Analyzing the regulation and function of ATM.
We describe here the cloning of full-length ataxia-telangiectasia mutated (ATM) cDNA and characterization of its activity. Full-length ATM cDNA is cloned into an inducible EBV-based vector (pMEP4) and its expression analyzed in a stably transfected cell line. ATM protein induction is monitored by immunoblotting with antibodies against both ATM and a FLAG sequence tag in the recombinant protein. Extracts from irradiated cells are immunoprecipitated with anti-ATM antibodies, and protein kinase activity is measured using p53(1-44)-specific substrate or by immunoblotting extracts with an anti-phosphoserine 15 p53-specific antibody. Missense mutations affecting ATM kinase activity are detected using in vitro mutagenesis of ATM cDNA followed by the procedures outlined above.
Lavin
Martin F
MF
The Queensland Cancer Fund Research Unit, The Queensland Institute of Medical Research, Royal Brisbane Hospital, Herston, Australia.
Scott
Shaun P
SP
Kozlov
Sergei
S
Gueven
Nuri
N
D001260:Ataxia Telangiectasia; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins; D003001:Cloning, Molecular / Q000379:methods*; D004268:DNA-Binding Proteins; D005786:Gene Expression Regulation; D006801:Humans; D015151:Immunoblotting; D008214:Lymphocytes / Q000502:physiology; D016296:Mutagenesis; D020125:Mutation, Missense / Q000235:genetics*; D010768:Phosphoserine / Q000737:chemistry / Q000378:metabolism; D010957:Plasmids / Q000302:isolation & purification; D011233:Precipitin Tests; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D025521:Tumor Suppressor Proteins
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D010768:Phosphoserine; D011494:Protein Kinases; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases