Application of phosphoinositide-binding domains for the detection and quantification of specific phosphoinositides.
In mammals, seven phosphoinositides are known to play crucial roles as signaling molecules in a variety of cellular processes. Their synthesis and degradation are thought to be strictly controlled by metabolic enzymes such as phosphoinositide kinases and phosphatases, and their aberrant activities cause diseases. Thus, there is great interest in convenient and high-throughput measurement of such activities for the screening of drugs that enhance or block them. To date, radioactive labeling and colorimetric detection of released inorganic phosphates are mainly used to measure phosphoinositide kinase and phosphatase activities, respectively. Here, we describe a novel method for detecting and quantifying individual phosphoinositides via phosphoinositide-binding domains that exhibit high specificity and affinity toward this lipid. Enzyme-linked immunosorbent assay wells are modified with alkyl chains (C16), which enables more uniform and quantitative immobilization of phosphoinositide-containing liposomes onto the well surfaces. Phosphoinositides, as the substrate or the product, are detected by pleckstrin homology domains that specifically bind to each phosphoinositide. By this method, phosphoinositide contents are measured with higher sensitivities than those by conventional methods. More importantly, both phosphoinositide kinase and phosphatase activities can be measured for purified enzymes and crude cellular lysates. This assay is easy, sensitive, and quantitative and thus may have a variety of applications in the development of diagnostic tests or the screening of therapeutic treatments for diseases such as cancer and diabetes which may be caused by abnormal phosphoinositide metabolism.
Furutani
Masahiro
M
Department of Biochemistry, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
Tsujita
Kazuya
K
Itoh
Toshiki
T
Ijuin
Takeshi
T
Takenawa
Tadaomi
T
D019870:1-Phosphatidylinositol 4-Kinase / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002457:Cell Extracts / Q000032:analysis; D045744:Cell Line, Tumor; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D008954:Models, Biological; D018129:Phosphatidylinositol Phosphates / Q000032:analysis / Q000378:metabolism; D010716:Phosphatidylinositols / Q000032:analysis* / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D002457:Cell Extracts; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols; D011994:Recombinant Proteins; D019870:1-Phosphatidylinositol 4-Kinase; D010744:Phosphoric Monoester Hydrolases