Identifying the substrate proteins of U-box E3s E4B and CHIP by orthogonal ubiquitin transfer

Karan Bhuripanyo, Yiyang Wang, Xianpeng Liu, Li Zhou, Ruochuan Liu, Duc Duong, Bo Zhao, Yingtao Bi, Han Zhou, Geng Chen, Nicholas T. Seyfried, Walter J. Chazin, Hiroaki Kiyokawa*, Jun Yin

*Corresponding author for this work

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

E3 ubiquitin (UB) ligases E4B and carboxyl terminus of Hsc70-interacting protein (CHIP) use a common U-box motif to transfer UB from E1 and E2 enzymes to their substrate proteins and regulate diverse cellular processes. To profile their ubiquitination targets in the cell, we used phage display to engineer E2-E4B and E2-CHIP pairs that were free of cross-reactivity with the native UB transfer cascades. We then used the engineered E2-E3 pairs to construct “orthogonal UB transfer (OUT)” cascades so that a mutant UB (xUB) could be exclusively used by the engineered E4B or CHIP to label their substrate proteins. Purification of xUB-conjugated proteins followed by proteomics analysis enabled the identification of hundreds of potential substrates of E4B and CHIP in human embryonic kidney 293 cells. Kinase MAPK3 (mitogen-activated protein kinase 3), methyltransferase PRMT1 (protein arginine N-methyltransferase 1), and phosphatase PPP3CA (protein phosphatase 3 catalytic subunit alpha) were identified as the shared substrates of the two E3s. Phosphatase PGAM5 (phosphoglycerate mutase 5) and deubiquitinase OTUB1 (ovarian tumor domain containing ubiquitin aldehyde binding protein 1) were confirmed as E4B substrates, and b-catenin and CDK4 (cyclin-dependent kinase 4) were confirmed as CHIP substrates. On the basis of the CHIP-CDK4 circuit identified by OUT, we revealed that CHIP signals CDK4 degradation in response to endoplasmic reticulum stress.

Original languageEnglish (US)
Article numbere1701393
JournalScience Advances
Volume4
Issue number1
DOIs
StatePublished - Jan 1 2018

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HSC70 Heat-Shock Proteins
Ubiquitin
Cyclin-Dependent Kinase 4
Proteins
Phosphoric Monoester Hydrolases
Protein-Arginine N-Methyltransferases
Phosphoglycerate Mutase
MAP Kinase Kinase 3
Catenins
Endoplasmic Reticulum Stress
Ubiquitin-Protein Ligases
Calcineurin
Ubiquitination
Methyltransferases
Proteomics
Bacteriophages
Carrier Proteins
Kidney

ASJC Scopus subject areas

  • General

Cite this

Bhuripanyo, Karan ; Wang, Yiyang ; Liu, Xianpeng ; Zhou, Li ; Liu, Ruochuan ; Duong, Duc ; Zhao, Bo ; Bi, Yingtao ; Zhou, Han ; Chen, Geng ; Seyfried, Nicholas T. ; Chazin, Walter J. ; Kiyokawa, Hiroaki ; Yin, Jun. / Identifying the substrate proteins of U-box E3s E4B and CHIP by orthogonal ubiquitin transfer. In: Science Advances. 2018 ; Vol. 4, No. 1.
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title = "Identifying the substrate proteins of U-box E3s E4B and CHIP by orthogonal ubiquitin transfer",
abstract = "E3 ubiquitin (UB) ligases E4B and carboxyl terminus of Hsc70-interacting protein (CHIP) use a common U-box motif to transfer UB from E1 and E2 enzymes to their substrate proteins and regulate diverse cellular processes. To profile their ubiquitination targets in the cell, we used phage display to engineer E2-E4B and E2-CHIP pairs that were free of cross-reactivity with the native UB transfer cascades. We then used the engineered E2-E3 pairs to construct “orthogonal UB transfer (OUT)” cascades so that a mutant UB (xUB) could be exclusively used by the engineered E4B or CHIP to label their substrate proteins. Purification of xUB-conjugated proteins followed by proteomics analysis enabled the identification of hundreds of potential substrates of E4B and CHIP in human embryonic kidney 293 cells. Kinase MAPK3 (mitogen-activated protein kinase 3), methyltransferase PRMT1 (protein arginine N-methyltransferase 1), and phosphatase PPP3CA (protein phosphatase 3 catalytic subunit alpha) were identified as the shared substrates of the two E3s. Phosphatase PGAM5 (phosphoglycerate mutase 5) and deubiquitinase OTUB1 (ovarian tumor domain containing ubiquitin aldehyde binding protein 1) were confirmed as E4B substrates, and b-catenin and CDK4 (cyclin-dependent kinase 4) were confirmed as CHIP substrates. On the basis of the CHIP-CDK4 circuit identified by OUT, we revealed that CHIP signals CDK4 degradation in response to endoplasmic reticulum stress.",
author = "Karan Bhuripanyo and Yiyang Wang and Xianpeng Liu and Li Zhou and Ruochuan Liu and Duc Duong and Bo Zhao and Yingtao Bi and Han Zhou and Geng Chen and Seyfried, {Nicholas T.} and Chazin, {Walter J.} and Hiroaki Kiyokawa and Jun Yin",
year = "2018",
month = "1",
day = "1",
doi = "10.1126/sciadv.1701393",
language = "English (US)",
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Bhuripanyo, K, Wang, Y, Liu, X, Zhou, L, Liu, R, Duong, D, Zhao, B, Bi, Y, Zhou, H, Chen, G, Seyfried, NT, Chazin, WJ, Kiyokawa, H & Yin, J 2018, 'Identifying the substrate proteins of U-box E3s E4B and CHIP by orthogonal ubiquitin transfer', Science Advances, vol. 4, no. 1, e1701393. https://doi.org/10.1126/sciadv.1701393

Identifying the substrate proteins of U-box E3s E4B and CHIP by orthogonal ubiquitin transfer. / Bhuripanyo, Karan; Wang, Yiyang; Liu, Xianpeng; Zhou, Li; Liu, Ruochuan; Duong, Duc; Zhao, Bo; Bi, Yingtao; Zhou, Han; Chen, Geng; Seyfried, Nicholas T.; Chazin, Walter J.; Kiyokawa, Hiroaki; Yin, Jun.

In: Science Advances, Vol. 4, No. 1, e1701393, 01.01.2018.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Identifying the substrate proteins of U-box E3s E4B and CHIP by orthogonal ubiquitin transfer

AU - Bhuripanyo, Karan

AU - Wang, Yiyang

AU - Liu, Xianpeng

AU - Zhou, Li

AU - Liu, Ruochuan

AU - Duong, Duc

AU - Zhao, Bo

AU - Bi, Yingtao

AU - Zhou, Han

AU - Chen, Geng

AU - Seyfried, Nicholas T.

AU - Chazin, Walter J.

AU - Kiyokawa, Hiroaki

AU - Yin, Jun

PY - 2018/1/1

Y1 - 2018/1/1

N2 - E3 ubiquitin (UB) ligases E4B and carboxyl terminus of Hsc70-interacting protein (CHIP) use a common U-box motif to transfer UB from E1 and E2 enzymes to their substrate proteins and regulate diverse cellular processes. To profile their ubiquitination targets in the cell, we used phage display to engineer E2-E4B and E2-CHIP pairs that were free of cross-reactivity with the native UB transfer cascades. We then used the engineered E2-E3 pairs to construct “orthogonal UB transfer (OUT)” cascades so that a mutant UB (xUB) could be exclusively used by the engineered E4B or CHIP to label their substrate proteins. Purification of xUB-conjugated proteins followed by proteomics analysis enabled the identification of hundreds of potential substrates of E4B and CHIP in human embryonic kidney 293 cells. Kinase MAPK3 (mitogen-activated protein kinase 3), methyltransferase PRMT1 (protein arginine N-methyltransferase 1), and phosphatase PPP3CA (protein phosphatase 3 catalytic subunit alpha) were identified as the shared substrates of the two E3s. Phosphatase PGAM5 (phosphoglycerate mutase 5) and deubiquitinase OTUB1 (ovarian tumor domain containing ubiquitin aldehyde binding protein 1) were confirmed as E4B substrates, and b-catenin and CDK4 (cyclin-dependent kinase 4) were confirmed as CHIP substrates. On the basis of the CHIP-CDK4 circuit identified by OUT, we revealed that CHIP signals CDK4 degradation in response to endoplasmic reticulum stress.

AB - E3 ubiquitin (UB) ligases E4B and carboxyl terminus of Hsc70-interacting protein (CHIP) use a common U-box motif to transfer UB from E1 and E2 enzymes to their substrate proteins and regulate diverse cellular processes. To profile their ubiquitination targets in the cell, we used phage display to engineer E2-E4B and E2-CHIP pairs that were free of cross-reactivity with the native UB transfer cascades. We then used the engineered E2-E3 pairs to construct “orthogonal UB transfer (OUT)” cascades so that a mutant UB (xUB) could be exclusively used by the engineered E4B or CHIP to label their substrate proteins. Purification of xUB-conjugated proteins followed by proteomics analysis enabled the identification of hundreds of potential substrates of E4B and CHIP in human embryonic kidney 293 cells. Kinase MAPK3 (mitogen-activated protein kinase 3), methyltransferase PRMT1 (protein arginine N-methyltransferase 1), and phosphatase PPP3CA (protein phosphatase 3 catalytic subunit alpha) were identified as the shared substrates of the two E3s. Phosphatase PGAM5 (phosphoglycerate mutase 5) and deubiquitinase OTUB1 (ovarian tumor domain containing ubiquitin aldehyde binding protein 1) were confirmed as E4B substrates, and b-catenin and CDK4 (cyclin-dependent kinase 4) were confirmed as CHIP substrates. On the basis of the CHIP-CDK4 circuit identified by OUT, we revealed that CHIP signals CDK4 degradation in response to endoplasmic reticulum stress.

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U2 - 10.1126/sciadv.1701393

DO - 10.1126/sciadv.1701393

M3 - Article

VL - 4

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