Determination of the catalytic activities of mTOR and other members of the phosphoinositide-3-kinase-related kinase family.
Members of the phosphoinositide-3-kinase-related kinase (PIKK) family, which includes mTOR, ATM, ATR, and hSMG-1, play important roles in regulating the cellular response to environmental stimuli. Despite the similarity of their catalytic domain to that of phosphoinositide-3-kinase, these extremely large (>250 kDa) polypeptides function as serine/threonine protein kinases. The catalytic activities of these PIKK family members can now be measured in immune-complex kinase assays. This assay involves isolation of the kinase by immunoprecipitation and the in vitro phosphorylation of a specific substrate in the presence of radio-labeled ATP. Here we describe, in detail, the determination of PIKK catalytic activity with a standardized immune-complex kinase assay protocol.
Chiang
Gary G
GG
Program in Signal Transduction Research, The Burnham Institute, La Jolla, CA, USA.
Abraham
Robert T
RT
D019870:1-Phosphatidylinositol 4-Kinase / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism*; D000954:Antigens, Surface; D064007:Ataxia Telangiectasia Mutated Proteins; D002384:Catalysis; D018797:Cell Cycle Proteins / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D002478:Cells, Cultured; D004268:DNA-Binding Proteins; D006801:Humans; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011233:Precipitin Tests; D011494:Protein Kinases / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D017346:Protein Serine-Threonine Kinases / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D013379:Substrate Specificity; D058570:TOR Serine-Threonine Kinases; D025521:Tumor Suppressor Proteins
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D008565:Membrane Proteins; D010761:Phosphorus Radioisotopes; C099014:SPAG8 protein, human; D025521:Tumor Suppressor Proteins; D000255:Adenosine Triphosphate; D011494:Protein Kinases; C546842:MTOR protein, human; D019870:1-Phosphatidylinositol 4-Kinase; C576324:ATM protein, human; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; D058570:TOR Serine-Threonine Kinases