A Conserved Proliferating Cell Nuclear Antigen-interacting Protein Sequence in Chk1 Is Required for Checkpoint Function

Jennifer Scorah,Meng-Qiu Dong,John R Yates,Mary Scott,David Gillespie,Clare H Mcgowan

doi:10.1074/jbc.m800369200

Abstract

Human checkpoint kinase 1 (Chk1) is an essential kinase required for cell cycle checkpoints and for coordination of DNA synthesis. To gain insight into the mechanisms by which Chk1 carries out these functions, we used mass spectrometry to identify previously uncharacterized interacting partners of Chk1. We describe a novel interaction between Chk1 and proliferating cell nuclear antigen (PCNA), an essential component of the replication machinery. Binding between Chk1 and PCNA was reduced in the presence of hydroxyurea, suggesting that the interaction is regulated by replication stress. A highly conserved PCNA-interacting protein (PIP) box motif was identified in Chk1. The intact PIP box is required for efficient DNA damage-induced phosphorylation and release of activated Chk1 from chromatin. We find that the PIP box of Chk1 is crucial for Chk1-mediated S-M and G(2)-M checkpoint responses. In addition, we show that mutations in the PIP box of Chk1 lead to decreased rates of replication fork progression and increased aberrant replication. These findings suggest an additional mechanism by which essential components of the DNA replication machinery interact with the replication checkpoint apparatus.

Highlights

The checkpoint kinase 1 (Chk1) kinase is required for the intra-S, S-M and G2-M checkpoint responses in vertebrate cells [1,2,3,4,5,6,7,8]
We discovered a conserved proliferating cell nuclear antigen (PCNA)-interacting protein (PIP) box in the carboxyl terminus of Chk1 that is required for efficient DNA damage-induced phosphorylation and chromatin release of Chk1
Chk1 and PCNA Interact in Vivo—To identify novel interacting partners of Chk1, HeLa cells were transfected with a plasmid encoding FLAG-tagged Chk1, and associated proteins were immunopurified using FLAG M2 affinity gel

Summary

Introduction

The Chk1 kinase is required for the intra-S, S-M and G2-M checkpoint responses in vertebrate cells [1,2,3,4,5,6,7,8]. The intact PIP box is required for efficient DNA damageinduced phosphorylation and release of activated Chk1 from chromatin. The replication factor C-related checkpoint protein Rad17 is required to load the 9-1-1 complex onto chromatin, and it interacts with Claspin to promote the phosphorylation of Chk1 [30, 35, 36].

Results

Conclusion