Nbr1 Is a Novel Inhibitor of Ligand-Mediated Receptor Tyrosine Kinase Degradation

Abstract

Neighbor of BRCA1 (Nbr1) is a highly conserved multidomain scaffold protein with proposed roles in endocytic trafficking and selective autophagy. However, the exact function of Nbr1 in these contexts has not been studied in detail. Here we investigated the role of Nbr1 in the trafficking of receptor tyrosine kinases (RTKs). We report that ectopic Nbr1 expression inhibits the ligand-mediated lysosomal degradation of RTKs, and this is probably done via the inhibition of receptor internalization. Conversely, the depletion of endogenous NBR1 enhances RTK degradation. Analyses of truncation mutations demonstrated that the C terminus of Nbr1 is essential but not sufficient for this activity. Moreover, the C terminus of Nbr1 is essential but not sufficient for the localization of the protein to late endosomes. We demonstrate that the C terminus of Nbr1 contains a novel membrane-interacting amphipathic α-helix, which is essential for the late endocytic localization of the protein but not for its effect on RTK degradation. Finally, autophagic and late endocytic localizations of Nbr1 are independent of one another, suggesting that the roles of Nbr1 in each context might be distinct. Our results define Nbr1 as a negative regulator of ligand-mediated RTK degradation and reveal the interplay between its various regions for protein localization and function.

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Articles of Significant Interest Selected from This Issue by the Editors

We thank Susan Brewer for the bacterial expression and purification of the GST-tagged C-terminal-only Nbr1 mutation. We also thank Sharon Tooze for 293A LC3-GFP stable cells and Steve Dove for his help with vesicle preparation. Finally, we give special thanks to all our present and past laboratory members for useful discussions and feedback.

This work was funded by Cancer Research UK (J.K.H.) and the Biotechnology and Biosciences Research Council (J.Z.R.). G.A. is funded through a Medical Research Council Ph.D. studentship, and F.K.M. is funded through a Cancer Research UK Ph.D. studentship. The Nikon A1R/TIRF microscope used in this research was obtained through the Birmingham Science City Translational Medicine Clinical Research and Infrastructure Trials Platform, with support from Advantage West Midlands (AWM).