Control of cellular phosphatidylinositol 4,5-bisphosphate levels by adhesion signals and rho GTPases in NIH 3T3 fibroblasts involvement of both phosphatidylinositol-4-phosphate 5-kinase and phospholipase C.
The involvement of small GTPases of the Rho family in the control of phosphoinositide metabolism by adhesion signals was examined in NIH 3T3 fibroblasts. Abrogation of adhesion signals by detachment of cells from their substratum resulted in a time-dependent decrease in the cellular level of PtdIns(4,5)P2 by approximately 50%. This effect could be mimicked by treatment of adherent cells with Clostridium difficile toxin B and toxin B-1470, which inhibit specific subsets of Rho and Ras GTPases. Detachment of cells that had been pretreated with the clostridial toxins did not cause a further reduction in PtdIns(4,5)P2 levels, suggesting that the target GTPases are integrated into the control of phosphoinositide levels by adhesion signals. The reduction in PtdIns(4,5)P2 levels could be attributed to reduced activity of the major PtdIns(4, 5)P2-producing enzyme, PtdIns4P 5-kinase. Unexpectedly, both cell detachment and toxin treatment resulted in a twofold to threefold increase in inositol phosphate production in intact cells. In lysates of these cells, in vitro phospholipase C activity was found to be elevated by 30-50%. The effects of cell detachment and toxin treatment on inositol phosphate formation could be mimicked by expression of dominant-negative N17 Rac1. Taken together, these data suggest that adhesion-controlled small GTPases of the Rho family are involved in the regulation of the cellular PtdIns(4,5)P2 levels in NIH 3T3 fibroblasts, by controlling the activities of both PtdIns4P 5-kinase and phospholipase C.
Institut für Pharmakologie, Universitätsklinikum Essen, Germany; Institut für Medizinische Mikrobiologie und Hygiene, Universität Mainz, Germany. paschal.oudeweernink@uni-essen.de
P A
PA
Weernink
Y
Y
Guo
C
C
Zhang
M
M
Schmidt
C
C
Von Eichel-Streiber
K H
KH
Jakobs
D016475:3T3 Cells; D000818:Animals; D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000494:pharmacology; D002118:Calcium / Q000502:physiology; D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology*; D003603:Cytotoxins / Q000494:pharmacology; D007700:Kinetics; D051379:Mice; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000378:metabolism*
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D003603:Cytotoxins; D019269:Phosphatidylinositol 4,5-Diphosphate; D011994:Recombinant Proteins; C031815:toxB protein, Clostridium difficile; D017853:Phosphotransferases (Alcohol Group Acceptor); C020077:1-phosphatidylinositol-4-phosphate 5-kinase; D010727:Phosphoric Diester Hydrolases; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; D020830:rac1 GTP-Binding Protein; D020741:rho GTP-Binding Proteins; D002118:Calcium