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

PHA - Life and Pharmaceutical Science

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

54 Citations (Scopus)

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
Publication status	Published - 2008 Dec 23

Keywords

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

Access to Document

10.1073/pnas.0810461105

Cite this

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

@article{ae097892527d47ec9d2e4dc5a6391cb3,

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",

pages = "20197--20202",

journal = "Proceedings of the National Academy of Sciences of the United States of America",

issn = "0027-8424",

publisher = "National Academy of Sciences",

number = "51",

}

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

TY - JOUR

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)

UR - http://www.scopus.com/inward/record.url?scp=58149520089&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=58149520089&partnerID=8YFLogxK

U2 - 10.1073/pnas.0810461105

DO - 10.1073/pnas.0810461105

M3 - Article

C2 - 19091944

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

Abstract

Keywords

Access to Document

Other files and links

Fingerprint

Cite this