The Yes-associated protein 1 stabilizes p73 by preventing Itch-mediated ubiquitination of p73

Abstract

Upon DNA damage signaling, p73, a member of the p53 tumor suppressor family, accumulates to support transcription of downstream apoptotic genes. p73 interacts with Yes-associated protein 1 (Yap1) through its PPPY motif, and increases p73 transactivation of apoptotic genes. The ubiquitin E3 ligase Itch, like Yap1, interacts with p73. Given the fact that both Itch and Yap1 bind p73 via the PPPY motif, we hypothesized that Yap may also function to stabilize p73 by displacing Itch binding to p73. We show that the interaction of Yap1 and p73 was necessary for p73 stabilization. Yap1 competed with Itch for binding to p73, and prevented Itch-mediated ubiquitination of p73. Treatment of cells with cisplatin leads to an increase in p73 accumulation and induction of apoptosis, but both were dramatically reduced in the presence of Yap1 siRNA. Altogether, our findings attribute a central role to Yap1 in regulating p73 accumulation and function under DNA damage signaling.

c-Abl antagonizes the YAP oncogenic function

“…13,24 In addition, the phosphorylation of YAP by c-Abl preferentially promotes its coactivation of pro-apoptotic genes by p73, as opposed to coactivation of Runx on non-apoptotic target genes, such as osteocalcin or Itch. 26 However, the question of how c-Abl-mediated YAP phosphorylation regulates TEAD coactivation remained open. To this end, we first asked whether DNA damage affects YAP-TEAD transcriptional output.…”

“…All transfections were done by the calcium phosphate method as previously described. 26 Total RNA was extracted using the Perfect Pure RNA cultured cell kit (5 PRIME, Gaithersburg, MD, USA) and then reverse transcribed by iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA). Quantitative RT-PCR was performed with SYBR Green PCR Master Mix (Kapa Biosystems, Woburn, MA, USA) using the LightCycler 480 Instrument (Roche Diagnostics, Basel, Switzerland).…”

“…Immunoblots and immunoprecipitations (IPs) were done as previously described. 26 The antibodies used were: anti-HA, monoclonal anti-β-actin, anti-Flag M2, and anti-Flag M5 (Sigma-Aldrich); anti c-Abl K12 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); and anti-E-cadherin (BD Biosciences, Franklin Lakes, NJ, USA). For IP of Flagtagged proteins, anti-Flag M2 agarose beads (Sigma-Aldrich) were used.…”

The Hippo pathway kinase Lats2 prevents DNA damage-induced apoptosis through inhibition of the tyrosine kinase c-Abl

“…Flow cytometry analyses were done as previously described. 55 For flow cytometry analysis, at least 20 000 cells were collected for each sample.…”

“…All transfections were done by the calcium phosphate method as described. 55 Total RNA was extracted using the TRI-Reagent solution (Molecular Research Center, Cincinnati, OH, USA), DNase treated using the RNAse-free DNAse set (Qiagen, Hilden, Germany) and then reverse-transcribed by iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA). Quantitative RT-PCR was performed with SYBR Green PCR Master Mix (Kapa Biosystems, Woburn, MA, USA) using the LightCycler 480 Instrument (Roche Diagnostics, Basel, Switzerland).…”