Involvement of RhoA and its interaction with protein kinase C and Src in CCK-stimulated pancreatic acini

Abstract

We evaluated intracellular pathways responsible for the activation of the small GTP-binding protein Rho p21 in rat pancreatic acini. Intact acini were incubated with or without CCK and carbachol, and Triton X-100-soluble and crude microsomes were used for Western immunoblotting. When a RhoA-specific antibody was used, a single band at the location of 21 kDa was detected. CCK (10 pM–10 nM) and carbachol (0.1–100 μM) dose dependently increased the amount of immunodetectable RhoA with a peak increase occurring at 3 min. High-affinity CCK-A-receptor agonists JMV-180 and CCK-OPE (1–1,000 nM) did not increase the intensities of the RhoA band, suggesting that stimulation of RhoA is mediated by the low-affinity CCK-A receptor. Although an increase in RhoA did not require the presence of extracellular Ca2+, the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid-AM abolished the appearance of the RhoA band in response to CCK and carbachol. The Gq protein inhibitor G protein antagonist-2A (10 μM) and the phospholipase C (PLC) inhibitor U-73122 (10 μM) markedly reduced RhoA bands in response to CCK. The protein kinase C (PKC) activator phorbol ester (10–1,000 nM) dose dependently increased the intensities of the RhoA band, which were inhibited by the PKC inhibitor K-252a (1 μM). The pp60c- src inhibitor herbimycin A (6 μM) inhibited the RhoA band in response to CCK, whereas the calmodulin inhibitor W-7 (100 μM) and the phosphoinositide 3-kinase inhibitor wortmannin (6 μM) had no effect. RhoA was immunoprecipitated with Src, suggesting association of RhoA with Src. Increases in mass of this complex were observed with CCK stimulation. In permeabilized acini, the Rho inhibitor Clostridium botulinum C3 exoenzyme dose dependently inhibited amylase secretion evoked by a Ca2+ concentration with an IC50 of C3 exoenzyme at 1 ng/ml. We concluded that the small GTP-binding protein RhoA p21 exists in pancreatic acini and appears to be involved in the mediation of pancreatic enzyme secretion evoked by CCK and carbachol. RhoA pathways are involved in the activation of PKC and Src cascades via Gq protein and PLC.

IL-6 induces AGS gastric cancer cell invasionvia activation of the c-Src/RhoA/ROCK signaling pathway

“…Because Rho possesses the forming homology (FH) 1 domain, which is enriched in proline, this domain may directly interact with the Src homology (SH) 3 domain of Src. 47 To our knowledge, this is the first report to demonstrate that the induction of RhoA activity by IL-6 was mediated by Src phosphorylation and then, IL-6 may act through Src phosphorylation on cancer cells to promote and accelerate cancer cell migration and invasion. Although more IL-6 production was noted in clone 5 ( Fig.…”

“…46 Besides, another study also found that RhoA was coimmunoprecipitated with Src, so RhoA may interact with Src. 47 Because Rho does not possess Src homology domains, this interaction may be mediated by Rho-GAP, which possesses Src homology domains. Indeed, p190…”

The Role of Protein Kinase D in Neurotensin Secretion Mediated by Protein Kinase C-α/-δ and Rho/Rho Kinase

“…For example, the interaction between PKC and Rho regulates enzyme secretion in pancreatic acini (64). In epithelial and endothelial cells, treatment with C3 toxin prevented PKC translocation and activation (65).…”

Role of Phospholipase C-β in the Modulation of Epithelial Tight Junction Permeability

“…Rho may also be involved in this pathway, since inhibition of rhoA with C3 transferase induced disassembly of the cytoskeletal actin, including the perijunctional actin myosin ring, and increased tight junction permeability (Nusrat et al, 1995). Furthermore, U73122 was found to inhibit Rho activity (Nozu et al, 1999).…”