COP9 Signalosome Interacts ATP-dependently with p97/Valosin-containing Protein (VCP) and Controls the Ubiquitination Status of Proteins Bound to p97/VCP

Abstract

Ubiquitinated proteins can alternatively be delivered directly to the proteasome or via p97/VCP (valosin-containing protein). Whereas the proteasome degrades ubiquitinated proteins, the homohexameric ATPase p97/VCP seems to control the ubiquitination status of recruited substrates. The COP9 signalosome (CSN) is also involved in the ubiquitin/proteasome system (UPS) as exemplified by regulating the neddylation of ubiquitin E3 ligases. Here, we show that p97/VCP colocalizes and directly interacts with subunit 5 of the CSN (CSN5) in vivo and is associated with the entire CSN complex in an ATP-dependent manner. Furthermore, we provide evidence that the CSN and in particular the isopeptidase activity of its subunit CSN5 as well as the associated deubiquitinase USP15 are required for proper processing of polyubiquitinated substrates bound to p97/VCP. Moreover, we show that in addition to NEDD8, CSN5 binds to oligoubiquitin chains in vitro. Therefore, CSN and p97/VCP could form an ATP-dependent complex that resembles the 19 S proteasome regulatory particle and serves as a key mediator between ubiquitination and degradation pathways.Many fundamental cellular functions such as membrane fusion, gene transcription, DNA replication, and repair are controlled by the covalent linkage of ubiquitin (Ub) 5 to substrate proteins (1). Different Ub modifications serve as signaling-dependent regulators of protein interaction networks (2). Substrates that are polyubiquitinated via Lys 48 are recognized by the 19 S regulatory particle of the proteasome that consists of a lid and a base complex. Upon recognition, substrates are unfolded by the base complex and hydrolyzed within the 20 S proteolytic chamber (3).The COP9 signalosome (CSN) plays an essential role as mediator between signaling pathways and downstream mechanisms controlling developmental processes. This involves ubiquitin-dependent protein degradation of key regulatory molecules like the cell cycle inhibitor p27Kip1 , the tumor suppressor p53, and IkB␣. The CSN is a highly conserved complex found in all higher eukaryotes (4). Like the proteasome lid, it contains eight core subunits (CSN1-8), and for each of them exists a paralogous subunit in the proteasome lid. CSN5 (also known as Jab1) and CSN6 possess a MOV34/PAD N-terminal (MPN) domain (5). The MPN domain of CSN5 harbors a metalloprotease motif referred to as the Jab1/MPN domain-associated metallopeptidase (JAMM) motif that regulates the activity of E3 Ub-ligases by deneddylation of the cullin component (6).The chaperone p97 or valosin-containing protein (p97/VCP) has been recognized as another key player within the ubiquitin/ proteasome system (UPS). p97/VCP extracts mono-or oligoubiquitinated substrates from complexes and presents them to the UPS. It is a member of the family of ATPases associated with various cellular activities (AAA ϩ ) and forms a homohexamer. Two AAA cassettes of each monomer build two consecutive stacked rings (D1 and D2 domain) in the hexamer underneath a ring of flexible N-terminal domains (7...

“Depupylation” of Prokaryotic Ubiquitin-like Protein from Mycobacterial Proteasome Substrates

“…It is able to interact with Ub conjugation machinery and either promote or inhibit ubiquitylation, thus influencing the stability of proteins. It was recently shown that p97 forms a complex with the COP9 signalosome (CSN) and the isopeptidase and deubiquitinase activities of CSN regulate the amount of ubiquitylated substrates bound to p97/VCP (Cayli et al, 2009). Perhaps functionally similar to CSN association with p97/VCP, our preliminary in vivo data suggest that Dop, possibly via other factors, interacts with Mpa to regulate the pupylation status of certain substrates.…”

Control of p97 function by cofactor binding

“…This domain neither displays structural similarity to any of the known p97 interacting domains nor does it contain any of the linear binding motifs. Other examples are the CSN5 subunit of the COP9 signalosome, which is proposed to interact via its MPN domain with the N domain [99] or SelS/VIMP, which instead of using its VIM binds via two proline residues [100].…”

Vesnarinone Suppresses TNFα mRNA Expression by Inhibiting Valosin-Containing Protein

“…at ASPET Journals on May 11, 2018 molpharm.aspetjournals.org 1998; Yen et al, 2000;Dai and Li, 2001;Asai et al, 2002;Alexandru et al, 2008;Cayli et al, 2009). Vesnarinone induced the accumulation of ubiquitinated IkBa by inhibiting the interaction between VCP and the 26S proteasome, which was essential for the degradation of IkBa and the activation of NFkB, implying that vesnarinone inhibited the function of VCP.…”

“…Because VCP is known to bind to ubiquitinated proteins such as IkBa, cyclin E, and hypoxiainducible factor (HIF)1a (Dai et al, 1998;Yen et al, 2000;Dai and Li, 2001;Asai et al, 2002;Alexandru et al, 2008;Cayli et al, 2009;) and contributes to their degradation, we examined whether vesnarinone inhibits the interaction of VCP with ubiquitinated IkBa or the 26S proteasome. An expression vector encoding VCP-His-FLAG was transfected into 293T cells, and a coimmunoprecipitation assay was performed using an anti-FLAG antibody in the presence of MG132.…”