PHYSICAL ASSOCIATION OF THE SMALL GTPASE RHO WITH A 68-KDA PHOSPHATIDYLINOSITOL 4-PHOSPHATE 5-KINASE IN SWISS 3T3 CELLS

Our previous work showed that post-translationally modified Rho in its GTP-bound state stimulated phosphatidylinositol 4-phosphate 5-kinase (PIP5K) activity in mouse fibroblast lysates. To investigate whether R ho physically interacts with PIP5K, we incubated immobilized Rho-GST w ith Swiss 3T3 cell lysates and tested for retained PIP5K activity. Rho -GST, but not Ras-GST or GST alone, bound significant PIP5K activity. The binding of PIP5K was independent of whether Rho was in a GTP- or G DP-bound state. An antibody against a 68-kDa human erythrocyte type I PIP5K recognized a single 68-kDa protein eluted from Rho-GST column. T he Rho-associated PIP5K responded to phosphatidic acid differentially from the erythrocyte type I PIP5K, suggesting that it could be a disti nct isoform not reported previously. Rho co-immunoprecipitated with th e 68-kDa PIP5K from Swiss 3T3 lysates, demonstrating that endogenous R ho also interacts with PIP5K. ADP-ribosylation of Rho with C3 exoenzym e enhanced PIP5K binding by approximately eightfold, consistent with t he ADP-ribosylated Rho functioning as a dominant negative inhibitor. T hese results demonstrate that Rho physically interacts with a 68-kDa P IP5K, although whether the association is direct or indirect is unknow n.