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

phopho-p38

phopho-JNK

phopho-ERK

1 2 3 4

1: Vehicle2: DON 5 min3: DON 15 min4: DON 30 min

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.

U9K-C2

HCK

RPS6 (40S)

PKR

RPL7 (60S)

40s

60s

80s

polysome

U9K-A1

HCK

PKR

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.

Conformational change

Activated kinases move to cytoplasm→ Signal pathway

Polysomeformation

Selective mRNA TranslationGene TranscriptionmRNA Stabilization

Ribosome

Toxin

Ribosome

Activation of protein kinases

PKR, HCK, ERK, JNK, p38