Binding to
eIF3c interferes with the formation of the 48S
pre-initiation complex, consisting of the ternary
complex, eIF3, the 40S ribosomal subunit, eIF4F,
and mRNA.
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Translation Elongation Factors
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Elongation Cycle of Eukaryotic Protein Synthesis
aa
aa
aa
aa
aa
aa
aa
aa
aa
aa
EF1A ? GTP
An
5'
An
5'
A
P
A
P
P

EF2 ? GDP
EF1A ? GTP
aminoacyl-tRNA binding
Translocation
aa
EF1B???
aa
aa
EF2 ? GTP
aa
aa
peptidyl transfer
aa
aa
aa
aa
aa
EF1A ? GDP
P
5'
An
An
5'
A
P
A
P
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eEF1A and eEF1B?,?,? are all phospho-proteins eEF
1A exists in two isoforms, eEF1A1 and eEF1A2 (92
identical, 98 similar) eEF1A1 is widely
expressed, eEF1A2 is normally expressed only in
neurons and muscle
eEF1A
MS6K
Protein kinase C (PKC)
Casein kinase 2 (CK2)
(eIF4B, eIF4G, ribosomal protein S6)
phosphorlyation stimulates activity no apparent
effect on activity
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eEF1A and eEF1B??? eEF1A2 is expressed in 30 of
ovarian tumours, but not in normal
ovaries eEF1A1 (the widely-expressed isoform)
was recently shown by microarray analysis to be
up-regulated in the infiltrating edge of invasive
breast tumours compared with the tumour
core eEF1A2 expression is barely detectable in
normal human breast tissue, but that the gene is
moderately to strongly expressed in 63 of
breast tumours examined. strong correlation
between eEF1A2 over-expression and estrogen
receptor (ER) positivity
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eEF2
Active form
Guanine nucleotide exchange factor not required
to regenerate GTP-bound form eEF2 has a low
affinity for GDP which spontaneously dissociates
Translocation of peptidyl- tRNA from A to P site
eEF2 ? GDP
Inactive form
eEF2 is inhibited by phosphorylation at Thr 56
Inactive form
P
Protein phosphatase 2A (PP2A)
eEF2 kinase
(Ca/calmodulin-dependent kinase III)
Active form
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phosphorylation
mild energy depletion, anoxia
translation (elongation)
(Ca/calmodulin regulated)
Protein kinase A (PKA)
Ser499
P
increased eEF2 phosphorylation translation inhibi
ted
autophosphorylation
P
P
P
P
(Ca/calmodulin independent)
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increased eEF2 phosphorylation translation inhibi
ted
Protein kinase A (PKA)
Ser499
P
eEF2 kinase activity inhibited
stress-activated protein kinase 4 (SAPK4)
Ser396
P
Ser359
P
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ribosomal protein S6 kinase-1 (S6K1)
eEF2 kinase activity inhibited
or,
Ser366
P
ribosomal protein S6 kinase (RSK)
AMP-activated protein kinase (AMPK)
eEF2 kinase activity stimulated
Ser398
P
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Ribosomal protein S6 (rpS6) In 1970, the
ribosomal protein S6 was found to be a
phosphoprotein, and later it was shown to be
phosphorylated when cells are stimulated with
growth factors and hormones. S6 is part of the
small ribosomal subunit (one copy per ribosome).
It binds to the 18S rRNA very early in the
assembly pathway leading to the 40S ribosome.
In the 80S ribosome it is localized at the
interface between the subunits in a region where
tRNA and protein factors catalyze mRNA
translation. thought to play a role in the
regulation of translation of a subset of mRNAs
containing a 5 terminal tract of
oligopyrimidines (5-TOP mRNAs) 5TOP mRNAs
mammalian ribosomal proteins, eEF1A, eEF2 and
PAPBP rpS6 phosphorylated by S6Ks S6Ks are
activated by phosphorylation mTOR
signalling Translational apparatus
preferentially synthesises its own components in
response to anabolic / proliferative stimuli
?? Proud, C. (2002). Regulation of mammalian
translation factors by nutrients. Eur. J.
Biochem. 269, 5338-5349.
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Cells are transformed by over-expression of a
dominant negative mutant of PKR over-expression
of the non-phosphorylatable (mutant) form eIF2A
(Ser51Ala). over-expression of eIF4E relative to
the 4E-BPs. Explanation (?) Failure to
down-regulate protein synthesis results in the
relatively more efficient translation of weak
mRNAs such as those for growth factors and
oncogenes.
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Translational control is exerted largely, but
certainly not exclusively, at the initiation
stage of translation. signalling pathways affect
the phosphorylation status of a number of
translation factors These controls are rapid and
reversible. They also operate in cells where
transcription is not occurring..

oocytes, reticulocytes They
form part of the defence against viruses They
may play a role in the spatial control of
translational activity.

pattern formation, neurones