Phosphoinositide 3-kinase (PI3K) mediates the formation of D-3 phosphoinositide lipids by transferring the terminal phosphate of ATP to the D-3 position of the inositol head group of phosphoinositide lipids, producing three lipid products: PtdIns(3)P, PtdIns(3,4)P₂ and PtdIns(3,4,5)P₃. These lipids bind to structural lipid binding motifs (e.g. pleckstrin homology domains) within proteins and control the activity and subcellular localisation of a diverse array of signal transduction molecules (Dowler et al., 2000). Such molecules have been implicated in a wide range of cellular processes, including cell motility, cell adhesion, cell growth, cell survival and cell cycle progession (Vanhaesebroeck et al., 2001; Cantley, 2002). There are multiple isoforms of PI3K which have been divided into three classes.

Here, we characterise the sensitivity of Class 1 ( , ß and p110 catalytic subunit isoforms) and Class II (PI3K-C2 and PI3K-C2ß ) isoforms to the widely employed PI3K inhibitors Wortmannin and LY294002. A specific monoclonal antibody to the p85 regulatory subunit was used to immunoprecipatate Class IA catalytic isoforms from the leukemic T cell line Jurkat. Similarly, specific antibodies were used to immunoprecipitate and ß isoforms of the Class II PI3Ks from Jurkats. Immunoprecipitates were assayed for associated lipid kinase activity using PtdIns as a substrate in the presence of 5 µCi -(³²P)-ATP.

The rank order of potency of Wortmannin against PI3K isoforms was Class IA (IC₅₀ =8.7 nM; n=3) > PI3K-C2ß (IC₅₀ = 51.2 nM; n=3) >> PI3K-C2 (IC₅₀> 500 nM; n=3). In contrast, the rank order of potency of LY294002 against PI3K isoforms was Class IA (IC₅₀ = 6.5 µM) = PI3K-C2(IC₅₀ 7.3 m M; n=3) > PI3K-C2ß (IC₅₀ = 23.1 µM; n=3). These results correlate with the known sensitivity of PI3K isoforms to Wortmannin, but reveal a hitherto unknown disparate sensitivity of PI3K-C2 and PI3K-C2ß to LY294002, a widely used tool with which to study biological readouts of PI3K. This work therefore reveals that studies based on the exclusive use of LY294002 may have underestimated the role of PI3K-C2ß in some biological processes.

Cantley, L.C. (2002). Science, 296:1655-1657
Dowler., S. et al. (2000). Biochem J. 351:19-27
Vanhaesebroeck, B. et al. (2001). Annu Rev Biochem, 70:535-602