Galpha11 induces caspase-mediated proteolytic activation of Rho-associated kinase, ROCK-I, in HeLa cells.
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.
Ueda
H
H
Department of Biochemistry, Institute for Developmental Research, Aichi Human Service Center, Kasugai, Aichi 480-0392, Japan.
Morishita
R
R
Itoh
H
H
Narumiya
S
S
Mikoshiba
K
K
Kato
K
K
Asano
T
T
D002118:Calcium / Q000378:metabolism; D020169:Caspases / Q000502:physiology*; D004789:Enzyme Activation; D006367:HeLa Cells; D020962:Heterotrimeric GTP-Binding Proteins / Q000502:physiology*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D011493:Protein Kinase C / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D054460:rho-Associated Kinases
D047908:Intracellular Signaling Peptides and Proteins; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D011493:Protein Kinase C; D020169:Caspases; D020962:Heterotrimeric GTP-Binding Proteins; D002118:Calcium