INIGEM   23989
INSTITUTO DE INMUNOLOGIA, GENETICA Y METABOLISMO
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Protein Kinase D1 (Pkd1) Mediates -Catenin Signaling and Phosphorylation at Ser552 in Intestinal Epithelial Cells
Autor/es:
JAMES SINNETT-SMITH; STEVEN H YOUNG; NORA ROZENGURT; ROBERT KUI; OSVALDO REY; ENRIQUE ROZENGURT
Reunión:
Congreso; Digestive Diseases Week; 2012
Institución organizadora:
AGA
Resumen:
Rationale: -catenin signaling plays a critical role in promoting normal and abnormal intestinal epithelial cell proliferation in response to Wnt ligands, pro-inflammatory cytokines and growth factors. Our recent results demonstrated that protein kinase D1 (PKD1) signaling stimulates intestinal epithelial cell proliferation, as revealed by using epithelial cells in culture and a novel PKD1 transgenic mouse model (J Biol Chem, 286, 511-520, 2011). We hypothesize that crosstalk between PKD1 and -catenin signaling provides a novel mechanism leading to intestinal epithelial cell proliferation and migration. Results: As a first step to test this hypothesis, we determined the effect of PKD1 expression on TCF/LEF-1 luciferase reporter TOP-Flash in HEK 293. Expression of PKD1 enhanced endogenous -catenin transcriptional activity in HEK 293 cells, as measured by TOP-Flash activity. Cell stimulation with the active phorbol ester PDBu to induce PKC/PKD activation caused a further increase in endogenous -catenin transcriptional activity in these cells. Next, we examined PKD1/ -catenin crosstalk in untrasformed intestinal epithelial cells. In unstimulated IEC-6 or IEC- 18 cells, -catenin was predominantly located at the plasma membrane, most likely bound to E-cadherin and/or -catenin. Stimulation with the Gq-coupled receptor agonist angiotensin II (ANG II) induced a marked redistribution of -catenin from the surface to the cytosol and nuclear compartment. The redistribution of -catenin induced by ANGII was prevented by PKD family inhibition with kb NB 142-70. These novel results suggest crosstalk between GPCR/PKD1 and -catenin signaling systems in intestinal epithelial cells. Given that - catenin phosphorylation at Ser552 promotes its dissociation from cell-cell contacts, induces its nuclear translocation and stimulates its transcriptional activity, we examined whether GPCR agonists stimulate phosphorylation of -catenin at this important regulatory site. Stimulation of IEC-18 cells with the Gq-coupled receptor agonist ANG II induced striking -catenin phosphorylation at Ser552, an effect prevented by either inhibitors of PKD family activity (kb NB 142-70 or CRT006610) or by siRNA-mediated knockdown of PKD1 expres- sion. In contrast, -catenin was constitutively phosphorylated at Ser675 in these cells and the phosphorylation of this residue did not change in response to GPCR stimulation. PKD1/ -catenin crosstalk also occurs in intestinal epithelial cell In Vivo, since an increase in - catenin nuclear localization was demonstrated by inmunohistochemistry in PKD1 transgenic mice. Conclusion: Our results support the hypothesis that PKD1 mediates crosstalk between PKD1 and -catenin signaling, thus providing a novel mechanism in the regulation of intestinal epithelial cell proliferation.