A Macrophage Migration Inhibitory Factor interactome screen identifies a complex of Jab1/CSN5 and Valosin-containing protein as an important mediator in the ubiquitin proteasome system
Macrophage migration inhibitory factor (MIF) is a conserved 12.5 kD protein that serves many critical functions in the regulation of gene expression, proliferation, apoptosis and development. Over the last few years evidence has accumulated that MIF may function in the inhibition of the deneddylase activity of the COP9 signalosome (CSN) by interacting with its subunit Jab1/CSN5. The target of the CSN deneddylase activity is the cullin component of SCF E3 ubiquitin ligases that are significantly more active when neddylated. These ligases are responsible for the selective ubiquitinylation of substrates which are hydrolysed in the 26S proteasome. The latter consists of a 19S reguatory and a 20S core particle. Thus, ubiquitinylating enzymes and the proteasome form the two main pillars of the ubiquitin proteasome system (UPS). To better understand the biological role of MIF in the UPS, a systematic interactome screen was performed in NIH3T3 cells that are constitutively expressing biotinylated MIF.
Biotinylated MIF and its associated proteins were purified by streptavidin binding. Subsequent identification of MIF interacting proteins by mass spectrometry detected a number of already known MIF interacting proteins including MIF itself, peroxiredoxin 1 and RPS19, thus emphasizing the validity of the approach. Besides other new candidates, endoplasmic reticulum (ER) associated chaperones amongst them BiP, ERp57 Sec61ß and the AAA ATPase valosin-containing protein (VCP), which are all involved in ER-associated degradation were identified. As many new MIF interacting partners are involved in the UPS, further investigations were concentrated on the association of MIF with Jab1/CSN5 and VCP. Whilst the binding of MIF to VCP seems to be indirectly mediated by Jab1/CSN5, the interaction of VCP with Jab1/CSN5 is direct and robust as was verified independently by co-immunoprecipitation (co-IP), in vitro pull-down assays and fluorescence resonance energy transfer experiments.
The interaction interface between VCP and Jab1/CSN5 was investigated by transient transfection of wild type and mutant proteins followed by co-IP. It emerged that Jab1/CSN5 associates with VCP via the core of its MPN domain that is contacting the N-terminal domain and the D1 domain of VCP. Its binding is independent of the JAMM motif harboring the NEDD8 isopeptidase activity. The JAMM motif was also shown to bind, but not to depolymerize poly-ubiquitinylated chains. Using gel filtration chromatography, VCP comigrated with the CSN and co-IP experiments confirmed that the interaction of VCP is not restricted to Jab1/CSN5, but involves the whole CSN complex in the association. The CSN consists of eight proteins that all have paralogs in the 19S regulatory particle of the proteasome. VCP is a homohexameric ATPase with structural homology to the heterohexameric ring of ATPases in the 19S regulatory particle.
RNA interference mediated knockdowns demonstrated that the JAMM motif of CSN5, a functional CSN complex and the CSN-associated deubiquitinase USP15 are all required for deubiquitinylation of substrates bound to VCP.
Taken together, these results suggest that the CSN together with VCP forms a complex. Apart from interacting with at least one deubiquitinylase, the CSN is also known to be associated with three kinases that could regulate the connection of substrate-processing cofactors with VCP via phosphorylation. Therefore, we hypothesize that the CSN together with VCP could have the function to extract ubiquitinylated and abnormal folded proteins from larger protein complexes or membranes and to determine their fate by means of associated substrate-processing cofactors and regulatory enzymes.
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