title: Assembly Dynamics and Stoichiometry of the Apoptosis Signal-regulating Kinase (ASK) Signalosome in Response to Electrophile Stress.
abstract: Apoptosis signal-regulating kinase 1 (ASK1) is a key sensor kinase in the mitogen-activated protein kinase pathway that transduces cellular responses to oxidants and electrophiles. ASK1 is regulated by a large, dynamic multiprotein signalosome complex, potentially including over 90 reported ASK1-interacting proteins. We employed both shotgun and targeted mass spectrometry assays to catalogue the ASK1 protein-protein interactions in HEK-293 cells treated with the prototypical lipid electrophile 4-hydroxy-2-nonenal (HNE). Using both epitope-tagged overexpression and endogenous expression cell systems, we verified most of the previously reported ASK1 protein-protein interactions and identified 14 proteins that exhibited dynamic shifts in association with ASK1 in response to HNE stress. We used precise stable isotope dilution assays to quantify protein stoichiometry in the ASK signalosome complex and identified ASK2 at a 1:1 stoichiometric ratio with ASK1 and 14-3-3 proteins (YWHAQ, YWHAB, YWHAH, and YWHAE) collectively at a 0.5:1 ratio with ASK1 as the main components. Several other proteins, including ASK3, PARK7, PRDX1, and USP9X were detected with stoichiometries of 0.1:1 or less. These data support an ASK signalosome comprising a multimeric core complex of ASK1, ASK2, and 14-3-3 proteins, which dynamically engages other binding partners needed to mediate diverse stress-response signaling events. This study further demonstrates the value of combining global and targeted MS approaches to interrogate multiprotein complex composition and dynamics.
authors.affiliation: From the ‡Department of Biochemistry, Vanderbilt University School of Medicine;
authors.forename: Joel D
authors.initials: JD
authors.lastname: Federspiel
authors.affiliation: From the ‡Department of Biochemistry, Vanderbilt University School of Medicine;
authors.forename: Simona G
authors.initials: SG
authors.lastname: Codreanu
authors.affiliation: §Neuroscience Graduate Program, Vanderbilt Brain Institute, Vanderbilt University;
authors.forename: Amy M
authors.initials: AM
authors.lastname: Palubinsky
authors.affiliation: ¶Department of Neurology, Vanderbilt University, Nashville, Tennessee, 37232.
authors.forename: Ama J
authors.initials: AJ
authors.lastname: Winland
authors.affiliation: From the ‡Department of Biochemistry, Vanderbilt University School of Medicine;
authors.forename: Carlos Morales
authors.initials: CM
authors.lastname: Betanzos
authors.affiliation: ¶Department of Neurology, Vanderbilt University, Nashville, Tennessee, 37232.
authors.forename: BethAnn
authors.initials: B
authors.lastname: McLaughlin
authors.affiliation: From the ‡Department of Biochemistry, Vanderbilt University School of Medicine; daniel.liebler@vanderbilt.edu.
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Liebler
mesh_terms: D048948:14-3-3 Proteins / Q000378:metabolism; D000447:Aldehydes / Q000494:pharmacology*; D000939:Epitopes / Q000032:analysis; D057809:HEK293 Cells; D006801:Humans; D007553:Isotope Labeling; D048848:MAP Kinase Kinase Kinase 5 / Q000378:metabolism*; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods; D060066:Protein Interaction Maps / Q000187:drug effects*; D040901:Proteomics / Q000379:methods*; D015398:Signal Transduction
chemical_list: D048948:14-3-3 Proteins; D000447:Aldehydes; D000939:Epitopes; D048848:MAP Kinase Kinase Kinase 5; D020930:MAP Kinase Kinase Kinases; C550123:MAP3K15 protein, human; C482858:MAP3K5 protein, human; C027576:4-hydroxy-2-nonenal