HCLK2 is required for activity of the DNA damage response kinase ATR

TY - JOUR

T1 - HCLK2 is required for activity of the DNA damage response kinase ATR

AU - Rendtlew Danielsen, Jannie M

AU - Larsen, Dorthe Helena

AU - Schou, Kenneth Bødtker

AU - Freire, Raimundo

AU - Falck, Jacob

AU - Bartek, Jiri

AU - Lukas, Jiri

N1 - Keywords: Carrier Proteins; Cell Cycle Proteins; Chromosomal Proteins, Non-Histone; DNA Damage; DNA-Binding Proteins; Enzyme Induction; Enzyme Stability; Hela Cells; Humans; Nuclear Proteins; Protein Kinases; Protein-Serine-Threonine Kinases; Protein-Tyrosine Kinases

PY - 2008

Y1 - 2008

N2 - ATR is a protein kinase that orchestrates the cellular response to replication problems and DNA damage. HCLK2 has previously been reported to stabilize ATR and Chk1. Here we provide evidence that human HCLK2 acts at an early step in the ATR signaling pathway and contributes to full-scale activation of ATR kinase activity. We show that HCLK2 forms a complex with ATR-ATRIP and the ATR activator TopBP1. We demonstrate that HCLK2-induced ATR kinase activity toward substrates requires TopBP1 and vice versa and provides evidence that HCLK2 facilitates efficient ATR-TopBP1 association. Consistent with its role in ATR activation, HCLK2 depletion severely impaired phosphorylation of multiple ATR targets including Chk1, Nbs1, and Smc1 after DNA damage. We show that HCLK2 is required for and stimulates ATR autophosphorylation and activity toward different substrates in vitro. Furthermore, HCLK2 depletion abrogated the G(2) checkpoint and decreased survival of cells after exposure to DNA damaging agents and replicative stress. Overall, our data suggest that HCLK2 facilitates ATR activation and, therefore, contributes to ATR-mediated checkpoint signaling. Importantly, our results suggest that HCLK2 functions in the same pathway as TopBP1 but that the two proteins regulate different steps in ATR activation.

AB - ATR is a protein kinase that orchestrates the cellular response to replication problems and DNA damage. HCLK2 has previously been reported to stabilize ATR and Chk1. Here we provide evidence that human HCLK2 acts at an early step in the ATR signaling pathway and contributes to full-scale activation of ATR kinase activity. We show that HCLK2 forms a complex with ATR-ATRIP and the ATR activator TopBP1. We demonstrate that HCLK2-induced ATR kinase activity toward substrates requires TopBP1 and vice versa and provides evidence that HCLK2 facilitates efficient ATR-TopBP1 association. Consistent with its role in ATR activation, HCLK2 depletion severely impaired phosphorylation of multiple ATR targets including Chk1, Nbs1, and Smc1 after DNA damage. We show that HCLK2 is required for and stimulates ATR autophosphorylation and activity toward different substrates in vitro. Furthermore, HCLK2 depletion abrogated the G(2) checkpoint and decreased survival of cells after exposure to DNA damaging agents and replicative stress. Overall, our data suggest that HCLK2 facilitates ATR activation and, therefore, contributes to ATR-mediated checkpoint signaling. Importantly, our results suggest that HCLK2 functions in the same pathway as TopBP1 but that the two proteins regulate different steps in ATR activation.

U2 - 10.1074/jbc.M808174200

DO - 10.1074/jbc.M808174200

M3 - Journal article

C2 - 19097996

SN - 0021-9258

VL - 284

SP - 4140

EP - 4147

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 7

ER -