Analysis of nondegradative protein ubiquitylation with a monoclonal antibody specific for lysine-63-linked polyubiquitin

Haopeng Wang; Atsushi Matsuzawa; Scott A. Brown; Jingran Zhou; Cliff S. Guy; Ping Hui Tseng; Karen Forbes; Thomas P. Nicholson; Paul W. Sheppard; Hans Häcker; Michael Karin; Dario A.A. Vignali

doi:10.1073/pnas.0810461105

Analysis of nondegradative protein ubiquitylation with a monoclonal antibody specific for lysine-63-linked polyubiquitin

Haopeng Wang, Atsushi Matsuzawa, Scott A. Brown, Jingran Zhou, Cliff S. Guy, Ping Hui Tseng, Karen Forbes, Thomas P. Nicholson, Paul W. Sheppard, Hans Häcker, Michael Karin^*, Dario A.A. Vignali

^*Corresponding author for this work

Institute of Biochemistry and Molecular Biology

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55 Scopus citations

Abstract

Modification of proteins by the addition of lysine (K)-63-linked polyubiquitin (polyUb) chains is suggested to play important roles in a variety of cellular events, including DNA repair, signal transduction, and receptor endocytosis. However, identifying such modifications in living cells is complex and cumbersome. We have generated a monoclonal antibody (mAb) that specifically recognizes K63-linked polyUb, but not any other isopeptide-linked (K6, K11, K27, K29, K33, or K48) polyUb or monoubiquitin. We demonstrate the sensitivity and specificity of this K63Ub-specific mAb to detect K63Ub-modified proteins in cell lysates by Western blotting and in cells by immunofluorescence, and K63Ub-modified TRAF6 and MEKK1 in vitro and ex vivo. This unique mAb will facilitate the analysis of K63-linked polyubiquitylation ex vivo and presents a strategy for the generation of similar reagents against other forms of polyUb.

Original language	English
Pages (from-to)	20197-20202
Number of pages	6
Journal	Proceedings of the National Academy of Sciences of the United States of America
Volume	105
Issue number	51
DOIs	https://doi.org/10.1073/pnas.0810461105
State	Published - 23 Dec 2008

Keywords

MAP kinase kinase kinase 1 (MEKK1)
TNF receptor-associated factor 6 (TRAF6)

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10.1073/pnas.0810461105

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Wang, H., Matsuzawa, A., Brown, S. A., Zhou, J., Guy, C. S., Tseng, P. H., Forbes, K., Nicholson, T. P., Sheppard, P. W., Häcker, H., Karin, M., & Vignali, D. A. A. (2008). Analysis of nondegradative protein ubiquitylation with a monoclonal antibody specific for lysine-63-linked polyubiquitin. Proceedings of the National Academy of Sciences of the United States of America, 105(51), 20197-20202. https://doi.org/10.1073/pnas.0810461105

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title = "Analysis of nondegradative protein ubiquitylation with a monoclonal antibody specific for lysine-63-linked polyubiquitin",

abstract = "Modification of proteins by the addition of lysine (K)-63-linked polyubiquitin (polyUb) chains is suggested to play important roles in a variety of cellular events, including DNA repair, signal transduction, and receptor endocytosis. However, identifying such modifications in living cells is complex and cumbersome. We have generated a monoclonal antibody (mAb) that specifically recognizes K63-linked polyUb, but not any other isopeptide-linked (K6, K11, K27, K29, K33, or K48) polyUb or monoubiquitin. We demonstrate the sensitivity and specificity of this K63Ub-specific mAb to detect K63Ub-modified proteins in cell lysates by Western blotting and in cells by immunofluorescence, and K63Ub-modified TRAF6 and MEKK1 in vitro and ex vivo. This unique mAb will facilitate the analysis of K63-linked polyubiquitylation ex vivo and presents a strategy for the generation of similar reagents against other forms of polyUb.",

keywords = "MAP kinase kinase kinase 1 (MEKK1), TNF receptor-associated factor 6 (TRAF6)",

author = "Haopeng Wang and Atsushi Matsuzawa and Brown, {Scott A.} and Jingran Zhou and Guy, {Cliff S.} and Tseng, {Ping Hui} and Karen Forbes and Nicholson, {Thomas P.} and Sheppard, {Paul W.} and Hans H{\"a}cker and Michael Karin and Vignali, {Dario A.A.}",

year = "2008",

month = dec,

day = "23",

doi = "10.1073/pnas.0810461105",

language = "English",

volume = "105",

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Wang, H, Matsuzawa, A, Brown, SA, Zhou, J, Guy, CS, Tseng, PH, Forbes, K, Nicholson, TP, Sheppard, PW, Häcker, H, Karin, M & Vignali, DAA 2008, 'Analysis of nondegradative protein ubiquitylation with a monoclonal antibody specific for lysine-63-linked polyubiquitin', Proceedings of the National Academy of Sciences of the United States of America, vol. 105, no. 51, pp. 20197-20202. https://doi.org/10.1073/pnas.0810461105

Analysis of nondegradative protein ubiquitylation with a monoclonal antibody specific for lysine-63-linked polyubiquitin. / Wang, Haopeng; Matsuzawa, Atsushi; Brown, Scott A. et al.
In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 105, No. 51, 23.12.2008, p. 20197-20202.

Research output: Contribution to journal › Article › peer-review

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T1 - Analysis of nondegradative protein ubiquitylation with a monoclonal antibody specific for lysine-63-linked polyubiquitin

AU - Wang, Haopeng

AU - Matsuzawa, Atsushi

AU - Brown, Scott A.

AU - Zhou, Jingran

AU - Guy, Cliff S.

AU - Tseng, Ping Hui

AU - Forbes, Karen

AU - Nicholson, Thomas P.

AU - Sheppard, Paul W.

AU - Häcker, Hans

AU - Karin, Michael

AU - Vignali, Dario A.A.

PY - 2008/12/23

Y1 - 2008/12/23

N2 - Modification of proteins by the addition of lysine (K)-63-linked polyubiquitin (polyUb) chains is suggested to play important roles in a variety of cellular events, including DNA repair, signal transduction, and receptor endocytosis. However, identifying such modifications in living cells is complex and cumbersome. We have generated a monoclonal antibody (mAb) that specifically recognizes K63-linked polyUb, but not any other isopeptide-linked (K6, K11, K27, K29, K33, or K48) polyUb or monoubiquitin. We demonstrate the sensitivity and specificity of this K63Ub-specific mAb to detect K63Ub-modified proteins in cell lysates by Western blotting and in cells by immunofluorescence, and K63Ub-modified TRAF6 and MEKK1 in vitro and ex vivo. This unique mAb will facilitate the analysis of K63-linked polyubiquitylation ex vivo and presents a strategy for the generation of similar reagents against other forms of polyUb.

AB - Modification of proteins by the addition of lysine (K)-63-linked polyubiquitin (polyUb) chains is suggested to play important roles in a variety of cellular events, including DNA repair, signal transduction, and receptor endocytosis. However, identifying such modifications in living cells is complex and cumbersome. We have generated a monoclonal antibody (mAb) that specifically recognizes K63-linked polyUb, but not any other isopeptide-linked (K6, K11, K27, K29, K33, or K48) polyUb or monoubiquitin. We demonstrate the sensitivity and specificity of this K63Ub-specific mAb to detect K63Ub-modified proteins in cell lysates by Western blotting and in cells by immunofluorescence, and K63Ub-modified TRAF6 and MEKK1 in vitro and ex vivo. This unique mAb will facilitate the analysis of K63-linked polyubiquitylation ex vivo and presents a strategy for the generation of similar reagents against other forms of polyUb.

KW - MAP kinase kinase kinase 1 (MEKK1)

KW - TNF receptor-associated factor 6 (TRAF6)

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DO - 10.1073/pnas.0810461105

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AN - SCOPUS:58149520089

SN - 0027-8424

VL - 105

SP - 20197

EP - 20202

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

IS - 51

ER -

Analysis of nondegradative protein ubiquitylation with a monoclonal antibody specific for lysine-63-linked polyubiquitin

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Keywords

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