Neuregulin-induced ErbB3 downregulation is mediated by a protein stability cascade involving the E3 ubiquitin ligase Nrdp1

Abstract

The molecular mechanisms underlying epidermal growth factor (EGF) receptor tyrosine kinase down-regulation in response to growth factor binding are coming into focus and involve cbl-mediated receptor ubiquitination followed by lysosomal degradation. However, mechanisms underlying the ligand-stimulated degradation of the related receptor tyrosine kinases of the ErbB family do not involve cbl and remain unexplored. Previous studies have demonstrated that the E3 ubiquitin ligase Nrdp1 contributes to the maintenance of steady-state ErbB3 levels by mediating its growth factor-independent degradation. Here we demonstrate that treatment of cells with the ErbB3 ligand neuregulin-1 (NRG1) stabilizes the deubiquitinating enzyme USP8, which in turn stabilizes Nrdp1. The catalytic activity of USP8 is required for NRG1-induced Nrdp1 stabilization. We provide evidence that Akt-mediated phosphorylation of USP8 threonine residue T907 contributes to USP8 stability. Finally, we demonstrate that Nrdp1 or USP8 knockdown suppresses NRG1-induced ErbB3 ubiquitination and degradation in MCF7 breast cancer cells. We conclude that an NRG1-induced protein stability cascade involving USP8 and Nrdp1 mediates the down-regulation of ErbB3. Our observations raise the possibility that the ligand-induced augmentation of pathways involved in the maintenance of basal levels of receptor tyrosine kinases can contribute to ligand-stimulated down-regulation.

FIG. 1.

NRG1 stimulates Nrdp1 accumulation. (A) MCF7 cells were treated without or with NRG1 for 6 h, and lysates were immunoblotted with antibodies to ErbB3, Nrdp1, and actin. (B) 293T cells were cotransfected with both ErbB2 and ErbB3, GFP, or FLAG-Nrdp1, as indicated. Cells were treated without and with NRG1 for 6 h, and lysates were blotted with antibodies to phosphotyrosine (pY), phospho-Akt (pAkt), GFP, FLAG, and actin.

FIG. 2.

USP8-deubiquitinating activity mediates NRG1-induced Nrdp1 accumulation. (A) 293T cells were cotransfected with ErbB2/3 and FLAG-Nrdp1, without and with V5-USP8 as indicated. Cells were treated without and with NRG1, and lysates were blotted with the indicated antibodies. (B) 293T cells were cotransfected with ErbB2/ErbB3 and FLAG-Nrdp1 and V5-tagged versions of either wild-type (wt) or inactive mutant (C748A) USP8.

FIG. 3.

USP8 knockdown suppresses NRG1-stimulated Nrdp1 accumulation. (A) 293T cells were cotransfected with V5-USP8 or its vector and USP8 shRNA or its vector, and lysates were blotted with antibodies to V5 epitope and actin. (B) 293T cells were cotransfected with ErbB2/ErbB3 and FLAG-Nrdp1 without and with USP8 shRNA plasmid, and lysates were blotted with antibodies to FLAG tag or actin. Blots were quantified, and the graph depicts the average (±standard error [SE]) stimulation of NRG1-induced Nrdp1 accumulation over three independent experiments. (C) MCF7 cells stably transduced with an empty vector or the USP8 shRNA vector were treated with NRG1, and lysates were blotted with the indicated antibodies.

FIG. 4.

USP8 is phosphorylated on threonine 907 in response to NRG1 treatment. (A) 293T cells were transfected with ErbB2/ErbB3 and FLAG-Nrdp1 and simultaneously treated for 6 h without and with LY294001 and NRG1, as indicated. Lysates were blotted with the indicated antibodies. (Lower panel) Lysates from NRG1-stimulated cells treated without and with LY294001 were blotted for phosphorylated Akt. (B) 293T cells were cotransfected with FLAG-Nrdp1 without or with HA-tagged dominant negative Akt (HA-DN-Akt). The graph depicts the average (±SE) stimulation of NRG1-induced Nrdp1 accumulation from three independent experiments. (C) 293T cells were transfected with either wild type (wt) or T907A USP8, with or without ErbB2 and ErbB3, as indicated. Cells were treated with NRG1 for 6 h, and anti-V5 immunoprecipitates were blotted with phosphothreonine and V5 antibodies. Lysates were blotted for phosphotyrosine (pY) and actin.

FIG. 5.

NRG1-stimulated T907 phosphorylation augments Nrdp1 accumulation. (A) Cells were cotransfected with FLAG-Nrdp1 and either wild-type or T907A mutant USP8, without and with HA-tagged constitutively active Akt (HA-CA-Akt), as indicated. (B) Cells were cotransfected with FLAG-Nrdp1 and either wild-type or T907A mutant USP8, with or without constitutively active Akt (CA-HA-Akt), and blotted with the indicated antibodies. The graphs in each panel depict the average (±SE) stimulation of NRG1-induced Nrdp1 accumulation from three independent experiments.

FIG. 6.

USP8 association with Akt. (A) Lysates from 293T cells cotransfected with HA-Akt and V5-USP8 were immunoprecipitated (IP) with anti-V5. Lysates and precipitates were blotted with the indicated antibodies. (B) Serum-starved MCF7 cells were treated without and with NRG1 for 15 min, and endogenous USP8 was visualized by immunofluorescence. (C) V5-USP8 (green) and HA-Akt (red) in cotransfected COS7 cells were visualized by deconvolving immunofluorescence microscopy. White arrows point to regions of colocalization at the cell surface and in a perinuclear structure.

FIG. 7.

Regulation of Nrdp1 and ErbB3 ubiquitination by USP8. (A) 293T cells were cotransfected with Nrdp1 and HA-ubiquitin (HA-Ub) along with wild-type USP8, the C748A mutant, or their vector pcDNA3.1 or with shRNA USP8 or its vector. Cells were treated with MG132 to stabilize Nrdp1, and Nrdp1 immunoprecipitates (IP) were blotted with antibodies to the HA epitope and ubiquitin. (B) MCF7 cells were treated for various times with NRG1, and ErbB3 immunoprecipitates were blotted with antibodies to ubiquitin and ErbB3. (C) MCF7 cells stably transduced with the pSuper vector, Nrdp1 shRNA, or USP8 shRNA were treated for the indicated times with NRG1, and ErbB3 immunoprecipitates were blotted for ubiquitin and ErbB3.

FIG. 8.

NRG1 induces ErbB3 but not ErbB2 down-regulation in MCF7 cells. (A) MCF7 cells were treated without and with 0.1 μM concanamycin (Con), as indicated. Cells were then treated with NRG1 for the indicated times, and lysates were blotted with antibodies to ErbB2 and ErbB3. (B) MCF7 cells were treated with cycloheximide to inhibit protein synthesis and then treated without and with NRG1 for various times. Lysates were blotted with anti-ErbB3 (upper panel), and bands were quantified and plotted (lower panel). ErbB3 half-life was calculated by fitting the data to a single exponential.

FIG. 9.

Nrdp1 and USP8 augment NRG1-induced ErbB3 down-regulation. (A) MCF7 cells stably transduced with the vector, Nrdp1 shRNA, or USP8 shRNA were treated with NRG1 for the indicated times and blotted with antibodies to ErbB3. (B) Bands were quantified, ErbB3 half-lives for the three cell lines were calculated as described for Fig. 8, and the averages (±SEs) from three independent experiments were plotted.

FIG. 10.

Comparison of known EGF receptor and ErbB3 down-regulation pathways. EGF stimulation causes recruitment of cbl to the EGF receptor. Tyrosine phosphorylation of cbl then promotes receptor ubiquitination and trafficking to the lysosome. In contrast, NRG1 stimulates the PI3K/Akt pathway, resulting in USP8 stabilization through the phosphorylation of T907. USP8 in turn stabilizes Nrdp1, which ubiquitinates ErbB3 to promote its degradation.