Limitation of the PIK Assay

A limitation of the PI3K assay described herein is that it might not reflect what is happening to 3'-phosphoinositide levels in some cells or tissues. For example, the activity of PI3K in a multiprotein complex may not correlate with the enzyme's activity in an intact cell. Alternatively, because 3'-phosphoinosi-tides can be regulated by other mechanisms (e.g., dephosphorylation by a lipid phosphatase), an increase in PI3-kinase activity may not be indicative of an increase in cellular 3'-phosphoinositides. Wherever possible, the PI3K enzyme assay described herein should be complemented with studies designed to meas ure the intracellular production of 3'-phosphoinositides. One such method is to label cells or tissues with [3H]inositol or [32PO4], extract and deacylate the lipids, and resolve the phosphoinositide head groups by high-performance liquid chromatography (HPLC) (4,5). Unfortunately, this method is both time-consuming and labor intensive. An alternative method for determining PI(3,4,5)P3 levels quantifies the displacement of 32P-labeled inositol(1,3,4,5)P4 (IP4) from a specific binding protein by IP4 obtained from the alkaline hydrolysis of PI(3,4,5)P3 in a cellular lipid extract (6,7). This mass assay is capable of detecting PI(3,4,5)P3 at subpicomole levels. A final method involves transfecting cells with specific PH domains coupled to green fluorescent protein to label membrane domains enriched in particular 3'-phosphoinositides (5,8).

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