PARENT SESSION
9:00 AM to 11:00 AM
Wednesday, April 25, 2001
Symposium 28 Cell Cycle
Room: Auditorium
Chair: H. Lieberman, Co-Chair:
D. Bulavin, R. Muschel, C. Canman, H. Lieberman

(S28-3) The Role of ATM and Chk2 in DNA damage responses.

Canman, Christine¹, ¹

ABSTRACT-
The ATM protein kinase participates in cellular responses to ionizing radiation (IR) including the induction of p53, transient arrest of DNA replication, and rapid growth arrest in the G2 phase of the cell cycle. We are currently focusing our efforts on the cell cycle checkpoint kinase Chk2 as a direct target of ATM. We have found that ATM directly phosphorylates T68 within the SQ/TQ-rich domain of Chk2 in vitro and this site is phosphorylated in vivo in response to IR in an ATM-dependent manner. T68 phosphorylation is required for full activation of Chk2 following IR suggesting that this event contributes to ATM-dependent activation of Chk2. Chk2 has been genetically linked to p53 in humans with the recent finding that several Li-Fraumeni syndrome variants possess heterozygous germ line mutations in Chk2 (Science, 286: 2528, 1999). Two mutations located within the forkhead homology-associated (FHA) domain of Chk2 were identified suggesting functional importance for this phosphoprotein-binding domain. We analyzed the effects of LFS mutations (I157T and R145W) and an additional FHA domain mutation (N166A) known to disrupt phosphoprotein-binding function. We found that the R145W and N166A mutants were not activated by IR as assessed by in vitro kinase assays and lack of IR-induced mobility shifts after SDS-PAGE. Unexpectedly, the I157T Chk2 behaved just like wild type suggesting that this mutation produced a defect that may not detectable by our assays. Overall, these data indicate that both T68 phosphorylation and an intact FHA domain are important for activation of Chk2 by ATM in irradiated cells.

KEYWORDS: checkpoint, ATM, Chk2