TAp73 regulates the spindle assembly checkpoint by modulating BubR1 activity

Abstract

The role of various p73 isoforms in tumorigenesis has been controversial. However, as we have recently shown, the generation of TAp73-deficient (TAp73(-/-)) mice reveals that TAp73 isoforms exert tumor-suppressive functions, indicating an emerging role for Trp-73 in the maintenance of genomic stability. Unlike mice lacking all p73 isoforms, TAp73(-/-) mice show a high incidence of spontaneous tumors. Moreover, TAp73(-/-) mice are infertile and produce oocytes exhibiting spindle abnormalities. These data suggest a link between TAp73 activities and the common molecular machinery underlying meiosis and mitosis. Previous studies have indicated that the spindle assembly checkpoint (SAC) complex, whose activation leads to mitotic arrest, also regulates meiosis. In this study, we demonstrate in murine and human cells that TAp73 is able to interact directly with several partners of the SAC complex (Bub1, Bub3, and BubR1). We also show that TAp73 is involved in SAC protein localization and activities. Moreover, we show that decreased TAp73 expression correlates with increases of SAC protein expression in patients with lung cancer. Our results establish TAp73 as a regulator of SAC responses and indicate that TAp73 loss can lead to mitotic arrest defects. Our data suggest that SAC impairment in the absence of functional TAp73 could explain the genomic instability and increased aneuploidy observed in TAp73-deficient cells.

Fig. 1.

TAp73 deficiency causes mis-localization of SAC components. (A and B) Mis-localization of Bub1 in ovulated oocytes (A) and 3 h after initiation of in vitro maturation (B). Blue staining (arrows), chromosomes; green staining (arrowheads), Bub1 staining; red staining, tubulin. (Magnification: 100 × 10.) (C) Decreased expression of BubR1. Ovulated oocytes from TAp73^+/+ and TAp73^−/− mice were immunostained for tubulin (green), DNA (blue), and BubR1 (red). (Magnification: 40 × 10; Insets, 100 × 10.) Arrows indicate specific localization of BubR1. (D) Increased total BubR1 protein. Total BubR1 and tubulin protein levels in TAp73^+/+ and TAp73^−/− MII oocytes were determined by using immunostaining and deconvolution imaging software. Results shown are mean ± SE for the indicated number (n) of oocytes per group (**P < 0.001, Student t test). (E) Decreased BubR1 localization at the metaphase plate area. The DNA concentration/BubR1 protein ratio in TAp73^+/+ and TAp73^−/− ovulated oocytes was determined by using immunostaining and deconvolution imaging software. Results shown are the mean DNA/BubR1 ratio ± SE for the indicated number (n) of oocytes per group (**P < 0.001, Student t test). (F) Mis-localization of BubR1. HeLa cells treated for 48 h with control siRNA (siCt) or TAp73 siRNA (siTAp73) were exposed for 8 h to 0.6 μM nocodazole and stained with anti-BubR1 and DAPI. Arrows indicate BubR1 staining; arrowheads indicate co-localization (siCt) and non-co-localization (siTAp73) of BubR1 with chromosomes. (Magnification: 63 × 10.)

Fig. 2.

Interaction of TAp73 with SAC components. (A) Only TAp73 (not ΔNp73) interacts with BubR1 in vivo. Human cancer cell lines MDA-MB-231 and SW480 were treated (+) or not treated (-) with nocodazole for 16 h and extracts were immunoprecipitated with BubR1 and blotted with either TAp73-specific antibody (Left, Center), or antibody recognizing both TAp73 and ΔNp73 (Right). (B) In vitro interaction of TAp73 with Bub1 determined as in A. OD, oligomerization domain.

Fig. 3.

TAp73 binding modifies BubR1 activity. TAp73 depletion decreases BubR1 activity. TAp73^+/+ and TAp73^−/− MEFs (A) or HeLa cells treated for 48 h with TAp73 siRNA (B) were treated with nocodazole, and extracts were immunoprecipitated with anti-p55/cdc20 and blotted with anti-BubR1 or anti-Mad2. (C) Increase in BubR1 activity induced by TAp73 overexpression. HeLa cells overexpressing TAp73 or ΔNp73 isoforms were treated and immunoprecipitated as in A and B. (D) Decreased TAp73 correlates with reduced SAC activity. Levels of phospho-BubR1 (P) and phospho-histone H3 (p-histh3) (representing SAC activity) were determined by Western blotting of HeLa cells treated with either control (siC) or TAp73 siRNA and subjected to nocodazole for the indicated number of hours. (E) TAp73 potentiates BubR1 kinase activity. HeLa cells overexpressing control, TAp73α, TAp73β, ΔNp73β, or p53 DNA were assayed for BubR1 kinase activity using histone-H1 as the substrate.

Fig. 4.

Correlation of TAp73, ΔNp73, and BubR1 expression in human lung cancer. Correlation of expression levels of TAp73, ΔNp73, and hBubR1 in human lung cancers. (Top) Orange bars indicate that the expression of the indicated mRNA was decreased in the tumor sample compared with the paired normal tissue. Green bars indicate that the expression of the indicated mRNA was increased in the tumor sample compared with the paired normal tissue. Pearson R, P = 0.001.