Cholecystokinin-Mediated RhoGDI Phosphorylation via PKCα Promotes both RhoA and Rac1 Signaling

Maria Eugenia Sabbatini,John A Williams

doi:10.1371/journal.pone.0066029

Abstract

RhoA and Rac1 have been implicated in the mechanism of CCK-induced amylase secretion from pancreatic acini. In all cell types studied to date, inactive Rho GTPases are present in the cytosol bound to the guanine nucleotide dissociation inhibitor RhoGDI. Here, we identified the switch mechanism regulating RhoGDI1-Rho GTPase dissociation and RhoA translocation upon CCK stimulation in pancreatic acini. We found that both Gα13 and PKC, independently, regulate CCK-induced RhoA translocation and that the PKC isoform involved is PKCα. Both RhoGDI1 and RhoGDI3, but not RhoGDI2, are expressed in pancreatic acini. Cytosolic RhoA and Rac1 are associated with RhoGDI1, and CCK-stimulated PKCα activation releases the complex. Overexpression of RhoGDI1, by binding RhoA, inhibits its activation, and thereby, CCK-induced apical amylase secretion. RhoA translocation is also inhibited by RhoGDI1. Inactive Rac1 influences CCK-induced RhoA activation by preventing RhoGDI1 from binding RhoA. By mutational analysis we found that CCK-induced PKCα phosphorylation on RhoGDI1 at Ser96 releases RhoA and Rac1 from RhoGDI1 to facilitate Rho GTPases signaling.

Highlights

RhoA and Rac1 are small GTP-binding proteins and cycle between two forms: an inactive GDP-bound form and an active GTP-bound form
We found that protein kinase C (PKC) is involved in cholecystokinin octapeptide (CCK)-induced RhoA translocation because the phorbol ester phorbol 12-myristate 13-acetate (PMA) increased RhoA translocation to the same extent as CCK (Fig. 1A)
When PKC activity was inhibited by GF-109203X, CCK-induced translocation was inhibited whereas when PKA activity was inhibited by H-89, CCK-induced RhoA translocation was not modified (Fig. 1B)

Summary

Introduction

RhoA and Rac are small GTP-binding proteins and cycle between two forms: an inactive GDP-bound form and an active GTP-bound form. A less studied regulatory protein is the Rho guanine nucleotide dissociation inhibitor (RhoGDI), which regulates the activity of Rho GTPases, and their partitioning between the cytosol and membranes as reviewed in [1]. A number of intracellular signals, including protein kinase C (PKC), calcium, and PKA, have been implicated in the regulation of the dissociation-association cycle of Rho GTPase-RhoGDI complexes. PKCa [4,5], atypical PKCs [6,7], p21-activated kinase [8,9], Src [10], PKA [11], PKG [12] and Ser/Thr kinase Ste20-related kinase (SLK) [13] have been shown to phosphorylate either RhoGDI or Rho GTPases and induce a dissociation or association of Rho GTPases-RhoGDI complexes

Methods

Results

Conclusion