Monitoring Agonist-Induced Activity of PI3-Kinase in HEK-293 with a Genetically Encoded Sensor

In non-excitable cells, IP3-driven Ca²⁺ release plays a pivotal role in agonist-induced Ca²⁺ signaling. The efficiency of the phosphoinositide cascade, which couples diverse cell surface receptors to Ca²⁺ mobilization, is modulated by a number of kinases, including phosphoinositide 3-kinase (PI3K) that phosphorylates PIP2 to generate the phospholipid PIP3. We have previously shown that the PI3K inhibitor wortmannin does not affect acetylcholine-induced Ca²⁺ signaling in HEK-293 cells, while PI828, a PI3K inhibitor of distinct chemical nature, completely suppressed cellular responses to the agonist. As a possible reason for the different effectivity of wortmannin and PI828, PI3K isoforms functioning in HEK-293 could be much more sensitive to PI828. To clarify this issue, we generated a monoclonal line of HEK-293 cell, which expresses two genetically encoded sensors, namely, cytosolic Ca²⁺ sensor R-GECO1 and PIP3 sensor PH(Akt)-Venus. The cells of this line allowed for simultaneous monitoring of Ca²⁺ signals and PI3K activity. While R-GECO1 fluorescence is directly stimulated by Ca²⁺ binding, generation of PIP3 by PI3K initiates the translocation of PH(Akt)-Venus from the cytosol to the plasmalemma. It turned out that acetylcholine initiated a transient increase in the intracellular Ca²⁺ but did not affect the distribution of the PIP3 sensor in the cell cytosol. This indicated that acetylcholine did not stimulate PI3K activity. At the same time, insulin, which stimulates PI3K through tyrosine kinase receptors, caused the cytosol/plasmalemma translocation of PH(Akt)-Venus, thus demonstrating insulin-induced PI3K activity. This insulin-evoked translocation of PH(Akt)-Venus was canceled by wortmannin and PI828, suggesting that the inhibition of PI3K activity by these compounds was rather effective. Thus, being capable of stimulating intracellular Ca²⁺ signaling in HEK-293 cells, acetylcholine did not stimulate the PI3K pathway, which, therefore, was not involved in cholinergic transduction. Although the inhibition of PI3K by wortmannin and PI828 was undoubtable, the results of the present work suggest that PI828 suppressed acetylcholine induced Ca²⁺ signaling nonspecifically, that is, not involving PI3K, but acting on some other cellular target.