TY - JOUR
T1 - Ca2+-Stimulated AMPK-Dependent Phosphorylation of Exo1 Protects Stressed Replication Forks from Aberrant Resection
AU - Li, Shan
AU - Lavagnino, Zeno
AU - Lemacon, Delphine
AU - Kong, Lingzhen
AU - Ustione, Alessandro
AU - Ng, Xuewen
AU - Zhang, Yuanya
AU - Wang, Yingchun
AU - Zheng, Bin
AU - Piwnica-Worms, Helen
AU - Vindigni, Alessandro
AU - Piston, David W.
AU - You, Zhongsheng
N1 - Funding Information:
We thank Qin Liu for the gift of the GCaMP6s reporter and Abigael Cheruiyot and Sharad Paudyal for technical support and critical discussions. This work was supported by NIH (GM098535 to Z.Y. DK098659 to D.W.P. GM108648 to A.V. CA166717 to B.Z.), an American Cancer Society Research Scholar Grant (RSG-13-212-01-DMC to Z.Y.), a Siteman Investment Program grant from the Siteman Cancer Center of Washington University (4036 to Z.Y.), a DOD BRCP Breakthrough Award (BC151728 to A.V.), and a Juvenile Diabetes Research Foundation Ltd Postdoctoral Fellowship (3-APF-2018-573-A-N to Z.L.), Z.Y. conceived and supervised the study. S.L. performed most of the experiments with contributions from L.K. Z.L. X.N. A.U. and S.L. performed the FRET experiment and analyzed the results in Figure 5C with supervision from D.P. and Z.Y. D.L. and S.L. performed the DNA fiber experiments and analyzed the results in Figures 1C, 1I, 3C, and 4C with supervision from A.V. and Z.Y. Y.Z. and Y.W. assisted in the identification of phosphorylation sites in Exo1. H.P.-W. D.P. A.U. and B.Z. provided key reagents and critical discussions on the project. S.L. and Z.Y. analyzed all the data and wrote the manuscript. All authors read and approved the manuscript. The authors declare no competing financial interests.
Funding Information:
We thank Qin Liu for the gift of the GCaMP6s reporter and Abigael Cheruiyot and Sharad Paudyal for technical support and critical discussions. This work was supported by NIH ( GM098535 to Z.Y., DK098659 to D.W.P., GM108648 to A.V., CA166717 to B.Z.), an American Cancer Society Research Scholar Grant ( RSG-13-212-01-DMC to Z.Y.), a Siteman Investment Program grant from the Siteman Cancer Center of Washington University ( 4036 to Z.Y.), a DOD BRCP Breakthrough Award ( BC151728 to A.V.), and a Juvenile Diabetes Research Foundation Ltd Postdoctoral Fellowship ( 3-APF-2018-573-A-N to Z.L.)
Publisher Copyright:
© 2019 Elsevier Inc.
PY - 2019/6/20
Y1 - 2019/6/20
N2 - Abnormal processing of stressed replication forks by nucleases can cause fork collapse, genomic instability, and cell death. Despite its importance, it is poorly understood how the cell properly controls nucleases to prevent detrimental fork processing. Here, we report a signaling pathway that controls the activity of exonuclease Exo1 to prevent aberrant fork resection during replication stress. Our results indicate that replication stress elevates intracellular Ca2+ concentration ([Ca2+]i), leading to activation of CaMKK2 and the downstream kinase 5′ AMP-activated protein kinase (AMPK). Following activation, AMPK directly phosphorylates Exo1 at serine 746 to promote 14-3-3 binding and inhibit Exo1 recruitment to stressed replication forks, thereby avoiding unscheduled fork resection. Disruption of this signaling pathway results in excessive ssDNA, chromosomal instability, and hypersensitivity to replication stress inducers. These findings reveal a link between [Ca2+]i and the replication stress response as well as a function of the Ca2+-CaMKK2-AMPK signaling axis in safeguarding fork structure to maintain genome stability.
AB - Abnormal processing of stressed replication forks by nucleases can cause fork collapse, genomic instability, and cell death. Despite its importance, it is poorly understood how the cell properly controls nucleases to prevent detrimental fork processing. Here, we report a signaling pathway that controls the activity of exonuclease Exo1 to prevent aberrant fork resection during replication stress. Our results indicate that replication stress elevates intracellular Ca2+ concentration ([Ca2+]i), leading to activation of CaMKK2 and the downstream kinase 5′ AMP-activated protein kinase (AMPK). Following activation, AMPK directly phosphorylates Exo1 at serine 746 to promote 14-3-3 binding and inhibit Exo1 recruitment to stressed replication forks, thereby avoiding unscheduled fork resection. Disruption of this signaling pathway results in excessive ssDNA, chromosomal instability, and hypersensitivity to replication stress inducers. These findings reveal a link between [Ca2+]i and the replication stress response as well as a function of the Ca2+-CaMKK2-AMPK signaling axis in safeguarding fork structure to maintain genome stability.
KW - 14-3-3
KW - AMPK
KW - Ca
KW - CaMKK2
KW - Exo1
KW - calcium signaling
KW - genome maintenance
KW - protein phosphorylation
KW - replication fork resection
KW - replication stress response
UR - http://www.scopus.com/inward/record.url?scp=85067078083&partnerID=8YFLogxK
U2 - 10.1016/j.molcel.2019.04.003
DO - 10.1016/j.molcel.2019.04.003
M3 - Article
C2 - 31053472
AN - SCOPUS:85067078083
SN - 1097-2765
VL - 74
SP - 1123-1137.e6
JO - Molecular Cell
JF - Molecular Cell
IS - 6
ER -