Morphological Changes and Detachment of Adherent Cells Induced by p122, a GTPase-activating Protein for Rho

Abstract

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 ␦1 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 Ca 2؉ microscopy, we found that p122 evoked a rapid elevation of intracellular Ca 2؉ levels, suggesting that p122 stimulates the phosphatidylinositol 4,5-bisphosphate-hydrolyzing activity of phospholipase C ␦1. These results demonstrate that p122 synergistically functions as a GTPase-activating protein specific for Rho and an activator of phospholipase C ␦1 in vivo and induces morphological changes and detachment through cytoskeletal reorganization.Recent studies have indicated a close association between the regulation of cytoskeletal assembly and phosphatidylinositol (PI) 1 metabolism. That is, a number of PI 4,5-bisphosphate (PIP 2 )-binding proteins including gelsolin, cofilin, profilin, and ␣-actinin are known to bind to actin and regulate cytoskeletal assembly (1-4). In addition, Rho has been shown to enhance the activity of PI 4-phosphate 5-kinase, the PIP 2 -synthesizing enzyme (5), and the overexpression of PI 4-phosphate 5-kinase induces massive actin polymerization in COS-7 cells (6). Alternatively, microinjection of antibodies against PIP 2 inhibits the formation of stress fibers and focal adhesions (7). These results strongly suggest that PIP 2 newly synthesized by PI 4-phosphate 5-kinase, which exists downstream of Rho, binds to PIP 2 -binding proteins (i.e. actin-binding protein) to release free Gactins.These reactions increase the intracellular concentrations of free G-actins, resulting in their reorganization to form actin fibers (8).On the other hand, Rho is inactivated by GTPase-activating proteins (GAPs) (9 -12). It is possible that Rho GAPs, as downstream components of Rho, regulate cytoskeletal disassembly. We recently cloned a GAP as a novel molecule that interacts with phospholipase C ␦1 (PLC␦1) (13). In addition to the GAP activity specific for Rho in vitro, p122 possesses the ability to enhance the PIP 2 -hydrolyzing activity of PLC␦1 in vitro. Therefore, it is possible that p122 is an ideal link between Rho and cytoskeletal disassembly. To address this issue, we tried to isolate cells stably overexpressing p122. However, we have never succeeded in obtaining such cells, sug...

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