Abstract
Ubiquitin conjugation (ubiquitylation) plays important roles not only in protein degradation but also in many other cellular functions. However, the sites of proteins that are targeted for such modification have remained poorly characterized at the proteomic level. We have now developed a method for the efficient identification of ubiquitylation sites in target proteins with the use of an engineered form of ubiquitin (K0-Ub), in which all seven lysine residues are replaced with arginine. K0-Ub is covalently attached to lysine residues of target proteins via an isopeptide bond, but further formation of a polyubiquitin chain does not occur on K0-Ub. We identified a total of 1392 ubiquitylation sites of 794 proteins from HEK293T cells. Profiling of ubiquitylation sites indicated that the sequences surrounding lysine residues targeted for ubiquitin conjugation do not share a common motif or structural feature. Furthermore, we identified a critical ubiquitylation site of the cyclin-dependent kinase inhibitor p27 Kip1. Mutation of this site thus inhibited ubiquitylation of and stabilized p27 Kip1, suggesting that this lysine residue is the target site of p27 Kip1 for ubiquitin conjugation in vivo. In conclusion, our method based on K0-Ub is a powerful tool for proteome-wide identification of ubiquitylation sites of target proteins.
| Original language | English |
|---|---|
| Pages (from-to) | 796-807 |
| Number of pages | 12 |
| Journal | Journal of Proteome Research |
| Volume | 11 |
| Issue number | 2 |
| DOIs | |
| Publication status | Published - Feb 3 2012 |
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All Science Journal Classification (ASJC) codes
- Biochemistry
- Chemistry(all)
Cite this
Proteome-wide identification of ubiquitylation sites by conjugation of engineered lysine-less ubiquitin. / Oshikawa, Kiyotaka; Matsumoto, Masaki; Oyamada, Koji; Nakayama, Keiichi I.
In: Journal of Proteome Research, Vol. 11, No. 2, 03.02.2012, p. 796-807.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Proteome-wide identification of ubiquitylation sites by conjugation of engineered lysine-less ubiquitin
AU - Oshikawa, Kiyotaka
AU - Matsumoto, Masaki
AU - Oyamada, Koji
AU - Nakayama, Keiichi I.
PY - 2012/2/3
Y1 - 2012/2/3
N2 - Ubiquitin conjugation (ubiquitylation) plays important roles not only in protein degradation but also in many other cellular functions. However, the sites of proteins that are targeted for such modification have remained poorly characterized at the proteomic level. We have now developed a method for the efficient identification of ubiquitylation sites in target proteins with the use of an engineered form of ubiquitin (K0-Ub), in which all seven lysine residues are replaced with arginine. K0-Ub is covalently attached to lysine residues of target proteins via an isopeptide bond, but further formation of a polyubiquitin chain does not occur on K0-Ub. We identified a total of 1392 ubiquitylation sites of 794 proteins from HEK293T cells. Profiling of ubiquitylation sites indicated that the sequences surrounding lysine residues targeted for ubiquitin conjugation do not share a common motif or structural feature. Furthermore, we identified a critical ubiquitylation site of the cyclin-dependent kinase inhibitor p27 Kip1. Mutation of this site thus inhibited ubiquitylation of and stabilized p27 Kip1, suggesting that this lysine residue is the target site of p27 Kip1 for ubiquitin conjugation in vivo. In conclusion, our method based on K0-Ub is a powerful tool for proteome-wide identification of ubiquitylation sites of target proteins.
AB - Ubiquitin conjugation (ubiquitylation) plays important roles not only in protein degradation but also in many other cellular functions. However, the sites of proteins that are targeted for such modification have remained poorly characterized at the proteomic level. We have now developed a method for the efficient identification of ubiquitylation sites in target proteins with the use of an engineered form of ubiquitin (K0-Ub), in which all seven lysine residues are replaced with arginine. K0-Ub is covalently attached to lysine residues of target proteins via an isopeptide bond, but further formation of a polyubiquitin chain does not occur on K0-Ub. We identified a total of 1392 ubiquitylation sites of 794 proteins from HEK293T cells. Profiling of ubiquitylation sites indicated that the sequences surrounding lysine residues targeted for ubiquitin conjugation do not share a common motif or structural feature. Furthermore, we identified a critical ubiquitylation site of the cyclin-dependent kinase inhibitor p27 Kip1. Mutation of this site thus inhibited ubiquitylation of and stabilized p27 Kip1, suggesting that this lysine residue is the target site of p27 Kip1 for ubiquitin conjugation in vivo. In conclusion, our method based on K0-Ub is a powerful tool for proteome-wide identification of ubiquitylation sites of target proteins.
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UR - http://www.scopus.com/inward/citedby.url?scp=84856637551&partnerID=8YFLogxK
U2 - 10.1021/pr200668y
DO - 10.1021/pr200668y
M3 - Article
C2 - 22053931
AN - SCOPUS:84856637551
VL - 11
SP - 796
EP - 807
JO - Journal of Proteome Research
JF - Journal of Proteome Research
SN - 1535-3893
IS - 2
ER -