Involvement of phosphoinositide 3-kinase and its association with pp60src 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 activated by a host of protein tyrosine kinases. PI3K has been demonstrated to play an important role in mitogenesis and cell transformation in several cell systems. However, the functional roles of PI3K in pancreatic acinar cells remain to be determined. The objective of this study was to identify and characterize the PI3K 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 10000 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-PI3K antibody (clone UB93-3) which recognizes the N-terminal SH2 domain of the p85 regulatory subunit of PI3K was used, CCK (10 pM-10 nM) dose-dependently increased the amount of the immunodetectable PI3K band with a peak occurring at 5 min. The increase in the immunodetectable PI3K band elicited by CCK did not require the presence of extracellular Ca2+. The pp60src inhibitor, herbimycin A (6 microM), and the PI3K inhibitor, wortmannin (6 microM), both decreased intensities of the PI3K 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 PI3K. Wortmannin (3-6 microM) abolished CCK-stimulated pancreatic amylase secretion. Immunoprecipitation studies using an anti-Src antibody (clone CD11) or PI3K antibody in conjunction with the anti-phosphotyrosine antibody showed that, in response to CCK, tyrosine phosphorylations of Src and PI3K were enhanced at the location of p60 and p85, respectively. Src was co-immunoprecipitated with PI3K following stimulation with CCK, suggesting that pp60src forms an immunocomplex with PI3K via the N-SH2 domain of the p85 regulatory subunit. Thus PI3K and its association with Src appear to be involved in mediating CCK-stimulated pancreatic exocytosis.
Nozu
F
F
Department of Internal Medicine, University of Michigan, Ann Arbor 48019, USA.
Owyang
C
C
Tsunoda
Y
Y
D000681:Amylases / Q000378:metabolism*; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D016227:Benzoquinones; D015153:Blotting, Western; D002457:Cell Extracts / Q000737:chemistry; D066298:In Vitro Techniques; D047029:Lactams, Macrocyclic; D008297:Male; D010179:Pancreas / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism*; D011809:Quinones / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D017828:Rifabutin / Q000031:analogs & derivatives; D012844:Sincalide / Q000494:pharmacology*; D000077191:Wortmannin
D000681:Amylases; D000730:Androstadienes; D016227:Benzoquinones; D002457:Cell Extracts; D047029:Lactams, Macrocyclic; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011505:Protein-Tyrosine Kinases; D016392:Proto-Oncogene Proteins pp60(c-src); D011809:Quinones; D017828:Rifabutin; D012844:Sincalide; D000077191:Wortmannin; C020754:herbimycin