ATM's leucine-rich domain and adjacent sequences are essential for ATM to regulate the DNA damage response

Abstract

The ATM protein kinase regulates the DNA damage response by phosphorylating proteins involved in cell cycle checkpoints and DNA repair. We report here on the function of the predicted leucine zipper (LZ) motif, and sequences adjacent to this, in regulating ATM activity. The predicted LZ sequence was deleted from ATM, generating ATMDLZ, and expressed in an ATM-negative AT cell line. ATM increased cell survival following exposure to ionizing radiation, whereas expression of ATMDLZ failed to increase cell survival. ATMDLZ retained in vitro kinase activity, but was unable to phosphorylate p53 in vivo. Leucine zippers mediate homoand heterodimerization of proteins. However, the predicted LZ of ATM did not mediate the formation of ATM dimers. We examined if the predicted LZ of ATM was a dominant-negative inhibitor of ATM function in SW480 cells. Expression of amino acids 769-1436 of ATM, including the predicted LZ, sensitized SW480 cells to ionizing radiation, but did not inhibit ATM's kinase activity or its ability to phosphorylate Brca1. Further, this dominant-negative activity was not dependent on the predicted LZ domain. The central region of the ATM protein therefore contains multiple sequences which regulate cell survival following DNA damage.

Activation of the Kinase Activity of ATM by Retinoic Acid Is Required for CREB-dependent Differentiation of Neuroblastoma Cells

“…The activity of ATM kinase was increased 2-fold by the radiomimetic agent bleomycin. This 2-fold increase of the kinase activity of ATM in response to DNA damage is similar to that previously reported (14,35). When cells were incubated with RA for 20 min, the kinase activity of ATM was increased to the same levels seen following DNA damage (Fig.…”

Involvement of Human MOF in ATM Function

“…3D). Such results are consistent with the fact that ATM⌬LZ retains kinase activity in vitro; however, it is unable to phosphorylate its substrates in vivo (10). IR enhances hMOF-mediated acetylation of histone H4 at K16.…”

“…Mutant forms of hMOF were created by using a PCR approach with appropriate primer pair combinations. ATM⌬LZ was generated as described previously (10). The wild or mutant forms tagged to GFP, HA, or Flag were cloned into the pIND(SP1)/Neo vector.…”

“…The Gal4 DNA-binding domain (DBD) was fused to bait (hMOF, hMOF fragments, or ATM fragments), and the activation domain (AD) of Gal4 was fused to prey (ATM fragments, hMOF, or hMOF fragments). DNA-binding domain 4, which lacks amino acids 1217 to 1239, was generated as described previously (10). (C) Plasmids in pairs were introduced into Saccharomyces cerevisiae HF7c cells, and ␤-gal activity was quantified by using an O-nitrophenyl ␤-D-galactoside assay.…”

Relation between genetic variants of the ataxia telangiectasia-mutated (ATM) gene, drug resistance, clinical outcome and predisposition to childhood T-lineage acute lymphoblastic leukaemia

“…[11][12][13] A predicted leucine zipper motif between amino acids 1217 and 1239 indicates that ATM is directly involved in DNA binding. 14 Cells that lack functional ATM exhibit a defect in cell cycle checkpoint control and an impaired DNA repair pathway. 15 Mutations that affect the kinase domain of ATM cause ataxia telangiectasia (AT), a degenerative neurological disorder which is characterized by immunodeficiency, genetic instability, radiosensitivity and an increased risk of developing haematological malignancies, particularly of T-cell origin.…”