ANG II and LPA induce Pyk2 tyrosine phosphorylation in intestinal epithelial cells: role of Ca²⁺, PKC, and Rho kinase

Abstract

2001.-The G protein-coupled receptor agonists angiotensin II (ANG II) and lysophosphatidic acid (LPA) rapidly induce tyrosine phosphorylation of the cytosolic proline-rich tyrosine kinase 2 (Pyk2) in IEC-18 intestinal epithelial cells. The combined Pyk2 tyrosine phosphorylation induced by phorbol 12,13-dibutyrate, a direct agonist of protein kinase C (PKC), and ionomycin, a Ca 2ϩ ionophore, was equal to that induced by ANG II. Inhibition of either PKC or Ca 2ϩ signaling attenuated the effect of ANG II and LPA, although simultaneous inhibition of both pathways failed to completely abolish Pyk2 tyrosine phosphorylation. Cytochalasin D, which disrupts stress fibers, strongly inhibited the response of Pyk2 to ANG II or LPA. The distinct Rho-associated kinase (ROK) inhibitors HA-1077 and Y-27632, as well as the Rho inhibitor Clostridium botulinum C3 exoenzyme, also significantly attenuated ANG II-and LPA-stimulated Pyk2 tyrosine phosphorylation. Simultaneous inhibition of PKC, Ca 2ϩ , and either actin assembly or ROK completely abolished the Pyk2 response. Together, these results show that ANG II and LPA rapidly induce Pyk2 tyrosine phosphorylation in intestinal epithelial cells via separate Ca 2ϩ -, PKC-, and Rho-mediated pathways.

Phosphospecific Site Tyrosine Phosphorylation of p125FAK and Proline-rich Kinase 2 Is Differentially Regulated by Cholecystokinin Receptor Type A Activation in Pancreatic Acini

“…However, our results have similarities and differences from studies investigating the magnitude of stimulation of the different sites in one of the kinases in a given cell. Our results with PYK2 differ from those reported with endothelin-1 in mesangial cells (45), in which all three sites had equal magnitude of phosphorylation, and from a number of cells in which the relative magnitude of stimulation of phosphorylation of the different sites differed from ours (angiotensin or lysophosphatidic acid in mesangial cells (57), histamine in platelets (43), thrombin in HUVEC cells (46), and hepatocyte growth factor in lung cancer cells (44)). Our results with the PYK2 phosphospecific sites were similar to the magnitude of stimulation at each site in HUVEC cells with thrombin (43).…”

Differential FAK phosphorylation at Ser-910, Ser-843 and Tyr-397 induced by angiotensin II, LPA and EGF in intestinal epithelial cells

“…Previous studies from our laboratory demonstrated that ANGII induces a rapid increase in [Ca 2+ ] i in IEC-18 cells [48] and that agonists of Gq-coupled receptors elicit FAK Ser-843 phosphorylation through a Ca 2+ -dependent pathway in Swiss 3T3 fibroblasts [37]. Here, we demonstrate that treatment of IEC-18 cells with the Ca 2+ ionophore ionomycin induced a striking, rapid and transient increase in FAK phosphorylation at Ser-843.…”

“…ANG II binds to specific endogenous receptors in intestinal epithelial IEC-18 cells [44] and induces multiple signaling pathways and biological responses in these cells, including DNA synthesis and proliferation [45][46][47][48][49]. As shown by the results presented in Fig.1 A (control), stimulation with ANGII also markedly increased the migration of IEC-18 cells into the denuded area of a razor blade wound.…”

“…ANGII, acting through the G q -coupled AT 1 receptor, stimulates activation of ERK, protein kinase D and Pyk2 through a protein kinase C (PKC)-dependent pathway [46,48]. Here, we examined whether the increase in FAK Ser-910 phosphorylation in response to ANGII in IEC-18 cells is mediated through a PKC/ERK-dependent pathway.…”

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

“…These cells endogenously express G␣ q/11 -coupled receptors for angiotensin II (ANG II) and vasopressin (23,39,40,(43)(44)(45)(46) and have been extensively used as a model system to examine signal transduction pathways in response to GPCR activation (23,36,38,39,(43)(44)(45)47). ANG II and vasopressin act as potent growth factors for IEC-18 and IEC-6 cells (23,39,(43)(44)(45)47). Because the levels of YAP/TAZ influence their association with transcriptional cofactors especially when expressed at elevated levels, we studied the localization, phosphorylation, and activity of the endogenous YAP protein.…”