TYROSINE-PHOSPHORYLATION-DEPENDENT AND RHO-PROTEIN-MEDIATED CONTROL OF CELLULAR PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE LEVELS

The polyphosphoinositide PtdIns(4,5)P-2, best known as a substrate for phospholipase C isozymes, has recently been recognized to be involved in a variety of other cellular processes. The aim of this study was t o examine whether the cellular levels of this versatile phospholipid a re controlled by tyrosine phosphorylation. The studies were performed in human embryonic kidney (HEK)-293 cells stably expressing the M-3 mu scarinic acetylcholine receptor. Inhibition of tyrosine phosphatases b y pervanadate induced an up-to-approx.-2.5-fold increase in the total cellular level of PtdIns(4,5)P-2, which was both time- and concentrati on-dependent. In contrast, the tyrosine kinase inhibitors, genistein a nd tyrphostin 23, caused a rapid and specific fall in the cellular Ptd Ins(4,5)P-2 level and prevented the stimulatory effect of pervanadate on PtdIns(4,5)P-2 formation. Inactivation of Rho proteins by Clostridi um difficile toxin B caused a similar fall in the HEK-293 cell PtdIns( 4,5)P-2 level, which was not altered by additional genistein treatment . Furthermore, toxin B treatment abolished the pervanadate-induced inc rease in PtdIns(4,5)P-2 levels. As PtdIns(4,5)P-2 is an essential stim ulatory cofactor for phospholipase D (PLD) enzymes, we finally examine d the effects of the agents regulating PtdIns(4,5)P-2 levels on PLD ac tivity in HEK-293 cells. Inhibition of tyrosine phosphatases by pervan adate caused an increase in PLD activity, which was susceptible to gen istein and tyrphostin 23, and which was abolished by prior treatment w ith toxin B. In conclusion, the data presented indicate that the cellu lar level of the multifunctional phospholipid, PtdIns(4,5)P-2, in HEK- 293 cells is controlled by a tyrosine-kinase-dependent mechanism and t hat this process apparently involves Rho proteins, as found similarly for tyrosine-phosphorylation-induced PLD activation.