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Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamAΔ41
Published on: March 25, 2010
Purification and characterization of ATM from human placenta. A manganese-dependent, wortmannin-sensitive
1Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada.
Abstract:
ATM is mutated in the human genetic disorder ataxia telangiectasia, which is characterized by ataxia, immune defects, and cancer predisposition. Cells that lack ATM exhibit delayed up-regulation of p53 in response to ionizing radiation. Serine 15 of p53 is phosphorylated in vivo in response to ionizing radiation, and antibodies to ATM immunoprecipitate a protein kinase activity that, in the presence of manganese, phosphorylates p53 at serine 15. Immunoprecipitates of ATM also phosphorylate PHAS-I in a manganese-dependent manner. Here we have purified ATM from human cells using nine chromatographic steps. Highly purified ATM phosphorylated PHAS-I, the 32-kDa subunit of RPA, serine 15 of p53, and Chk2 in vitro. The majority of the ATM phosphorylation sites in Chk2 were located in the amino-terminal 57 amino acids. In each case, phosphorylation was strictly dependent on manganese. ATM protein kinase activity was inhibited by wortmannin with an IC(50) of approximately 100 nM. Phosphorylation of RPA, but not p53, Chk2, or PHAS-I, was stimulated by DNA. The related protein, DNA-dependent protein kinase catalytic subunit, also phosphorylated PHAS-I, RPA, and Chk2 in the presence of manganese, suggesting that the requirement for manganese is a characteristic of this class of enzyme.
Insights
The ATM protein kinase phosphorylates key proteins like p53 and Chk2, crucial for DNA damage response. This ATM kinase activity, dependent on manganese, is vital for cellular repair mechanisms following radiation exposure.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Ataxia telangiectasia is a human genetic disorder linked to ATM mutations, causing immune defects and cancer predisposition.
- ATM-deficient cells show delayed p53 upregulation after ionizing radiation.
- p53 phosphorylation at serine 15 occurs in vivo following ionizing radiation.
Purpose of the Study:
- To purify and characterize the protein kinase activity of ATM.
- To identify substrates phosphorylated by ATM.
- To investigate the enzymatic properties and regulation of ATM.
Main Methods:
- Purification of ATM from human cells using nine chromatographic steps.
- In vitro kinase assays using purified ATM.
- Analysis of ATM phosphorylation sites on Chk2.
- Inhibition studies using wortmannin.
Main Results:
- Highly purified ATM phosphorylates PHAS-I, RPA, p53 (at serine 15), and Chk2 in vitro.
- ATM kinase activity is strictly dependent on manganese.
- Wortmannin inhibits ATM kinase activity with an IC50 of approximately 100 nM.
- DNA stimulates RPA phosphorylation by ATM, but not p53, Chk2, or PHAS-I.
Conclusions:
- ATM functions as a protein kinase that phosphorylates multiple substrates involved in DNA damage response.
- Manganese is essential for ATM kinase activity.
- ATM's substrate specificity and regulation provide insights into DNA repair pathways.
