CST Guide:

Section I: Research Areas
eIF4B is expressed in
HeLa cells and human
lung carcinoma.
Sin1, a component of
the mTORC2 complex,
is expressed in many
cell lines.
Translational Control
The synthesis of new proteins is a highly regulated process that allows rapid cellular responses to a
diverse set of stimuli. Two key events in the control of translational initiation are 1) the association
between 5’ capped mRNA and the preinitiation complex, and 2) the binding of initiator tRNA to the start
codon. Both events are mediated by multiple eukaryotic initiator factors (eIFs) that are regulated by
effector kinases and inhibitors.
Cap-dependent Initiation
Translation initiation requires a set of factors to facilitate the association of the 40S ribosomal subunit
with mRNA. The eIF4F complex, consisting of eIF4E, eIF4A, and eIF4G, binds to the 5ʹ cap structure of
mRNA. eIF4A is a helicase, and together with accessory protein eIF4B, serves to unwind the secondary
structure of mRNA at its 5’ untranslated region and promote formation of the preinitiation complex.
A
B
eIF4B (1F5) Mouse mAb #13088: Confocal
IF analysis of HeLa cells (A) using #13088
(green). Actin filaments were labeled with
DyLight 554 Phalloidin #13054 (red). Blue
pseudocolor = DRAQ5 ® #4084 (fluorescent
DNA dye). IHC analysis of paraffin-embedded
human lung carcinoma (B) using #13088.
Assembly of eIF4F is controlled by growth and survival factors that regulate activity of upstream kinase
effectors, including Akt, mTOR, p70 S6 kinase, and p90RSK. mTOR kinase complexes mTORC1 and
mTORC2 promote eIF4F cap-binding complex formation by activating upstream elements that favor
complex assembly and inhibiting proteins that block eIF4F formation. The mTORC1 complex includes
mTOR kinase bound by the adaptor raptor and several regulatory proteins (GβL, PRAS40, and DEPTOR),
while the mTORC2 complex contains mTOR kinase, rictor, GβL, DEPTOR, and Sin1. mTORC1 activates
p70 S6 kinase to relieve PDCD4 inhibition of eIF4A and activate eIF4B. Initiation factor eIF4B interacts
with both the eIF3 scaffold protein complex and eIF4A, stimulating eIF4A RNA helicase activity. Upstream
kinase pathways mediate the phosphorylation of eIF4B by p70 S6 kinase and p90RSK to increase the association
between eIF4B, eIF3, and eIF4A. Inhibition of translation repressor protein 4E-BP1 by mTORC1
phosphorylation causes release of cap-binding protein eIF4E and its incorporation into eIF4F.
Sin1 (D7G1A) Rabbit mAb #12860: WB analysis
of extracts from various cell lines using #12860.
Lanes
1. HeLa
2. MCF7
3. Hep G2
4. INS-1
5. KNRK
6. NBT-11
7. PANC-1
8. Vero
9. COS-7
10. U-87 MG
11. 293
kDa
200
140
100
80
60
50
40
30
1 2 3 4 5 6 7 8 9 10 11
Sin1.1
Sin1.2
chapter 01: GENE EXPRESSION, EPIGENETICS, AND NUCLEAR FUNCTION
In hypoxic environments, mTORC1 activity is inhibited, leading to down regulation of eIF4E capdependent
translation. Instead, protein synthesis is driven during low oxygen conditions by eIF4E2
(also known as 4EHP), which binds the 5ʹ cap and forms a complex with the hypoxia-inducible factor
HIF-2α and the RNA-binding protein RBM4. This complex stimulates translation of select RNAs,
including those implicated in cancer growth.
The 40S ribosomal subunit then binds to the 5ʹ mRNA cap and associated initiation factors and
searches along the mRNA for the initiation codon. eIF3 physically interacts with eIF4G, which may be
responsible for the association of the 40S ribosomal subunit with mRNA.
eIF3H is a core component of the eIF3 complex
that facilitates binding of mRNA to the 40S ribosomal
subunit.
eIF3H (D9C1) XP ® Rabbit mAb #3413: Confocal IF analysis of SK-N-MC cells using
#3413 (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor
= DRAQ5 ® #4084 (fluorescent DNA dye).
Initiator tRNA and the Start Codon
eIF2 mediates binding of initiator tRNA to the ribosome at the start codon to form the 43S preinitiation
complex. Trimeric eIF2 is made up of regulatory (α), tRNA/mRNA interacting (β), and GTP/GDP binding
(γ) proteins. Phosphorylation of eIF2α by multiple upstream kinases (including PKR, PERK, and GCN2)
is stimulated by environmental stress and the presence of dsDNA, and leads to inactive eIF2 and
translation inhibition. Additional control of eIF2 activity occurs through regulation of guanine nucleotide
exchange, which is catalyzed by eIF2B. Exchange of GDP for GTP promotes the essential association
between the eIF2 complex and tRNA. eIF2B activity is inhibited by GSK-3β phosphorylation and through
interaction with eIF5, which also acts as a GDP dissociation inhibitor by stabilizing eIF2 bound by GDP.
Treatment of cells with ER stress-inducing agent
thapsigargin results in phosphorylation of eIF2α at Ser51.
Phospho-eIF2α (Ser51) (D9G8) XP ® Rabbit mAb #3398: WB analysis of extracts from C2C12 cells,
untreated or treated with Thapsigargin #12758, using #3398 (upper) or eIF2α Antibody #9722 (lower).
eIF2, which transfers Met-tRNA to the 40S subunit
to form the 43S preinitiation complex, is expressed
in multiple cell lines.
eIF2α (D7D3) XP ® Rabbit mAb #5324: WB analysis
of extracts from various cell lines using #5324.
Lanes
1. MCF7
2. Hep G2
3. NIH/3T3
4. COS-7
kDa
140
100
80
60
50
40
30
1 2 3 4
elF2α
kDa
100
80
60
50
40
30
20
100
80
60
50
40
30
20
– +
PhosphoeIF2α
(Ser51)
eIF2α
Thapsigargin
S6 ribosomal protein
is phosphorylated
by p70 S6 kinase
at Ser235/236 in
response to growth
factors and mitogens.
Phospho-S6 Ribosomal Protein (Ser235/
Ser236) (D57.2.2E) XP ® Rabbit mAb
#4858: Confocal IF analysis of HeLa cells,
rapamycin-treated (left) or 20% serum-treated
(right), using #4858 (green). Actin filaments
were labeled with Alexa Fluor ® 555 Phalloidin
#8953 (red). Blue pseudocolor = DRAQ5 ®
#4084 (fluorescent DNA dye).
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32 For Research Use Only. Not For Use in Diagnostic Procedures. See pages 302 & 303 for Pathway Diagrams, Application, and Reactivity keys.
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