Biochemical characterization of the ataxia-telangiectasia mutated (ATM) protein from human cells.
Ataxia-telangiectasia mutated (ATM) is a serine/threonine protein kinase that plays a central role in controlling the cellular response to ionizing radiation and other DNA-damaging agents. ATM is a 3056 amino acid polypeptide that is present in low abundance in the nucleus of human cells. Here, we describe the purification and characterization of ATM from the nuclear fraction of HeLa cells. Microgram quantities of highly stable, kinase-active ATM were prepared. Purified ATM was phosphorylated on serine 1981 and was active towards a variety of known ATM substrates, including p53 and the Bloom Syndrome helicase, BLM. The protein kinase activity of ATM was selectively inhibited by wortmannin, caffeine and LY294002 and was stimulated by charged biological polymers, including single-stranded M13 DNA (ssDNA), sheared double-stranded calf thymus DNA, heparin sulfate and poly ADP-ribose (PAR), raising the possibility that charged structures may contribute to regulation of ATM activity. However, chemical inhibition of the formation of poly ADP-ribose in cells had no effect on the activation of ATM-dependent pathways by ionizing radiation. Using gel filtration chromatography, we also show that purified ATM, as well as ATM in crude nuclear extracts from unirradiated and irradiated cells elutes with an estimated native molecular weight of approximately 600 kDa. Moreover, dephosphorylation of serine 1981 did not affect the apparent molecular weight of ATM in irradiated extracts. Our results suggest that phosphorylation of serine 1981 alone may not directly regulate the subunit composition of ATM.
Goodarzi
Aaron A
AA
Departments of Biochemistry and Molecular Biology, and Biological Sciences, Cancer Biology Research Group, University of Calgary, 3330 Hospital Drive N.W., Calgary, AB, Canada .
Lees-Miller
Susan P
SP
D000595:Amino Acid Sequence; D000730:Androstadienes / Q000494:pharmacology; D001260:Ataxia Telangiectasia / Q000235:genetics* / Q000378:metabolism; D064007:Ataxia Telangiectasia Mutated Proteins; D002110:Caffeine / Q000494:pharmacology; D018797:Cell Cycle Proteins; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000737:chemistry; D002850:Chromatography, Gel; D002867:Chromones / Q000494:pharmacology; D004273:DNA, Neoplasm; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006367:HeLa Cells; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D008970:Molecular Weight; D009025:Morpholines / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011064:Poly Adenosine Diphosphate Ribose / Q000494:pharmacology; D011494:Protein Kinases / Q000187:drug effects / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism* / Q000528:radiation effects; D011839:Radiation, Ionizing; D012694:Serine / Q000737:chemistry / Q000378:metabolism; D025521:Tumor Suppressor Proteins; D000077191:Wortmannin
D000730:Androstadienes; D018797:Cell Cycle Proteins; D002867:Chromones; D004273:DNA, Neoplasm; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D009025:Morpholines; D010455:Peptides; D025521:Tumor Suppressor Proteins; D011064:Poly Adenosine Diphosphate Ribose; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D002110:Caffeine; D012694:Serine; D006493:Heparin; D011494:Protein Kinases; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; D000077191:Wortmannin