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Using Proteomics and Biochemical Approaches to Evaluate the Function of SAKS1 in p97‐associated Process
Author(s) -
Li Shan,
Chou TsuiFen
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.1069.2
Subject(s) - cofactor , mutant , ubiquitin , aaa proteins , immunoprecipitation , atpase , biochemistry , microbiology and biotechnology , chemistry , transfection , enzyme , biology , gene
p97 is an AAA (ATPase associated with a variety of activities) ATPase that consists of an N‐domain and two ATPase domains (D1 and D2). A group of p97 cofactors, called the Ubiquitin Regulatory X (UBX) domain‐containing cofactors, direct the function of p97 in many different cellular pathways. Among them, the cofactor SAKS1 is able to bind to both p97 and polyubiquitinated proteins through its UBX and UBA (ubiquitin‐associated) domains. SAKS1 enhances the ATPase activity of WT p97 by 2.4‐fold and of the D2‐active mutant by 1.3‐fold, whereas it has no effect on D1‐active mutant. The steady‐state kinetic study demonstrates that SAKS1 increases the catalytic efficiency (Kcat/Km) of both WT and the D2‐active mutant by more than 4‐fold, but does not affect the D1‐active mutant. Thus, SAKS1 regulates mainly the D2 ATPase activity of WT p97. We used co‐immunoprecipitation (IP) and LC‐MS/MS quantitation approaches to analyze interacting proteins of p97 and SAKS1. Analysis of SAKS1‐Flag IP from transient transfection with 293T and U2OS cells demonstrated that SAKS1 likely binds with four other p97 cofactors (p47, Ubxd1, NPL4 and Ufd1). This indicates that SAKS1 not only interacts with p97 directly but also forms mixed p97/cofactor complexes to mediate p97‐associated processes. Besides, we inhibited p97 activity using ATP‐competitive inhibitor (ML240) or noncompetitive inhibitor (NMS‐873) in SAKS1 expressing cells and carried out the IP and LC‐MS/MS analysis, which reveal significant changes in 76 SAKS1‐associated proteins and we are examining their functional role in p97‐associated processes.