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.
Subject(s)
Ataxia Telangiectasia/enzymology , Placenta/enzymology , Protein Serine-Threonine Kinases/isolation & purification , Androstadienes/pharmacology , Antibody Specificity , Ataxia Telangiectasia Mutated Proteins , Cell Cycle Proteins , Checkpoint Kinase 2 , Cross Reactions , DNA-Activated Protein Kinase , DNA-Binding Proteins/metabolism , Female , Humans , Manganese , Nuclear Proteins , Phosphorylation , Pregnancy , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/metabolism , Replication Protein A , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Proteins , WortmanninABSTRACT
STATEMENT OF PROBLEM: The existence of mandibular lateral translation and the approaches to its measurement and interpretation by using a pantograph are controversial. PURPOSE: This study evaluated the validity of using a pantograph to measure mandibular lateral translation and analyzed human pantographic tracings to determine whether they exhibited mandibular lateral translation. MATERIAL AND METHODS: A pantograph was modified by adding 2 posterior horizontal recording tables and styli at the transverse horizontal axis. Pantographic tracings of 25 human subjects were compared with the corresponding theoretically determined values for tracings that exhibited only rotation with no translation. Differences in the tracings at 2 pantographic recording table locations, relative to the transverse horizontal axis, were also compared. RESULTS: The character of the lateral component of 100 pantographic tracings all differed from the lateral component of theoretically determined values for pure rotation. In 64% of tracings, over 50% of the total mandibular lateral translation occurred by the first 1 mm of forward movement of the nonworking side condyle. In 94% of tracings, more than 50% of the translation had occurred in the first 3 mm of forward movement. For the pantographic system used, the amount of mandibular translation represented in the tracing was not changed by altering the posterior horizontal recording table position in the anterior-posterior direction, relative to the transverse horizontal axis. CONCLUSION: All subjects showed evidence of mandibular lateral translation. New definitions for timing of mandibular lateral translation are proposed. Of the tracings, 64% were classified as exhibiting early translation, 30% as intermediate, and 4% as late mandibular lateral translation.
