Stabilization of the E3 ubiquitin ligase Nrdp1 by the deubiquitinating enzyme USP8.
Nrdp1 is a RING finger-containing E3 ubiquitin ligase that physically interacts with and regulates steady-state 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.
UC Davis Cancer Center, Research Building III, Room 1400, 4645 2nd Ave., Sacramento, CA 95817, USA.
Xiuli
X
Wu
Lily
L
Yen
Lisa
L
Irwin
Colleen
C
Sweeney
Kermit L
KL
Carraway
D000818:Animals; D002460:Cell Line; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D056827:Endosomal Sorting Complexes Required for Transport; D004795:Enzyme Stability; D006801:Humans; D051379:Mice; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism; D043222:Ubiquitin Thiolesterase; D044767:Ubiquitin-Protein Ligases
D056827:Endosomal Sorting Complexes Required for Transport; D011506:Proteins; D025801:Ubiquitin; C452580:RNF41 protein, human; D044767:Ubiquitin-Protein Ligases; D010450:Endopeptidases; C541534:USP8 protein, human; D043222:Ubiquitin Thiolesterase; C541536:Usp8 protein, mouse; C091939:ubiquitin isopeptidase