WOX1 Is Essential for Tumor Necrosis Factor-, UV Light-, Staurosporine-, and p53-mediated Cell Death, and Its Tyrosine 33-phosphorylated Form Binds and Stabilizes Serine 46-phosphorylated p53

Joan Doherty,Nan-Shan Chang,Lori Schultz,Li-Jin Hsu,Qunying Hong,Amy Ensign

doi:10.1074/jbc.m505590200

Abstract

WW domain-containing oxidoreductase WOX1, also named WWOX or FOR, undergoes Tyr33 phosphorylation at its first N-terminal WW domain and subsequent nuclear translocation in response to sex steroid hormones and stress stimuli. The activated WOX1 binds tumor suppressor p53, and both proteins may induce apoptosis synergistically. Functional suppression of WOX1 by antisense mRNA or a dominant negative abolishes p53-mediated apoptosis. Here, we determined that UV light, anisomycin, etoposide, and hypoxic stress rapidly induced phosphorylation of p53 at Ser46 and WOX1 at Tyr33 (phospho-WOX1) and their binding interactions in several tested cancer cells. Mapping by yeast two-hybrid analysis and co-immunoprecipitation showed that phospho-WOX1 physically interacted with Ser46-phosphorylated p53. Knockdown of WOX1 protein expression by small interfering RNA resulted in L929 fibroblast resistance to apoptosis by tumor necrosis factor, staurosporine, UV light, and ectopic p53, indicating an essential role of WOX1 in stress stimuli-induced apoptosis. Notably, UV light could not induce p53 protein expression in these WOX1 knockdown cells, although p53 mRNA levels were not reduced. Suppression of WOX1 by dominant negative WOX1 (to block Tyr33 phosphorylation) also abolished UV light-induced p53 protein expression. Time course analysis showed that the stability of ectopic wild type p53, tagged with DsRed, was decreased in WOX1 knockdown cells. Inhibition of MDM2 by nutlin-3 increased the binding of p53 and WOX1 and stability of p53. Together, our data show that WOX1 plays a critical role in conferring cellular sensitivity to apoptotic stress and that Tyr33 phosphorylation in WOX1 is essential for binding and stabilizing Ser46-phosphorylated p53.

Highlights

The human WWOX gene, which comprises nine exons encoding the WWOX/WOX1 family proteins, is located on a fragile site on the chromosome ch16q23.3–24.1 (6 –9)
Exposure of Molt-4 T cells to UV light resulted in a timedependent phosphorylation of p53 at Ser46 and WOX1 at Tyr33 and their nuclear translocation (Fig. 1A)
UV light-induced nuclear translocation of p53 and WOX1 was further confirmed by immunofluorescence microscopy (Fig. 1B)

Summary

Introduction

The human WWOX gene, which comprises nine exons encoding the WWOX/WOX1 family proteins, is located on a fragile site on the chromosome ch16q23.3–24.1 (6 –9). We determined that WOX1 plays an essential role in TNF-, UV light-, staurosporine-, and p53-mediated cell death and that Tyr33-phosphorylated WOX1 binds and regulates the stability of Ser46-phosphorylated or activated p53. Precipitating with anti-WOX1 IgG showed an increased binding of WOX1 with p53 (data not shown; or see Fig. 8 from using HCT116 cells).

Results

Conclusion