An Inducible Retroviral Expression System for Tandem Affinity Purification Mass-Spectrometry-Based Proteomics Identifies Mixed Lineage Kinase Domain-like Protein (MLKL) as an Heat Shock Protein 90 (HSP90) Client.

Details

Serval ID
serval:BIB_5EACE3AC18B6
Type
Article: article from journal or magazin.
Collection
Publications
Title
An Inducible Retroviral Expression System for Tandem Affinity Purification Mass-Spectrometry-Based Proteomics Identifies Mixed Lineage Kinase Domain-like Protein (MLKL) as an Heat Shock Protein 90 (HSP90) Client.
Journal
Molecular & cellular proteomics
Author(s)
Bigenzahn J.W., Fauster A., Rebsamen M., Kandasamy R.K., Scorzoni S., Vladimer G.I., Müller A.C., Gstaiger M., Zuber J., Bennett K.L., Superti-Furga G.
ISSN
1535-9484 (Electronic)
ISSN-L
1535-9476
Publication state
Published
Issued date
03/2016
Peer-reviewed
Oui
Volume
15
Number
3
Pages
1139-1150
Language
english
Notes
Publication types: Journal Article ; Research Support, Non-U.S. Gov't
Publication Status: ppublish
Abstract
Tandem affinity purification-mass spectrometry (TAP-MS) is a popular strategy for the identification of protein-protein interactions, characterization of protein complexes, and entire networks. Its employment in cellular settings best fitting the relevant physiology is limited by convenient expression vector systems. We developed an easy-to-handle, inducible, dually selectable retroviral expression vector allowing dose- and time-dependent control of bait proteins bearing the efficient streptavidin-hemagglutinin (SH)-tag at their N- or C termini. Concomitant expression of a reporter fluorophore allows to monitor bait-expressing cells by flow cytometry or microscopy and enables high-throughput phenotypic assays. We used the system to successfully characterize the interactome of the neuroblastoma RAS viral oncogene homolog (NRAS) Gly12Asp (G12D) mutant and exploited the advantage of reporter fluorophore expression by tracking cytokine-independent cell growth using flow cytometry. Moreover, we tested the feasibility of studying cytotoxicity-mediating proteins with the vector system on the cell death-inducing mixed lineage kinase domain-like protein (MLKL) Ser358Asp (S358D) mutant. Interaction proteomics analysis of MLKL Ser358Asp (S358D) identified heat shock protein 90 (HSP90) as a high-confidence interacting protein. Further phenotypic characterization established MLKL as a novel HSP90 client. In summary, this novel inducible expression system enables SH-tag-based interaction studies in the cell line proficient for the respective phenotypic or signaling context and constitutes a valuable tool for experimental approaches requiring inducible or traceable protein expression.
Keywords
Animals, Cell Line, Chromatography, Affinity/methods, GTP Phosphohydrolases/genetics, GTP Phosphohydrolases/metabolism, Genes, Reporter, HEK293 Cells, HSP90 Heat-Shock Proteins/metabolism, HT29 Cells, Humans, K562 Cells, Membrane Proteins/genetics, Membrane Proteins/metabolism, Mice, Mutation, Protein Kinases/genetics, Protein Kinases/metabolism, Proteomics/methods, Retroviridae/genetics, Tandem Mass Spectrometry/methods
Pubmed
Web of science
Open Access
Yes
Create date
11/08/2020 17:06
Last modification date
13/08/2020 6:26
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