Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 Jul 20;107(29):12877-82.
doi: 10.1073/pnas.0911828107. Epub 2010 Jul 6.

Differential control of TAp73 and DeltaNp73 protein stability by the ring finger ubiquitin ligase PIR2

Berna S Sayan  1 , Ai Li Yang, Franco Conforti, Paola Tucci, Maria Cristina Piro, Gareth J Browne, Massimiliano Agostini, Sergio Bernardini, Richard A Knight, Tak W Mak, Gerry Melino
Affiliations

Differential control of TAp73 and DeltaNp73 protein stability by the ring finger ubiquitin ligase PIR2

Berna S Sayan et al. Proc Natl Acad Sci U S A. .

Abstract

p73 is a p53-related transcription factor with fundamental roles in development and tumor suppression. Transcription from two different promoters on the p73 gene results in generation of transcriptionally active TAp73 isoforms and dominant negative DeltaNp73 isoforms with opposing pro- and anti-apoptotic functions. Therefore, the relative ratio of each isoform is an important determinant of the cell fate. Proteasomal degradation of p73 is mediated by polyubiquitination-dependent and -independent processes both of which appear, thus far, to lack selectivity for the TAp73 and DeltaNp73 isoforms. Here, we describe the characterization of another transcriptional target of TAp73; a ring finger domain ubiquitin ligase p73 Induced RING 2 protein (PIR2). Although PIR2 was initially identified a p53-induced gene (p53RFP), low abundance of PIR2 transcript in mouse embryonic fibroblasts of TAp73 KO mice compared with WT mice and comparison of PIR2 mRNA and protein levels following TAp73 or p53 overexpression substantiate TAp73 isoforms as strong inducers of PIR2. Although PIR2 expression was induced by DNA damage, its expression did not alter apoptotic response or cell cycle profile per se. However, coexpression of PIR2 with TAp73 or DeltaNp73 resulted in an increase of the TA/DeltaNp73 ratio, due to preferential degradation of DeltaNp73. Finally, PIR2 was able to relieve the inhibitory effect of DeltaNp73 on TAp73 induced apoptosis following DNA damage. These results suggest that PIR2, by being induced by TAp73 and degrading DeltaNp73, differentially regulates TAp73/DeltaNp73 stability, and, hence, it may offer a therapeutic approach to enhance the chemosensitivity of tumor cells.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
p73 induces PIR2 expression. (A) Induction of PIR2 by TAp73. TAp73-SaOS-2 tet-on cells were treated with doxycyclin (dox) and cells were collected at the indicated time points. Quantitative analysis of PIR2 induction was performed by real-time PCR. The PIR2 mRNA level was up-regulated approximately 14-fold compared with basal level following 24–48 h of TAp73 expression. Induction of PIR2 detected by semiquantitative PCR is shown in the box. (B) TAp73 transactivates PIR2 promoter. Point mutations targeting the −211/−148 region caused a marked reduction of transcriptional activity of p73 on PIR2 promoter. (C) Expression of PIR2 in TAp73 KO MEFs. TAp73 KO MEFs have reduced PIR2 transcript compared with WT counterparts. cDNA from WT and TAp73 KO MEFs were used to quantitate PIR2 mRNA level by real-time PCR. (D) TAp73 induces PIR2 protein expression. Induction of PIR2 was also evident at protein level in TAp73 inducible SaOS-2 cells. PIR2 protein was detected in TAp73 expressing cells, whereas the uninduced cells had no detectable PIR2 protein.
Fig. 2.
Fig. 2.
PIR2 expression modulates TAp73α and ΔNp73α protein levels. (A) Effect of PIR2 expression on endogenous TAp73α was analyzed by using HCT116 cells stably transfected with the plasmid harboring PIR2 cDNA under the control of a doxycylin-regulated promoter (tet-on PIR2-HCT116). PIR2 expression resulted in twofold induction of TAp73α expression. (B) TAp73α protein levels during DNA damage. Tet-on PIR2-HCT116 cells were treated with 100 μM etoposide for 24 h in the presence or absence of PIR2. Endogenous TAp73α accumulation was more evident in cells expressing PIR2. (C) ΔNp73α-SaOS-2 tet-on cells were transfected with the myc-PIR2 plasmid, and 24 h after transfection cells were induced to express ΔNp73. Cells were harvested 24 or 48 h after induction, and total cell lysate was used to assess p73 levels by Western blotting. Less ΔNp73α protein was detected in cells that express PIR2. All Western blots were subjected to densitometric analysis and results were normalized based on actin expression levels and reported in graphical form (Right).
Fig. 3.
Fig. 3.
PIR2 binds p73 and affects its stability. (A) PIR2 can bind TAp73γ and ΔNp73α efficiently. HEK293 cells were transiently transfected to express myc-PIR2 alone or in different combinations with HA-tagged TAp73α, TAp73β, TAp73γ, or ΔNp73α. Cell extracts were immunoprecipitated with anti-Myc antibody. The immune complexes were subjected to Western blot analysis with anti-HA antibody. Aliquots of total cell extracts from unprocessed cells were directly subjected to immunoblot analysis with anti-HA antibody or anti-myc antibody (input). (B) PIR2 expresion modulates TAp73γ and ΔNp73α protein levels. Coexpression of PIR2 with TAp73α, TAp73β, TAp73γ, or ΔNp73α resulted in detection of less TAp73γ or ΔNp73α protein. H1299 cells were transfected with indicated plasmids and total protein was used to assess p73 and PIR2 levels.
Fig. 4.
Fig. 4.
PIR2 induces ubiquitin mediated degradation of ΔNp73α. (A) PIR2 ubiquitinates ΔNp73. HEK293 cells were transiently cotransfected with expression plasmids for Ub-HA, Flag-TAp73α, Flag-ΔNp73α and Myc-PIR2. Myc-Itch was used as a positive control. Forty-eight hours after transfection, cells were treated with MG132 and then collected. Lysates were subjected to IP using an anti-Flag antibody. Immune complexes were revealed with anti-HA antibody. (B) MG132 blocks PIR2 mediated ΔNp73 degradation. H1299 cells were transfected as in A in the ratio of 1:4. Cells were incubated with 10 μM MG132 for 6 h before harvesting. (C) 35S pulse chase of ΔNp73: effect of PIR2 on protein half-life. H1299 cells were transfected as in B. Forty-eight hours after transfection, cells were labeled for 1 h and chased for the indicated times. Levels of ΔNp73 were evaluated at the indicated time points. Following immunoprecipitation, autoradiography was performed; ΔNp73 bands were quantified and results normalized to both incorporation at time 0 and to total immunoprecipitated HA-p73 protein. The results of three independent experiments (**P < 0.001, *P < 0.05) are represented. A representative example of a 35S pulse-chase experiment showing autoradiographic and Western blotting results for HA-tagged ΔNp73α in the absence and presence of overexpressed myc-tagged PIR2 is shown (Left).
Fig. 5.
Fig. 5.
PIR2 modulates apoptotic response. (A) Induction of PIR2 following DNA damage in MDA-MB-231 cells is p73 dependent. MDA-MB-231 cells were transfected with scrambled (scr) or short-interfering RNA (siRNA) against p73. Twenty-four hours after transfection, cells were treated with 10 μM or 20 μM etoposide (etop) or cisplatin (cpt) for 24 h. PIR2 and p73 levels were measured by Western blotting. (B) PIR2 reverts ΔNp73 inhibition on TAp73 induced death. HA-TAp73 (1 μg), ΔNp73 (2 μg), and PIR2 (8 μg) expression plasmids were transfected to HeLa cells as indicated. Thirty-six hours after transfection, the medium was changed and cells were treated with 100 μM cisplatin (9 h), 20 mJ/cm2 UV (6 h), or 20 nM staurosporine (16 h). Cells were collected and subjected to flow cytometric analysis to detect mitochondrial membrane depolarization.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Melino G, Lu X, Gasco M, Crook T, Knight RA. Functional regulation of p73 and p63: Development and cancer. Trends Biochem Sci. 2003;28:663–670. - PubMed
    1. Melino G, De Laurenzi V, Vousden KH. p73: Friend or foe in tumorigenesis. Nat Rev Cancer. 2002;2:605–615. - PubMed
    1. Ishimoto O, et al. Possible oncogenic potential of DeltaNp73: A newly identified isoform of human p73. Cancer Res. 2002;62:636–641. - PubMed
    1. Stiewe T, Theseling CC, Pützer BM. Transactivation-deficient Delta TA-p73 inhibits p53 by direct competition for DNA binding: Implications for tumorigenesis. J Biol Chem. 2002;277:14177–14185. - PubMed
    1. Pozniak CD, et al. An anti-apoptotic role for the p53 family member, p73, during developmental neuron death. Science. 2000;289:304–306. - PubMed

Publication types

MeSH terms