Galpha11 induces caspase-mediated proteolytic activation of Rho-associated kinase, ROCK-I, in HeLa cells.

Hiroshi Ueda,Rika Morishita,Kanefusa Kato,Tomiko Asano,Katsuhiko Mikoshiba,Hiroshi Itoh,Shuh Narumiya

doi:10.1074/jbc.m102529200

Abstract

Expression of the constitutively active mutant of Galpha(11) (Galpha(11)QL) induces the formation of vinculin-containing focal adhesion-like structures in HeLa cells. This was found to be inhibited by Y-27632, a specific inhibitor of Rho-associated kinases (ROCK), but not by co-expression with a dominant negative mutant of RhoA, suggesting Rho-independent activation of ROCK by Galpha(11)QL. Investigation of trypan blue exclusion and immunocytochemistry with an antibody against cleaved caspase revealed the cellular phenotype of Galpha(11)QL-expressing cells to be identical to that displayed by cells undergoing apoptosis, and the caspase inhibitor zVAD-fmk blocked all morphological changes induced by Galpha(11)QL. Transfection of Galpha(11)QL induced cleavage of ROCK-I, and this proteolysis was also prevented by zVAD-fmk. ROCK-I C-terminally truncated at its authentic caspase sites also induced the formation of vinculin-containing focal adhesion-like structures. In addition, cleavage of ROCK-I was observed when cells overexpressing m1 muscarinic acetylcholine receptors were stimulated with carbachol. These results suggest that Galpha(11) induces proteolytic activation of ROCK-I by caspase and thereby regulates the actin cytoskeleton during apoptosis.

Highlights

Expression of the constitutively active mutant of G␣11 (G␣11QL) induces the formation of vinculincontaining focal adhesion-like structures in HeLa cells
These results suggest that G␣11 induces proteolytic activation of Rho-associated kinases (ROCK)-I by caspase and thereby regulates the actin cytoskeleton during apoptosis
We have previously demonstrated that G␤␥ and a constitutively active mutant of G␣12 caused the formation of actin stress fibers and focal adhesions in a Rho-dependent manner in HeLa cells [6]

Summary

Introduction

Expression of the constitutively active mutant of G␣11 (G␣11QL) induces the formation of vinculincontaining focal adhesion-like structures in HeLa cells. Transfection of a constitutively active mutant of G␣q into COS-7 and CHO cells was further found to cause apoptosis through a protein kinase C-dependent mechanism [17]. We have previously demonstrated that G␤␥ and a constitutively active mutant of G␣12 caused the formation of actin stress fibers and focal adhesions in a Rho-dependent manner in HeLa cells [6].

Results

Conclusion