Involvement of phosphoinositide 3-kinase and its association with pp60(src) in cholecystokinin-stimulated pancreatic acinar cells

Phosphoinositide 3-kinase (PI3K) is a lipid kinase which phosphorylates the D3 position of the phosphoinositide derivatives and is known to be activat ed by a host of protein tyrosine kinases, PI3K has been demonstrated to pla y an important role in mitogenesis and cell transformation in several cell systems, However, the functional roles of PI3K in pancreatic acinar cells r emain to be determined, The objective of this study was to identify and cha racterize the PI3K pathway and ifs relation to other non-receptor protein t yrosine kinases in mediating signal transduction of pancreatic acinar cells , Intact acini isolated from the rat pancreas were incubated with or withou t cholecystokinin octapeptide (CCK-8), A Triton X-100-soluble and 10000 rpm supernatant of the cell sonicates was used for immunoprecipitation and Wes tern immunoblotting, When a monoclonal anti-phosphotyrosine antibody (clone 4G10) was used, two major tyrosine-phosphorylated bands were observed at t he location of p85 and p60, CCK (10 pM and 10 nM) significantly enhanced th e tyrosine phosphorylation of these two bands, Furthermore, when a monoclon al anti-PI3K antibody (clone UB93-3) which recognizes the N-terminal SH2 do main of the p85 regulatory subunit of PI3K was used, CCK (10pM-10nM) dose-d ependently increased the amount of the immunodetectable PI3K band,vith a pe ak occurring at 5 min. The increase in the immunodetectable PI3K band elici ted by CCK did not require the presence of extracellular Ca2+. The pp(60src ) inhibitor, herbimycin A (6 muM), and the PI3K inhibitor, wortmannin (6 mu M), both decreased intensities of the PI3K band elicited by CCK, Herbimycin A abolished phosphotransferase activities of the Src kinase following stim ulation with CCK, whereas wortmannin had no effect, suggesting that Src is an upstream regulator of PI3K, Wortmannin (3-6 muM) abolished CCK-stimulate d pancreatic amylase secretion. Immunoprecipitation studies using an anti-S rc antibody (clone CD11) or PI3K antibody in conjunction with the anti-phos photyrosine antibody showed that, in response to CCK, tyrosine phosphorylat ions of Src and PI3K were enhanced at the location of p60 and p85, respecti vely. Src was co-immunoprecipitated,vith PI3K following stimulation with CC K, suggesting that pp(60src) forms an immunocomplex with PI3K via the N-SH2 domain of the p85 regulatory subunit, Thus PI3K and ifs association with S rc appear to be involved in mediating CCK-stimulated pancreatic exocytosis.