Characterization of a Soluble Adrenal Phosphatidylinositol 4-Kinase Reveals Wortmannin Sensitivity of Type III Phosphatidylinositol Kinases

Abstract

Phosphorylation of phosphatidylinositol (PtdIns) by PtdIns 4-kinases is the first step in the synthesis of polyphosphoinositides, the lipid precursors of intracellular signaling molecules. We have recently identified a cytosolic PtdIns 4-kinase (cPI4K) in the bovine adrenal cortex that is distinguished from previously known PtdIns 4-kinases by its sensitivity to the PtdIns 3-kinase inhibitor wortmannin (WT). The present study has further characterized this soluble enzyme and compared its properties to those of the membrane-bound, type II PtdIns 4-kinase activity of the adrenal cortex and the type III enzyme of bovine brain. The enzymatic activity of adrenal cPI4K was inhibited not only by WT (IC50 approximately 50 nM) but also by LY-294002 (IC50 approximately 100 microM), another inhibitor of PtdIns 3-kinase, and neither compound affected type II PtdIns 4-kinase at concentrations that inhibited cPI4K. In contrast to the type II enzyme, cPI4K had a significantly higher Km for ATP, was relatively insensitive to inhibition by adenosine (Ki approximately 800 microM vs approximately 40 microM), had lower affinity for PtdIns, and was not inhibited by Ca2+ ions. These properties identify the WT-sensitive adrenal cPI4K as a type III PtdIns 4-kinase that is distinct from the tightly membrane-bound, Ca2+- and adenosine-sensitive, type II PtdIns 4-kinase. The type III PtdIns 4-kinase prepared from bovine brain exhibited similar kinetic parameters as the adrenal cPI4K, and was also inhibited by WT with an IC50 of 30-50 nM. Since WT inhibits the synthesis of agonist-regulated phosphoinositide pools in intact cells at micromolar concentrations, these findings indicated that type III rather than type II PtdIns 4-kinases are responsible for the maintenance of the precursor phospholipids required for intracellular signaling through the inositol phosphate/Ca2+ pathway.

Inhibition of the Phosphoinositide 3-Kinase Pathway Induces a Senescence-like Arrest Mediated by p27Kip1

“…Recent studies have shown that other kinases, including PI4K (65)(66)(67)(68), are also susceptible to inhibition by both LY294002 and Wortmannin at concentrations in excess of those required for inhibition of PI3K. To further confirm our results and to gain further insight into the mechanism by which the PI3K modulates p27…”

Golgi and plasma membrane pools of PI(4)P contribute to plasma membrane PI(4,5)P 2 and maintenance of KCNQ2/3 ion channel current

“…PI4KIII types α and β are present in the cytoplasm, plasma membrane, the Golgi, and the nucleolus. Considering the importance of PI4KIIIα in maintaining PM PI(4)P (16) and its potent inhibition by wortmannin (44,45), the small size of the wortmannin effect on KCNQ2/3 currents (only a 15% decrease) is more puzzling. One possible explanation would be that following PI4KIII inhibition there is a compensatory slowing of lipid phosphatases.…”

“…We asked whether both classes of PI 4-kinase contribute precursor PI(4)P that ultimately enters the PM PI(4,5)P 2 pool. As general blockers of each class of PI 4-kinase, we used the type IIα and type IIβ inhibitor cytosolic adenosine (25,44) (13,14,16,23,30,45). In control experiments, the KCNQ2/3 current is quite stable during 500 s of whole-cell recording (Fig.…”

Role of the Phospholipase C-Inositol 1,4,5-Trisphosphate Pathway in Calcium Release-activated Calcium Current and Capacitative Calcium Entry

“…Concentrations of wortmannin 100-fold greater than those required to inhibit phosphatidylinositol 3-kinase (10 -100 nM) are reported to inhibit phosphatidylinositol 4-kinase (10 -100 M) (35). PIP is the immediate precursor of PIP 2 , which is the substrate for PLC and the precursor of IP 3 (36).…”