The SUMO (Small Ubiquitin-like Modifier) Ligase PIAS3 Primes ATR for Checkpoint Activation

Ching-Shyi Wu,Lee Zou

doi:10.1074/jbc.m115.691170

Abstract

The maintenance of genomic stability relies on the concerted action of DNA repair and DNA damage signaling pathways. The PIAS (protein inhibitor of activated STAT) family of SUMO (small ubiquitin-like modifier) ligases has been implicated in DNA repair, but whether it plays a role in DNA damage signaling is still unclear. Here, we show that the PIAS3 SUMO ligase is important for activation of the ATR (ataxia telangiectasia and Rad3 related)-regulated DNA damage signaling pathway. PIAS3 is the only member of the PIAS family that is indispensable for ATR activation. In response to different types of DNA damage and replication stress, PIAS3 plays multiple roles in ATR activation. In cells treated with camptothecin (CPT), PIAS3 contributes to formation of DNA double-stranded breaks. In UV (ultraviolet light)- or HU (hydroxyurea)-treated cells, PIAS3 is required for efficient ATR autophosphorylation, one of the earliest events during ATR activation. Although PIAS3 is dispensable for ATRIP (ATR-interacting protein) SUMOylation and the ATR-ATRIP interaction, it is required for maintaining the basal kinase activity of ATR prior to DNA damage. In the absence of PIAS3, ATR fails to display normal kinase activity after DNA damage, which accompanies with reduced phosphorylation of ATR substrates. Together, these results suggest that PIAS3 primes ATR for checkpoint activation by sustaining its basal kinase activity, revealing a new function of the PIAS family in DNA damage signaling.

Highlights

Multiple types of post-translational modifications (PTMs),2 including protein SUMOylation, are important for regulation of the DNA damage response (DDR) from yeast to human [1, 2]
PIAS3 Is the Only PIAS SUMO Ligase Indispensable for ATR Activation—While members of the PIAS family of SUMO ligases have been implicated in the DDR, whether and how they contribute to DNA damage signaling is still unclear
The effects of PIAS3 knockdown on Chk1 activation were seen in HeLa cells, and in U2OS cells (Fig. 2A). These results suggest that PIAS3 is the only PIAS family member that plays a non-redundant role in ATR activation, they do not exclude the possibility that other PIAS ligases function redundantly in this pathway

Summary

Introduction

Multiple types of post-translational modifications (PTMs),2 including protein SUMOylation, are important for regulation of the DNA damage response (DDR) from yeast to human [1, 2]. We found that loss of PIAS3 reduces the basal kinase activity of ATR prior to DNA damage, suggesting that PIAS3 primes ATR for the DDR. This question, we used siRNAs to knock down all 4 members of the PIAS family in HeLa cells and analyzed the effects on the CPT-induced, ATR-mediated Chk1 phosphorylation at Ser317.

Results

Conclusion