Related Experiment Video
Updated: Aug 10, 2026
Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay
Published on: June 9, 2017
Constitutive phosphorylation of ATM in lymphoblastoid cell lines from patients with ICF syndrome without downstream
Jimena V Goldstine1, Shareef Nahas, Kristin Gamo
1Department of Human Genetics, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA.
Abstract:
Double strand DNA breaks in the genome lead to the activation of the ataxia-telangiectasia mutated (ATM) kinase in a process that requires ATM autophosphorylation at serine-1981. ATM autophosphorylation only occurs if ATM is previously acetylated by Tip60. The activated ATM kinase phosphorylates proteins involved in arresting the cell cycle, including p53, and in repairing the DNA breaks. Chloroquine treatment and other manipulations that produce chromatin defects in the absence of detectable double strand breaks also trigger ATM phosphorylation and the phosphorylation of p53 in primary human fibroblasts, while other downstream substrates of ATM that are involved in the repair of DNA double strand breaks remain unphosphorylated. This raises the issue of whether ATM is constitutively activated in patients with genetic diseases that display chromatin defects. We examined lymphoblastoid cell lines (LCLs) generated from patients with different types of chromatin disorders: Immunodeficiency, Centromeric instability, Facial anomalies (ICF) syndrome, Coffin Lowry syndrome, Rubinstein Taybi syndrome and Fascioscapulohumeral Muscular Dystrophy. We show that ATM is phosphorylated on serine-1981 in LCLs derived from ICF patients but not from the other syndromes. The phosphorylated ATM in ICF cells did not phosphorylate the downstream targets NBS1, SMC1 and H2AX, all of which require the presence of double strand breaks. We demonstrate that ICF cells respond normally to ionizing radiation, ruling out the possibility that genetic deficiency in ICF cells renders activated ATM incapable of phosphorylating its downstream substrates. Surprisingly, p53 was also not phosphorylated in ICF cells or in chloroquine-treated wild type LCLs. In this regard the response to chromatin-altering agents differs between primary fibroblasts and LCLs. Our findings indicate that although phosphorylation at serine-1981 is essential in the activation of the ATM kinase, serine-1981 phosphorylation is insufficient to render ATM an active kinase towards downstream substrates, including p53.
Insights
ATM kinase activation is crucial for DNA repair. Phosphorylation at serine-1981 is necessary but insufficient for ATM kinase activity in chromatin defect disorders like ICF syndrome.
Area of Science:
- Molecular Biology
- Cellular Biology
- Genetics
Background:
- Double-strand DNA breaks activate ataxia-telangiectasia mutated (ATM) kinase via autophosphorylation at serine-1981.
- ATM activation requires prior acetylation by Tip60 and leads to cell cycle arrest and DNA repair.
- Chromatin defects, without DNA breaks, can trigger ATM and p53 phosphorylation in fibroblasts, raising questions about ATM activation in genetic chromatin disorders.
Purpose of the Study:
- To investigate ATM kinase activation and downstream substrate phosphorylation in lymphoblastoid cell lines (LCLs) from patients with various chromatin disorders.
- To determine if ATM is constitutively activated in genetic diseases characterized by chromatin defects.
- To compare the response of LCLs to chromatin-altering agents with that of primary fibroblasts.
Main Methods:
- Analysis of ATM serine-1981 phosphorylation in LCLs from patients with ICF syndrome, Coffin Lowry syndrome, Rubinstein Taybi syndrome, and Fascioscapulohumeral Muscular Dystrophy.
- Assessment of downstream ATM substrate phosphorylation (NBS1, SMC1, H2AX) in ICF patient LCLs.
- Evaluation of ICF LCL response to ionizing radiation.
- Examination of p53 phosphorylation in ICF LCLs and chloroquine-treated wild-type LCLs.
Main Results:
- ATM was phosphorylated at serine-1981 in LCLs from ICF syndrome patients, but not in LCLs from other chromatin disorder groups.
- Phosphorylated ATM in ICF cells failed to phosphorylate downstream targets NBS1, SMC1, and H2AX.
- ICF LCLs exhibited normal responses to ionizing radiation, indicating no general defect in ATM substrate phosphorylation.
- p53 was not phosphorylated in ICF LCLs or in chloroquine-treated wild-type LCLs, differing from primary fibroblast responses.
Conclusions:
- Serine-1981 phosphorylation is essential but not sufficient for ATM kinase activation towards downstream substrates, including p53.
- The response to chromatin-altering agents differs between LCLs and primary fibroblasts.
- ATM activation in ICF syndrome LCLs, indicated by serine-1981 phosphorylation, does not lead to functional kinase activity against key DNA repair substrates.
More Related Videos
11:23Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein
Published on: June 30, 2019
07:40Preparation of Peripheral Blood Mononuclear Cell Pellets and Plasma from a Single Blood Draw at Clinical Trial Sites for Biomarker Analysis
Published on: March 20, 2021
Related Concept Videos
01:02Phosphorylation
During phosphorylation, protein kinases transfer the terminal phosphate group of ATP to specific amino acid side chains of substrate proteins. Serine, threonine, and tyrosine are the most commonly...
02:54Protein Kinases and Phosphatases
Protein kinases
Many proteins in the cell are regulated by phosphorylation, the addition of a phosphate group. A family of enzymes called kinases...
01:13Actin Polymerization and Cell Motility
Actin cytoskeleton dynamics can produce pushing, pulling, and resistance forces that help the cell to migrate.
01:20The JAK-STAT Signaling Pathway
01:22PI3K/mTOR/AKT Signaling Pathway
01:16Calmodulin-dependent Signaling
The Ca2+-CaM complex does not have enzymatic activity by itself. Instead, the complex binds downstream target proteins, including membrane proteins or enzymes,...