Protein–protein interactions are essential for almost all cellular processes, hence understanding these processes mainly depends on the identification and characterization of the relevant protein–protein interactions. In the present paper, we introduce the concept of TRS (trans-SUMOylation), a new method developed to identify and verify protein–protein interactions in mammalian cells in vivo. TRS utilizes Ubc9-fusion proteins that trans-SUMOylate co-expressed interacting proteins. Using TRS, we analysed interactions of 65 protein pairs co-expressed in HEK (human embryonic kidney)-293 cells. We identified seven new and confirmed 16 known protein interactions, which were determined via endogenous SUMOylation sites of the binding partners or by using SUMOylation-site tags respectively. Four of the new protein interactions were confirmed by GST (glutathione transferase) pull-down and the p38α–Edr2 interaction was verified by co-localization analysis. Functionally, this p38α–Edr2 interaction could possibly be involved in the recruitment of p38α to the polycomb chromatin-remodelling complex to phosphorylate Bmi1. We also used TRS to characterize protein-interaction domains of the protein kinase pairs p38α–MK2 [MK is MAPK (mitogen-activated protein kinase)-activated protein kinase] and ERK3 (extracellular-signal-regulated kinase 3)–MK5 and of the p38α–p53 complex. The ability of TRS to monitor protein interactions in mammalian cells in vivo at levels similar to endogenous expression makes it an excellent new tool that can help in defining the protein interactome of mammalian cells.
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Research Article|
August 26 2011
Monitoring protein–protein interactions in mammalian cells by trans-SUMOylation
Ratnesh K. Srivastav;
Ratnesh K. Srivastav
1Institute for Physiological Chemistry/Biochemistry, Hannover Medical School, Carl-Neuberg Strasse 1, 30625 Hannover, Germany
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Susan Schwede;
Susan Schwede
1Institute for Physiological Chemistry/Biochemistry, Hannover Medical School, Carl-Neuberg Strasse 1, 30625 Hannover, Germany
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Malte Klaus;
Malte Klaus
1Institute for Physiological Chemistry/Biochemistry, Hannover Medical School, Carl-Neuberg Strasse 1, 30625 Hannover, Germany
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Jessica Schwermann;
Jessica Schwermann
1Institute for Physiological Chemistry/Biochemistry, Hannover Medical School, Carl-Neuberg Strasse 1, 30625 Hannover, Germany
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Matthias Gaestel;
Matthias Gaestel
1Institute for Physiological Chemistry/Biochemistry, Hannover Medical School, Carl-Neuberg Strasse 1, 30625 Hannover, Germany
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Rainer Niedenthal
Rainer Niedenthal
1
1Institute for Physiological Chemistry/Biochemistry, Hannover Medical School, Carl-Neuberg Strasse 1, 30625 Hannover, Germany
1To whom correspondence should be addressed (email Niedenthal.Rainer@MH-Hannover.de).
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Publisher: Portland Press Ltd
Received:
January 05 2011
Revision Received:
June 07 2011
Accepted:
June 15 2011
Accepted Manuscript online:
June 15 2011
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2011 Biochemical Society
2011
Biochem J (2011) 438 (3): 495–503.
Article history
Received:
January 05 2011
Revision Received:
June 07 2011
Accepted:
June 15 2011
Accepted Manuscript online:
June 15 2011
Citation
Ratnesh K. Srivastav, Susan Schwede, Malte Klaus, Jessica Schwermann, Matthias Gaestel, Rainer Niedenthal; Monitoring protein–protein interactions in mammalian cells by trans-SUMOylation. Biochem J 15 September 2011; 438 (3): 495–503. doi: https://doi.org/10.1042/BJ20110035
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