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Biochemistry. 1996 Mar 19;35(11):3587-94.

Characterization of a soluble adrenal phosphatidylinositol 4-kinase reveals wortmannin sensitivity of type III phosphatidylinositol kinases.

Author information

1
Endocrinology and Reproduction Research Branch, National Institutes of Health, Bethesda, Maryland 20892, USA.

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.

PMID:
8639510
DOI:
10.1021/bi9517493
[Indexed for MEDLINE]

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