Constitutive activation of Akt promotes senescence in a variety of cell types [16], including endothelial progenitors, mouse embryonic fibroblasts [59]C[61] and mouse prostate epithelial cell [60] and links diet-induced obesity with vascular senescence and cardiovascular disease [62]. NB 142C70 strikingly increased membrane accumulation of Akt-PH-GFP in response to ANG II. The translocation of the PIP3 sensor to the plasma membrane and the phosphorylation of Akt was completed prevented by prior exposure to the class I p110 specific inhibitor A66. ANG II markedly increased the phosphorylation of p85 detected by a PKD motif-specific antibody and enhanced the association of p85 with PTEN. Transgenic mice overexpressing PKD1 showed a reduced phosphorylation of Akt at Ser473 in intestinal epithelial cells compared to wild type littermates. Collectively these results show that PKD1 activation mediates opinions inhibition of PI3K/Akt signaling in intestinal epithelial cells and Confluent cultures of IEC-18 cells were incubated in 10-Deacetylbaccatin III the absence (?) or presence (kb) of either 3.5 M kb NB 142C70 or 5 M CRT0066101 (CRT) for 1 h prior to stimulation of the cells with 50 nM angiotensin II (ANGII) for 1 h. Cells CD121A were lysed and p85 immunoprecipitated as explained in Materials and Methods. Immunoblotting was peformed using the PKD substrate motif antibody (Cell Signling Technology). Fold increases in p85 phosphorylation was quantified using Multi Gauge V3.0 and plotted as bars, mean S.E; n?=?4. Panel B, Confluent cultures of IEC-18 cells were incubated in the absence (?) or presence (kb) of either 3.5 M kb NB 142C70 or 1 M AG1478 for 1 10-Deacetylbaccatin III h prior to stimulation of the cells with 50 nM angiotensin II (ANGII) for either 30 or 60 min, as indicated. Cells were lysed and p85 immunoprecipitates were analyzed by antiphosphotyrosine immunoblotting. Panel C, Confluent cultures of IEC-18 cells were incubated in the absence (?) or presence (kb) of 3.5 M kb NB 142C70 for 1 h prior to stimulation of the cells with 50 nM angiotensin II (ANGII) for either 30 or 60 min, as indicated. Cells were lysed and p85 immunoprecipitates were analyzed by immunoblotting with PTEN antibodies. Bars stand for the mean S.E n?=?4. Individual values are the ratio of PTEN band intensity to the corresponding p85 10-Deacetylbaccatin III band intensity in each experiment. *p 0.05 as compared with values at time 0 or values obtained in cells treated with kb NB 142C70 and stimulated with ANG II and at each time point. Panel D, Epithelial cells from your ileum of transgenic (Tg) mice and nontransgenic (NTg) littermates mice were isolated sequentially by timed incubations in a EDTA-PBS answer. Western blot was used to analyze lysates of these cells for Akt phosphorylated at Ser473, total Akt, PKD1 autophosphorylated at Ser916 and total PKD1 (PKD-C20). Comparative loading was verified by immunoblotting for tubulin. Results are shown for 2 transgenic mice and 2 nontransgenic littermates. Bars: represent Akt phosphorylated at Ser473 (means SE; n?=?4). *p 0.05. Akt phosphorylation in intestinal epithelial cells reporter of PIP3 [48]. In unstimulated cells, the PIP3 sensor was located primarily in the cytosolic compartment without any detectable accumulation at the plasma membrane ( Fig. 5 A). Treatment with ANG II induced detectable translocation of Akt-PH-GFP to the plasma membrane. Prior exposure of the cells to kb NB 142C70 strikingly increased membrane accumulation of the PIP3 sensor in response to subsequent activation with ANG II ( Fig. 5 A; quatification in Fig. 5 B). Translocation of Akt-PH-GFP to the plasma membrane was also detected at 5 min and 30 min after ANG II activation of IEC-18 cells treated with kb NB 142C70 (Fig. S2). Open in a separate window Physique 5 PKD1 inhibition potentiates PI3K-mediated production of PIP3 in response to angiotensin II activation.IEC-18 cells were transiently transfected with a plasmid encoding a fusion protein between GFP and the PH domain name of Akt (Akt-PH-GFP). The cultures were incubated in the in the absence (?) or presence of either 3.5 M kb NB 142C70 (kb) or 3.5 M kb NB 142C70 and 10-Deacetylbaccatin III 10 M A66 (kb + A66) in DMEM containing 10.