Stabilization of the E3 Ubiquitin Ligase Nrdp1 by the Deubiquitinating Enzyme USP8

Abstract

Nrdp1 is a RING finger-containing E3 ubiquitin ligase that physically interacts with and regulates steadystate cellular levels of the ErbB3 and ErbB4 receptor tyrosine kinases and has been implicated in the degradation of the inhibitor-of-apoptosis protein BRUCE. Here we demonstrate that the Nrdp1 protein undergoes efficient proteasome-dependent degradation and that mutations in its RING finger domain that disrupt ubiquitin ligase activity enhance stability. These observations suggest that Nrdp1 self-ubiquitination and stability could play an important role in regulating the activity of this protein. Using affinity chromatography, we identified the deubiquitinating enzyme USP8 (also called Ubpy) as a protein that physically interacts with Nrdp1. Nrdp1 and USP8 could be coimmunoprecipitated, and in transfected cells USP8 specifically bound to Nrdp1 but not cbl, a RING finger E3 ligase involved in ligand-stimulated epidermal growth factor receptor down-regulation. The USP8 rhodanese and catalytic domains mediated Nrdp1 binding. USP8 markedly enhanced the stability of Nrdp1, and a point mutant that disrupts USP8 catalytic activity destabilized endogenous Nrdp1. Our results indicate that Nrdp1 is a specific target for the USP8 deubiquitinating enzyme and are consistent with a model where USP8 augments Nrdp1 activity by mediating its stabilization.

Oligomerization of the Nrdp1 E3 Ubiquitin Ligase Is Necessary for Efficient Autoubiquitination but Not ErbB3 Ubiquitination

“…Nrdp1-32 encodes a naturally occurring splice variant lacking the RING finger and B-box domains and acts as a dominant negative to stabilize ErbB3 (13,16,17); the Nrdp1-32⌬cc derivative also lacks the coiled-coil region. Finally, Nrdp1-CSHQ is a double point mutant in zinc-binding residues 34 and 36 of the RING finger domain that lacks ligase activity (32).…”

“…When more than one plasmid was transfected at a time, equal amounts of each plasmid were used. Plasmids encoding ErbB3, wild type Nrdp1-FLAG, Nrdp1-32-FLAG, and Nrpd1-CHSQ-FLAG were described previously (17,32). HA-labeled ubiquitin has been described elsewhere (33).…”

Regulation of Epidermal Growth Factor Receptor Down-Regulation by UBPY-mediated Deubiquitination at Endosomes

“…UBPY also binds to several other proteins: CDC25 Mm , a GDP/GTP exchange factor for Ras (Gnesutta et al, 2001); GRAIL, an E3 Ub ligase involved in anergy induction in CD4 ϩ T-cells (Soares et al, 2004); and Nrdp1, another E3 ligase that regulates the steady-state level of ErbB3 and ErbB4 RTKs (Wu et al, 2004). UBPY is implicated in deubiquitinating and regulating the stability of these proteins (Gnesutta et al, 2001;Soares et al, 2004;Wu et al, 2004).…”

“…UBPY is implicated in deubiquitinating and regulating the stability of these proteins (Gnesutta et al, 2001;Soares et al, 2004;Wu et al, 2004). Therefore, through its deubiquitinating activity, UBPY probably participates in several different cellular processes by regulating the degradation or stability of distinct substrate proteins.…”

“…This might indicate that UBPY localizes to several different subcellular sites because it is implicated in the deubiquitination of several unrelated proteins (Gnesutta et al, 2001;Soares et al, 2004;Wu et al, 2004). When endosomes were exaggerated by impairing membrane traffic via the organelle, however, UBPY was accumulated on the aberrant endosomes.…”