Regulation of the Discs Large Tumor Suppressor by a Phosphorylation-dependent Interaction with the β-TrCP Ubiquitin Ligase Receptor

Fiamma Mantovani,Lawrence Banks

doi:10.1074/jbc.m302799200

Abstract

The discs large (hDlg) tumor suppressor is intimately involved in the control of cell contact, polarity, and proliferation by interacting with several components of the epithelial junctional complex and with the APC tumor suppressor protein. In epithelial cells, hDlg protein stability is regulated through the ubiquitin-proteasome pathway: hDlg is actively degraded in isolated cells, whereas it accumulates upon cell-cell contact. During neoplastic transformation of epithelial cells, loss of the differentiated morphology and progression toward a metastatic phenotype correlate with down-regulation of hDlg levels and loss of contact-dependent stabilization. Here we show that upon hyperphosphorylation, hDlg interacts with the beta-TrCP ubiquitin ligase receptor through a DSGLPS motif within its Src homology 3 domain. As a consequence, overexpression of beta-TrCP enhances ubiquitination of Dlg protein and decreases its stability, whereas a dominant negative beta-TrCP mutant inhibits this process. Furthermore, a mutant Dlg protein that is unable to bind beta-TrCP displays a higher protein stability and is insensitive to beta-TrCP. Using RNA interference, we also demonstrate that endogenous beta-TrCP regulates hDlg protein levels in epithelial cells. Finally, we show that beta-TrCP selectively induces the degradation of the membrane-cytoplasmic pool, without affecting the nuclear pool of hDlg.

Highlights

Ubiquitin-mediated proteolysis is a highly selective, temporally controlled and tightly regulated pathway that plays crucial roles in a broad array of basic cellular processes, including regulation of the cell cycle, control of signal transduction, differentiation, and development
HDlg protein stability is regulated through the ubiquitin-proteasome pathway: hDlg is actively degraded in isolated cells, whereas it accumulates upon cell-cell contact
We have shown previously that in differentiated epithelial cells, hDlg protein accumulates upon cell-cell contact, being rapidly degraded by the ubiquitin-proteasome pathway in isolated cells [22]

Summary

Introduction

Ubiquitin-mediated proteolysis is a highly selective, temporally controlled and tightly regulated pathway that plays crucial roles in a broad array of basic cellular processes, including regulation of the cell cycle, control of signal transduction, differentiation, and development. ␤-Catenin has a dual role: a membrane pool participates in formation of adherens junctions, whereas a short-lived soluble pool functions as signal transducer/transcription factor to promote proliferation, an activity that is tightly controlled by means of regulating its stability [5] This is achieved by a destruction complex containing the APC tumor suppressor protein, axin, the GSK3␤ kinase, and the F-box protein ␤-TrCP [4, 6]. The growth suppressive activities of hDlg represent a common target for several viral transforming proteins, including high risk human papilloma virus E6 [24, 25], human T-cell lymphotrophic virus-1 Tax [26], and adenovirus 9 E4ORF1 [25] All of these bind to the PDZ-2 domain of hDlg, thereby inhibiting its interaction with APC and the consequent block of cell cycle progression [26]. The findings that hDlg forms a complex with both ␤-catenin and APC [20] and that hyperphosphorylated hDlg is selectively degraded by the proteasome pathway [22] prompted us to investigate the possibility that hDlg could be regulated through a pathway similar to ␤-catenin, involving the SCF␤-TrCP ubiquitin ligase

Methods

Results

Conclusion