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

Abstract

Phosphoinositide 3-kinase (P13K) is a lipid kinase which phosphorylates the D3 position of the phosphoinositide derivatives and is known to be activated by a host of protein tyrosine kinases. P13K has been demonstrated to play an important role in mitogenesis and cell transformation in several cell systems. However, the functional roles of P13K in pancreatic acinar cells remain to be determined. The objective of this study was to identify and characterize the P13K pathway and its relation to other non-receptor protein tyrosine kinases in mediating signal transduction of pancreatic acinar cells. Intact acini isolated from the rat pancreas were incubated with or without cholecystokinin octapeptide (CCK-8). A Triton X-100-soluble and 10 000 rpm supernatant of the cell sonicates was used for immunoprecipitation and Western immunoblotting. When a monoclonal anti-phosphotyrosine antibody (clone 4G10) was used, two major tyrosine-phosphorylated bands were observed at the location of p85 and p60. CCK (10 pM and 10 nM) significantly enhanced the tyrosine phosphorylation of these two bands. Furthermore, when a monoclonal anti-P13K antibody (clone UB93-3) which recognizes the N-terminal SH2 domain of the p85 regulatory subunit of P13K was used, CCK (10 pM - 10 nM) dose-dependently increased the amount of the immunodetectable P13K band with a peak occurring at 5 min. The increase in the immunodetectable P13K band elicited by CCK did not require the presence of extracellular Ca2+. The pp60(src) inhibitor, herbimycin A (6 μM), and the P13K inhibitor, wortmannin (6 μM), both decreased intensities of the P13K band elicited by CCK. Herbimycin A abolished phosphotransferase activities of the Src kinase following stimulation with CCK, whereas wortmannin had no effect, suggesting that Src is an upstream regulator of P13K. Wortmannin (3-6 μM) abolished CCK-stimulated pancreatic amylase secretion. Immunoprecipitation studies using an anti-Src antibody (clone CD11) or P13K antibody in conjunction with the anti-phosphotyrosine antibody showed that, in response to CCK, tyrosine phosphorylations of Src and P13K were enhanced at the location of p60 and p85, respectively. Src was co-immunoprecipitated with P13K following stimulation with CCK, suggesting that pp60(src) forms an immunocomplex with P13K via the N-SH2 domain of the p85 regulatory subunit. Thus P13K and its association with Src appear to be involved in mediating CCK-stimulated pancreatic exocytosis.