Mechanisms and consequences of ATM activation

Mand, Michael Rodgers
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Mutations in the ataxia telangiectasia mutated (ATM) gene cause the disease ataxia-telangiectasia (A-T). Patients with this disease have multiple symptoms, including the eponymous ataxia and telangiectasia as well as immunodeficiency, radiation sensitivity, and increased cancer rates. The ATM protein is a kinase and is activated by multiple types of stress to affect many cellular processes. At sites of DNA double-strand breaks (DSBs), ATM is activated by the protein complex Mre11/Rad50/Nbs1 (MRN). As part of this complex, Rad50 binds and hydrolyzes ATP and causes large conformational changes in the complex. However, the importance of this enzymatic activity in the activation of ATM has been unknown. Here I show ATP binding by Rad50 is required for ATM activation while ATP hydrolysis is dispensable. ATM is also activated in the presence of oxidative stress. Separation-of-function mutations for the activation of ATM by DSBs and oxidative stress have been characterized in vitro. Here, the effects of expressing wild-type ATM or ATM with these different separation-of-function mutations in an ATM-deficient lymphoblast cell line have been characterized. Analysis of the proteomes of these cells and a control cell line revealed that non-functional ATM resulted in the loss of a large group of proteins by mass spectrometry. The levels of these proteins were similar in the cells, but in the presence of non-functional ATM they showed increased levels of aggregation. Thus my results suggest ATM may function to prevent aggregation in these conditions. Notably neurodegeneration is often associated with aggregation. In the phosphoproteomes of cells expressing the various ATM constructs, the parental cell line and cells with ATM unable to be activated by oxidative stress had lower levels of phosphopeptides predicted to be phosphorylated by CK2. This decrease in CK2 activity was also associated with increased aggregation, specifically a subunit of CK2 known as CK2β. This work provides insights into the mechanism of ATM activation by MRN and the potential involvement of ATM in the prevention of protein aggregation.