DNA damage causes rapid accumulation of phosphoinositides for ATR\xa0signaling

Yu-Hsiu Wang,Yusuke Toyama,Anushya Hariharan,Marco Foiani,G V Shivashankar,Giulia Bastianello,Michael P Sheetz

doi:10.1038/s41467-017-01805-9

Abstract

Phosphoinositide lipids (PPIs) are enriched in the nucleus and are accumulated at DNA damage sites. Here, we investigate roles of nuclear PPIs in DNA damage response by sequestering specific PPIs with the expression of nuclear-targeted PH domains, which inhibits recruitment of Ataxia telangiectasia and Rad3-related protein (ATR) and reduces activation of Chk1. PPI-binding domains rapidly (< 1 s) accumulate at damage sites with local enrichment of PPIs. Accumulation of PIP3 in complex with the nuclear receptor protein, SF1, at damage sites requires phosphorylation by inositol polyphosphate multikinase (IPMK) and promotes nuclear actin assembly that is required for ATR recruitment. Suppressed ATR recruitment/activation is confirmed with latrunculin A and wortmannin treatment as well as IPMK or SF1 depletion. Other DNA repair pathways involving ATM and DNA-PKcs are unaffected by PPI sequestration. Together, these findings reveal that nuclear PPI metabolism mediates an early damage response through the IPMK-dependent pathway to specifically recruit ATR.

Highlights

Phosphoinositide lipids (PPIs) are enriched in the nucleus and are accumulated at DNA damage sites
Evidence that a distinct pool of phosphoinositide exists in the nucleus was established by Smith and Wells[5,6], who demonstrated that phosphatidic acid (PA), phosphatidylinositol 4-phosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) were rapidly labeled by 32P when intact rat liver nuclei were isolated and incubated with [γ 32P]-Adenosine triphosphate (ATP)
In order to understand the role of PPIs in the DNA damage response, we overexpressed pleckstrin homology (PH) domains with triple nuclear localization signals (3xNLS) to complex nuclear PPIs and block normal PPI functions

Summary

Introduction

Phosphoinositide lipids (PPIs) are enriched in the nucleus and are accumulated at DNA damage sites. Other DNA repair pathways involving ATM and DNA-PKcs are unaffected by PPI sequestration Together, these findings reveal that nuclear PPI metabolism mediates an early damage response through the IPMK-dependent pathway to recruit ATR. Evidence that a distinct pool of phosphoinositide exists in the nucleus was established by Smith and Wells[5,6], who demonstrated that phosphatidic acid (PA), phosphatidylinositol 4-phosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) were rapidly labeled by 32P when intact rat liver nuclei were isolated and incubated with [γ 32P]-ATP This finding indicates that PPI can be synthesized in the nucleus in the absence of cytosolic PPI kinases. Cisplatin-induced DNA damage causes an increase in the nuclear PIP2 level through type I PI4P-5K activation, the PI4P-5K isoform and the mechanism involved remains unclear[10] Together, these early observations imply that nuclear phosphoinositides have a role in a. A similar strategy was used to investigate the role of PPIs in membrane-cytoskeleton adhesion[17], these nuclear-targeted domains enable the analysis of the roles of nuclear PPIs, in DNA damage response

Methods

Results

Conclusion