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 neutrophils stimulated with chemotactic agonists. Treatment of neutrophils with radiolabeled [H-3]HWT resulted in specific and saturable binding that paralleled the inhibition of the respiratory burst. Both half-maximal binding and half-maximal inhibition Here observed at 5 nM, and >90% of maximal binding and inhibition was observed at 20 nM HWT. Fluorography of subcellular fractions that were separated on NaDodSO(4)/PAGE showed that [H-3]HWT binds covalently to a 110-kDa cytosolic protein. The WT-binding protein was purified from human neutrophils and bovine brain homogenates by column chromatography. The pure protein was eluted from gel filtration columns with an apparent molecular mass of 200 kDa and showed a heterodimeric structure on Coomassie-stained NaDodSO(4)/PAGE. In addition to the 110 kDa wortmannin binding protein an equally intense band was seen migrating at 85 kDa. This band was identified on Western blots as p85 alpha, the regulatory subunit of phosphatidylinositol (PI) 3-kinase (ATP:1-phosphatidyl-1D-myo-inositol 3-phosphotransferase, EC 2.7.1.137). The purified protein contained PI 3-kinase activity that was enriched >20,000-fold from human neutrophil cytosol during preparation. The data impose a key role for PI 3-kinase-mediated signal transduction through guanine nucleotide-binding protein-coupled receptors and suggest that 3-phosphorylated inositol phospholipids are important second messengers for immediate responses in neutrophils. Furthermore, the results show that WT is a powerful and selective tool to study the function of PI 3-kinase.,