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Ribosome-Mediated Stress Response Ribosome-Mediated Stress Response ToDeoxynivalenol in the MacrophageHeekyong (Becky) Bae Food Science and Human NutritionCenter for Integrative Toxicology
Ribosome
PKR Hck
MAPK signaling pathway(p38, JNK, ERK)
How does DON activate MAPK signaling pathway?
DON
?
DON binding to the ribosome induces MAPK activation.
• MAPK activation occur subsequently after DON uptake
• When DON binding to the ribosome is inhibited by SG, MAPK activation by DON is inhibited.
Design and Methods:Uptake of [ 3H] DON
RAW 264.7 cells
Cell Lysate
[3H] DON
0, 250, 500, 1000 ng/ml0, 2.5, 5, 10, 15, 30 min
Scintillation counter
% Maximum DON uptake
Minutes
Kinetics of [ 3H] DON (250ng/ml) uptake into RAW 264.7 cells
DON is taken up rapidly within 15 minutes. MAPK activation is also maximized within 15 minutes.
120
100
80
60
40
20
0
50
0 5 10 15 20 25 30 352.5
SG(20ng/ml) was treated to Raw 264.7 cells for 1 hour and ribosomal fractions were separated using sucrose gradient system. Those fractions were reacted with additional SG and/or DON at 37oC for 1 hour. Each groups was centrifuged and washed using Centricon tubes.
1: ribosomal fractions from SG treated cells2: 1 + 20ng/ml SG3: 1 + 500ng/ml DON4: 1 + 20ng/ml SG + 500ng/ml DON
Satratoxin binding sites of the ribosome were saturated and the binding was recovered by endogenous DON.
aa
a
b
aa
a
b
RAW 264.7 cells
Phospho-p38 Phospho-JNK
p38 JNK
1 2 3 4 1 2 3 4
1: Vehicle2: SG 100ng/ml, 30min3: DON 100ng/ml, 30min4: SG+DON 100ng/ml, 30min
MAPK have direct interaction with the ribosome.
• Motif scan predicts MAPK interaction with the ribosomal proteins.
• MAPK interaction with the ribosome increases after DON treatment.
http://Scansite.mit.edu
Multiple ribosomal proteins might play scaffolding role for intracellular kinases
Example: Ribosomal Protein L3
Ribosome fraction(Sucrose gradient separation)
U937 cells DON
Western Blotting
2341
Monosome
Polysome
Ribosome fraction(Sucrose gradient separation)
U937 cells DON
Western Blotting
2341
Monosome
Polysome
1 2 3 4 5 6 7
40s
60s
80s
Fig. 4. P38 interacts with 40s ribosomal subunit and interaction is increased by DON-stimulated U937 cells. U937 cells (2 x 105/ml) were treated with DON (500 ng/ml) for 5 minutes compared with a vehicle.
p38
pp38
RPS6
RPL7
1 2 3 4 5 6 7 1 2 3 4 5 6 7
Vehicle DON
PKR and HCK mediate MAPK interaction with the ribosome.
• PKR and HCK are important for MAPK interaction with the ribosome .
• PKR can be upstream for HCK activation in the ribosome.
• Ribosomal protein S3 can mediate MAPK interaction with the ribosome.
p-p38
p38
2-AP - - + + - - + + - - + +
15 min 30 min
DON - - - - + + + + + + + +
p-p38
p38
PP2 - - + + - - + + - - + +
15 min 30 min
DON - - - - + + + + + + + +
(A)
(B)
pHCK
p38
- 5 15 30 min DON
Monosome(M)
pp38
Ribosomal protein S3 can mediate MAPK interaction with the ribosome.
Activated MAPK in the ribosome affect to transcription and translation.
• Activated MAPK via the ribosome can move to cytoplasm and affect to polysome formation.
• Activated MAPK can move to nucleus to change transcriptional level.
P38
pP38
RPL7
P38
pP38
RPL7
- 5 15 30 min
Polysome(P )
- 5 15 30 min
Polysome(P )
Activated MAPK via the ribosome can affect polysome formation.
Cytoplasmic p38 MAP kinase can be moved to the perinuclear region by ribotoxic stress response.
1 2 3 4 5
1 2 3 4 5
p-p38
p38
p-p38
p38
A.
B.
RAW 264.7 cells were treated with DON 250ng/ml with different time point (1: vehicle, 2: 5min, 3: 15min, 4: 30min, 5: 60min) and lysed in PEB lysis buffer. After centrifugation at 10,000g for 15min, supernatents were collected and analyzed by western blot , A. The pellets were resuspensed into hot SDS lysis buffer with sonication, then centrifuged and analyzed by western blot , B.
Calnexin
Calnexin
B.
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