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 Rho physically interacts with PIP5K, we incubated immobilized Rho-GST with 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 GDP-bound state. An antibody against a 68-kDa human erythrocyte type I PIP5K recognized a single 68-kDa protein eluted from Rho-GST column. The Rho-associated PIP5K responded to phosphatidic acid differentially from the erythrocyte type I PIP5K, suggesting that it could be a distinct isoform not reported previously. Rho co-immunoprecipitated with the 68-kDa PIP5K from Swiss 3T3 lysates, demonstrating that endogenous Rho also interacts with PIP5K. ADP-ribosylation of Rho with C3 exoenzyme enhanced PIP5K binding by approximately eightfold, consistent with the ADP-ribosylated Rho functioning as a dominant negative inhibitor. These results demonstrate that Rho physically interacts with a 68-kDa PIP5K, although whether the association is direct or indirect is unknown.
Ren
X D
XD
Department of Vascular Biology, Scripps Research Institute, La Jolla, California 92037, USA.
Bokoch
G M
GM
Traynor-Kaplan
A
A
Jenkins
G H
GH
Anderson
R A
RA
Schwartz
M A
MA
D016475:3T3 Cells; D000244:Adenosine Diphosphate; D000818:Animals; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D051379:Mice; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D020741:rho GTP-Binding Proteins
D000244:Adenosine Diphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C020077:1-phosphatidylinositol-4-phosphate 5-kinase; D019204:GTP-Binding Proteins; D020741:rho GTP-Binding Proteins