Binding of JAB1/CSN5 to MIF is mediated by the MPN domain but is independent of the JAMM motif.
Macrophage migration inhibitory factor (MIF) binds to c-Jun activation domain binding protein-1 (JAB1)/subunit 5 of COP9 signalosome (CSN5) and modulates cell signaling and the cell cycle through JAB1. The binding domain of JAB1 responsible for binding to MIF is unknown. We hypothesized that the conserved Mpr1p Pad1p N-terminal (MPN) domain of JAB1 may mediate binding to MIF. In fact, yeast two hybrid (YTH) and in vitro translation/coimmunoprecipitation (CoIP) analysis showed that a core MPN domain, which did not cover the functional JAB1/MPN/Mov34 metalloenzyme (JAMM) deneddylase sequence, binds to MIF comparable to full-length JAB1. YTH and pull-down analysis in conjunction with nanobead affinity matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry demonstrated that MIF(50-65) and MPN are sufficient to mediate MIF-JAB1 interaction, respectively. Finally, endogenous CoIP of MIF-CSN6 complexes from mammalian cells demonstrated that MPN is responsible for MIF-JAB1 binding in vivo, and, as CSN6 does not contain a functional JAMM motif, confirmed that the interaction does not require JAMM.
Burger-Kentischer
Anke
A
Fraunhofer Institute for Interfacial Engineering and Biotechnology, Nobelstrasse 12, D-70569 Stuttgart, Germany.
Finkelmeier
Doris
D
Thiele
Michael
M
Schmucker
Jürgen
J
Geiger
Georg
G
Tovar
Günter E M
GE
Bernhagen
Jürgen
J
D020816:Amino Acid Motifs / Q000235:genetics; D001665:Binding Sites; D000075686:COP9 Signalosome Complex; D002460:Cell Line; D017124:Conserved Sequence / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins; D008263:Macrophage Migration-Inhibitory Factors / Q000378:metabolism*; D010447:Peptide Hydrolases; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008263:Macrophage Migration-Inhibitory Factors; D014157:Transcription Factors; D010447:Peptide Hydrolases; C501488:COPS5 protein, human; D000075686:COP9 Signalosome Complex