WORTMANNIN BINDS SPECIFICALLY TO 1-PHOSPHATIDYLINOSITOL 3-KINASE WHILE INHIBITING GUANINE-NUCLEOTIDE-BINDING PROTEIN-COUPLED RECEPTOR SIGNALING IN NEUTROPHIL LEUKOCYTES

Wortmannin (WT) and its derivative 17-hydroxywortmannin (HWT) inhibit at nanomolar concentrations superoxide formation and exocytosis in neu trophils stimulated with chemotactic agonists. Treatment of neutrophil s with radiolabeled [H-3]HWT resulted in specific and saturable bindin g that paralleled the inhibition of the respiratory burst. Both half-m aximal binding and half-maximal inhibition Here observed at 5 nM, and >90% of maximal binding and inhibition was observed at 20 nM HWT. Fluo rography of subcellular fractions that were separated on NaDodSO(4)/PA GE showed that [H-3]HWT binds covalently to a 110-kDa cytosolic protei n. The WT-binding protein was purified from human neutrophils and bovi ne brain homogenates by column chromatography. The pure protein was el uted from gel filtration columns with an apparent molecular mass of 20 0 kDa and showed a heterodimeric structure on Coomassie-stained NaDodS O(4)/PAGE. In addition to the 110 kDa wortmannin binding protein an eq ually intense band was seen migrating at 85 kDa. This band was identif ied on Western blots as p85 alpha, the regulatory subunit of phosphati dylinositol (PI) 3-kinase (ATP:1-phosphatidyl-1D-myo-inositol 3-phosph otransferase, EC 2.7.1.137). The purified protein contained PI 3-kinas e activity that was enriched >20,000-fold from human neutrophil cytoso l during preparation. The data impose a key role for PI 3-kinase-media ted signal transduction through guanine nucleotide-binding protein-cou pled receptors and suggest that 3-phosphorylated inositol phospholipid s are important second messengers for immediate responses in neutrophi ls. Furthermore, the results show that WT is a powerful and selective tool to study the function of PI 3-kinase.,