PICT-1 is a key nucleolar sensor in DNA damage response signaling that regulates apoptosis through the RPL11-MDM2-p53 pathway.

Hongbo Chen,Wei Cao,Ning Xu,Hsiangi Tsai,Junhua Zhuang,Yanhong Duo,Xianzhang Huang,Yanping Wu,Lijun Chen,Zhiwei Wang,Zhirong Tan,Liqiao Han,Laiqiang Huang

doi:10.18632/oncotarget.13082

Abstract

PICT-1 is an essential ribosome biogenesis factor whose loss induces p53 accumulation and apoptosis. Here, we show that DNA damage changes PICT-1 localization and decreases PICT-1 protein levels via the proteasome pathway. Two important phosphatidylinositol 3-kinase-like kinases (PIKKs), ataxia-telangiectasia mutated (ATM) and the Ku70 subunit of DNA-dependent protein kinase (DNA-PK), co-localize and interact with PICT-1 in the nucleolus. Computational prediction of phosphorylation sites and detection using an anti-phospho-substrate antibody suggest that PICT-1 might be a substrate of PIKKs. PICT-1 S233 and T289 were identified as the key phosphorylation sites in this pathway, as mutating both to alanine abolished UVB-induced increase of PICT-1 phosporylation. Inhibition of PIKKs or ATM (with wortmannin and KU55933, respectively) prevented the agglomeration and degradation of PICT-1, suggesting that ATM is a key regulator of PICT-1. PICT-1(S233A, T289A) demonstrated marked resistance to DNA damage-induced agglomeration and loss of PICT-1. Phosphomimetic PICT-1 (S233D, T289D) showed a different nuclear distribution and was more rapidly degraded after DNA damage than wild-type PICT-1. Furthermore, both phosphorylation and degradation of PICT-1 released RPL11 from the nucleolus to the nucleoplasm, increased binding of RPL11 to MDM2, and promoted p53 accumulation and apoptosis in an ATM-dependent manner after DNA damage. These data indicate that PICT-1 is a major nucleolar sensor of the DNA damage repair response and an important upstream regulator of p53 via the RPL11-MDM2-p53 pathway.

Highlights

The protein interacting with carboxyl terminus 1 (PICT-1) is encoded by glioma tumor suppressor candidate region gene 2 (GLTSCR2), located at human chromosome 19q13.32 [1, 2]
To evaluate overall levels of PICT-1, HEK293 were exposed to UVB light or Mitomycin C (MMC), and immunoblot analysis was performed on cell lysates at different post-treatment time points
PICT-1 phosphorylation was significantly lower in cells transfected with mutant than in wild-type PICT-1 1 h after UVB radiation (Figure 3B). These findings demonstrate that PICT-1 S233 and T289 are phosphorylated by phosphatidylinositol 3-kinase-like kinases (PIKKs) in response to DNA damage

Summary

Introduction

The protein interacting with carboxyl terminus 1 (PICT-1) is encoded by glioma tumor suppressor candidate region gene 2 (GLTSCR2), located at human chromosome 19q13.32 [1, 2]. PICT-1 is frequently lost in gliomas and was identified as a candidate tumor suppressor [2]. In support of this role, PICT-1 expression is negatively correlated with the development and progression of ovarian cancer and glioblastomas [3,4,5,6]. PICT-1 achieves this function by directly interacting with and stabilizing the tumor suppressor phosphatase and tensin homolog protein (PTEN) in the cytoplasm, thereby inhibiting the PI3K/ PIP3 pathway [7, 8]. Many studies support a role for PICT-1 as a tumor suppressor, some contradictory findings have been reported. Further studies are needed to elucidate the function of PICT-1 in normal and cancerous cells

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Results

Conclusion