PKD1 Mediates Negative Feedback of PI3K/Akt Activation in Response to G Protein-Coupled Receptors

Abstract

We examined whether protein kinase D1 (PKD1) mediates negative feeback of PI3K/Akt signaling in intestinal epithelial cells stimulated with G protein-coupled receptor (GPCR) agonists. Exposure of intestinal epithelial IEC-18 cells to increasing concentrations of the PKD family inhibitor kb NB 142­70, at concentrations that inhibited PKD1 activation, strikingly potentiated Akt phosphorylation at Thr308 and Ser473 in response to the mitogenic GPCR agonist angiotensin II (ANG II). Enhancement of Akt activation by kb NB 142-70 was also evident in cells with other GPCR agonists, including vasopressin and lysophosphatidic acid. Cell treatment rovincial Hospital Affiliated to Shandong University, Jinan, China with the structurally unrelated PKD family inhibitor CRT0066101 increased Akt phosphorylation as potently as kb NB 142–70. Knockdown of PKD1 with two different siRNAs strikingly enhanced Akt phosphorylation in response to ANG II stimulation in IEC-18 cells. To determine whether treatment with kb NB 142–70 enhances accumulation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) in the plasma membrane, we monitored the redistribution of Akt-pleckstrin homology domain-green fluorescent protein (Akt-PH-GFP) in single IEC-18 cells. Exposure to kb NB 142–70 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 p110α specific inhibitor A66. ANG II markedly increased the phosphorylation of p85α detected by a PKD motif-specific antibody and enhanced the association of p85α 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 indicate that PKD1 activation mediates feedback inhibition of PI3K/Akt signaling in intestinal epithelial cells in vitro and in vivo.

Positive cross talk between protein kinase D and β-catenin in intestinal epithelial cells: impact on β-catenin nuclear localization and phosphorylation at Ser552

“…In addition, inhibition of PKD by kb NB 142-70 or CRT006610 prevented ␤-catenin phosphorylation at Ser 552 , but enhanced Akt activation, as shown by the increase in the phosphorylation of the Akt substrate PRAS40 at Ser 246 (data not shown). These results are consistent with the notion that PKD mediates negative feedback on PI3K/ Akt activation in GPCR-stimulated intestinal epithelial cells (36). Accordingly, treatment with the Akt inhibitor GSK690693 suppressed ANG II-induced phosphorylation of PRAS40 at Ser 246 (data not shown), but did not prevent ␤-catenin Ser 552 phosphorylation in response to ANG II (Fig.…”

“…These cells express G q-coupled receptors for angiotensin II (ANG II) and vasopressin (3-6, 44, 73, 78) and have been extensively used as a model system to examine signal transduction pathways in response to GPCR activation (3-6, 54, 56, 73, 79). Stock cultures of IEC-18 cells were maintained as described previously (36,54). Briefly, cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal bovine serum and penicillinstreptomycin and kept at 37°C in a humidified atmosphere containing 10% CO 2 and 90% air.…”

Biphasic Regulation of Yes-associated Protein (YAP) Cellular Localization, Phosphorylation, and Activity by G Protein-coupled Receptor Agonists in Intestinal Epithelial Cells

“…5, D sibility, we used two structurally unrelated selective inhibitors of PKD activity, CRT0066101 (58) and kb NB 142-70. In previous studies, we demonstrated that these inhibitors block PKD activation and DNA synthesis in IEC-18 cells (36,38,59,60). As shown in Fig.…”

“…These cells exhibit a number of features similar to normal cells in culture, including a normal rat diploid karyotype, a strong density inhibition of growth, a lack of growth in soft agar, and resemble stem/progenitor cells (74). Cultures of IEC-18, IEC-6, and CaCo-2 cells were maintained as described previously (36,60). Briefly, cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin/ streptomycin, and kept at 37°C in a humidified atmosphere containing 10% CO 2 and 90% air.…”

Mitotic Golgi translocation of ERK1c is mediated by PI4KIIIβ/14-3-3γ shuttling complex

“…7A,B). The importance of PKD phosphorylation to the assembly of the shuttling complex was verified by inhibition of PKD activity with 10 µM kb NB 142-70 (Ni et al, 2013), which indeed prevented the assembly of ERK1c with PI4KIIIβ but not with 14-3-3γ, as assessed by a co-immunoprecipitation assay (Fig. 7C,D) and by PLA (Fig.…”