Pancreas growth, tyrosine kinase, PtdIns 3-kinase, and PLD involve high-affinity CCK-receptor occupation

Pancreas growth, tyrosine kinase, PtdIns 3-kinase, and PLD involve high-affinity CCK-receptor occupation. which activates adenylate cyclase did not stimulate Yes, nor did pancreatic growth factors. CCK activation of Yes required both high- and low-affinity CCK1-receptor states. TPA?/CCK-stimulated Yes activation were completely inhibited by thapsigargin and the PKC inhibitor, GF109203X. CCK/TPA stimulated the association of Yes with focal adhesion kinases (Pyk2, FAK) and its autophosphorylated forms (pY397FAK, pY402Pyk2). Moreover, CCK/TPA stimulated Yes interacted with a number of other signaling proteins, including Shc, PKD, p130Cas, PI3K and PTEN. This study demonstrates that TAS 103 2HCl in rat pancreatic acini, the SFK member Yes is expressed and activated by CCK and other gastrointestinal hormones/neurotransmitters. Because its activation results in the direct activation of many cellular signaling cascades that have been shown to mediate CCKs effect in TAS 103 2HCl acinar cell function our results suggest it is one of the important pancreatic SFKs mediating these effects. CS). We observed a signal only in the lane corresponding to the Yes kinase human recombinant (Fig. 1). Open in a separate window Fig. 1 Specificity of the antibodies used in the studyLysates from pancreatic acini and equal amounts of human recombinant Src family members present in the pancreatic acinar cells (Lyn, Yes, and Src) or the related SFK, Fyn were immunoprecipitated (IP) in parallel with 4 g of anti-Yes BD antibody and the resultant immunocomplexes were detected by Western blotting (WB) with a specific anti-Yes antibody (Cell signaling= CS), Activation of pY416 Yes induced by CCK (100 nM), carbachol (10 M), bombesin (1 nM), secretin (10 nM), VIP (10 nM) in isolated pancreatic acini. Isolated pancreatic acini were incubated with the indicated agents for 1 minute, and then lysed. Yes kinase was immunoprecipitated as established in and Western blots were analyzed using anti-pY416 Src family and, as loading control, anti-Total Yes. Bands were visualized using chemiluminescence and quantified by densitometry. Action of CCK (100 nM, 1 min), insulin (1 M, 10 min), EGF (10 nM, 5 min), PDGF (100 ng/ml, 10 min), bFGF (100 ng/ml, 5 min), IGF (100 nM, 10 min) and HGF (1 Rabbit Polyclonal to GRP94 nM) upon the activation of Yes in the pancreatic acini. The experiment was performed as described in Part A above. Results of a representative blot of 6 independent experiments are shown. * Isolated pancreatic acini were incubated in the absence or presence of CCK (100 nM), gastrin (10 nM) or A71378 (30 nM) for 1 min, or preincubated for 5 min in the presence of L364,718 (1 M), YM022 (1 M), L365,260 (1 M) or SR27897 (1 M) and then in the additional presence of CCK (100 nM) for 1 min. Yes kinase activity was determined as established in Lanes 1C4). Gastrin did not produce any increase in pY416 Yes phosphorylation, and the CCK activation of Yes was mimicked by the incubation of the cells with the selective CCK1 receptor agonist, A71378. Moreover, when the acinar cells were incubated with CCK and two different CCK1 receptor antagonists [L364,718 or SR27897] [17,18] the increment in pY416 Yes phosphorylation observed in the sole presence of CCK was largely inhibited, but not in the presence of CCK2 receptor antagonists [L365,260 or YM022] [18] (Fig. 2, Lanes 5C10). These results demonstrate that the observed effect of CCK in pY416 Yes phosphorylation is only due to the activation of the CCK1 receptors. 3.3. Dose-response effect of CCK and CCK-JMV on Yes kinase Y416 phosphorylation in rat pancreatic acini As CCK has an important role in both the physiology and pathophysiology of the pancreas [10,19], we focused our study in the activation of Yes kinase exerted by this hormone in rat pancreatic acini. Increasing concentrations of CCK produced a monophasic increase in Y416 phosphorylation of Yes, detectable at 0.01 nM concentration (Fig. 3), maximal stimulation occurred with 100 nM CCK (28016% of control) and CCKs half-maximal effect (EC50) occurred with 2.11 nM0.15 nM (Fig. 3, Table 1). The CCK1 receptor in pancreatic acini can exist in two different activation states, a low and a high-affinity state, and the activation of the different states activates different cell signaling cascades [20C22]. In order to determine the contribution of each activation receptor state to the TAS 103 2HCl activation of Yes kinase by CCK, pancreatic acini were incubated in.