TopBP1 activates ATR through ATRIP and a PIKK regulatory domain.
The ATR (ATM and Rad3-related) kinase and its regulatory partner ATRIP (ATR-interacting protein) coordinate checkpoint responses to DNA damage and replication stress. TopBP1 functions as a general activator of ATR. However, the mechanism by which TopBP1 activates ATR is unknown. Here, we show that ATRIP contains a TopBP1-interacting region that is necessary for the association of TopBP1 and ATR, for TopBP1-mediated activation of ATR, and for cells to survive and recover DNA synthesis following replication stress. We demonstrate that this region is functionally conserved in the Saccharomyces cerevisiae ATRIP ortholog Ddc2, suggesting a conserved mechanism of regulation. In addition, we identify a domain of ATR that is critical for its activation by TopBP1. Mutations of the ATR PRD (PIKK [phosphoinositide 3-kinase related kinase] Regulatory Domain) do not affect the basal kinase activity of ATR but prevent its activation. Cellular complementation experiments demonstrate that TopBP1-mediated ATR activation is required for checkpoint signaling and cellular viability. The PRDs of ATM and mTOR (mammalian target of rapamycin) were shown previously to regulate the activities of these kinases, and our data indicate that the DNA-PKcs (DNA-dependent protein kinase catalytic subunit) PRD is important for DNA-PKcs regulation. Therefore, divergent amino acid sequences within the PRD and a unique protein partner allow each of these PIK kinases to respond to distinct cellular events.
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Daniel A
DA
Mordes
Gloria G
GG
Glick
Runxiang
R
Zhao
David
D
Cortez
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D020816:Amino Acid Motifs; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D002470:Cell Survival; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism*; D011485:Protein Binding; D012045:Regulatory Sequences, Nucleic Acid; D012441:Saccharomyces cerevisiae; D015398:Signal Transduction; D014162:Transfection
C521194:ATRIP protein, human; D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C110513:TOPBP1 protein, human