Morphological changes and detachment of adherent cells induced by p122, a GTPase-activating protein for Rho.
We recently cloned a novel signaling molecule, p122, that shows a GTPase-activating activity specific for Rho and the ability to enhance the phosphatidylinositol 4,5-bisphosphate-hydrolyzing activity of phospholipase C delta1 in vitro. Here we analyzed the in vivo function of p122. Microinjection of the GTPase-activating domain of p122 suppressed the formation of stress fibers and focal adhesions induced by lysophosphatidic acid, suggesting a GTPase-activating activity for Rho as in in vitro. Transfection of p122 also induced the disassembly of stress fibers and the morphological rounding of various adherent cells. Analyses using deletion and point mutants demonstrated that the GTPase-activating domain of p122 is responsible for the morphological changes and detachment and that arginine residues at positions 668 and 710 and a lysine residue at position 706 in the GTPase-activating domain are essential. Using Fluo-3-based Ca2+ microscopy, we found that p122 evoked a rapid elevation of intracellular Ca2+ levels, suggesting that p122 stimulates the phosphatidylinositol 4, 5-bisphosphate-hydrolyzing activity of phospholipase C delta1. These results demonstrate that p122 synergistically functions as a GTPase-activating protein specific for Rho and an activator of phospholipase C delta1 in vivo and induces morphological changes and detachment through cytoskeletal reorganization.
Sekimata
M
M
Department of Biomolecular Sciences, Institute of Biomedical Sciences, Fukushima Medical College, 1 Hikariga-oka, Fukushima 960-1295, Japan.
Kabuyama
Y
Y
Emori
Y
Y
Homma
Y
Y
D000595:Amino Acid Sequence; D000814:Aniline Compounds; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002448:Cell Adhesion / Q000187:drug effects; D002460:Cell Line; D048429:Cell Size / Q000187:drug effects; D003599:Cytoskeleton / Q000378:metabolism; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D020690:GTPase-Activating Proteins; D007527:Isoenzymes / Q000378:metabolism; D008246:Lysophospholipids / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D054803:Phospholipase C delta; D010957:Plasmids; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism; D014966:Xanthenes
D000814:Aniline Compounds; D020690:GTPase-Activating Proteins; D007527:Isoenzymes; D008246:Lysophospholipids; D019269:Phosphatidylinositol 4,5-Diphosphate; D014966:Xanthenes; C085379:rho GTPase-activating protein; C059715:Fluo-3; D010738:Type C Phospholipases; D054803:Phospholipase C delta; D019204:GTP-Binding Proteins; D002118:Calcium