Tyrosine-phosphorylation-dependent and Rho-protein-mediated control of cellular phosphatidylinositol 4,5-bisphosphate levels

Abstract

The polyphosphoinositide PtdIns(4,5)P # , 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 to examine whether the cellular levels of this versatile phospholipid are controlled by tyrosine phosphorylation. The studies were performed in human embryonic kidney (HEK)-293 cells stably expressing the M $ muscarinic acetylcholine receptor. Inhibition of tyrosine phosphatases by pervanadate induced an up-to-approx.-2.5-fold increase in the total cellular level of PtdIns(4,5)P # , which was both time-and concentration-dependent. In contrast, the tyrosine kinase inhibitors, genistein and tyrphostin 23, caused a rapid and specific fall in the cellular PtdIns(4,5)P # level and prevented the stimulatory effect of pervanadate on PtdIns(4,5)P # formation. Inactivation of Rho proteins by Clostridium difficile toxin B caused a similar fall in

Src modulates serotonin-induced calcium signaling by regulating phosphatidylinositol 4,5-bisphosphate

“…This effect could be explained by the ability of Src to modulate the amount of PLC substrate, PIP 2 . It has been previously reported that tyrosine kinase inhibition leads to a rapid decrease of cellular PIP 2 levels in HEK-293 cells (15). The resynthesis of PIP 2 occurs in a sustained fashion in nonstimulated cells and very rapidly after PLC hydrolysis (26).…”

“…Inositol phosphate production reflects PLC activity, which in turn depends on the availability of the substrate PIP 2 (5). Because tyrosine kinase inhibition may significantly reduce levels of PIP 2 (15), amounts of PIP 2 in control and PP1 (10 M)-treated cells were measured. No effect of PP1 was detected when the whole pool of phosphoinositides was assayed (data not shown).…”

“…However, the majority of GPCRs are known to be linked to the ␤-isoform of PLC (PLC-␤), and activation of 5-HT receptors in rat tracheal smooth muscle cells, as well as of several types of GPCRs in A10 vascular smooth muscle cells, does not appear to result in PLC-␥ tyrosine phosphorylation despite the observed reductions in Ca 2ϩ signals after tyrosine kinase inhibition (6,24). Regardless of how and which PLC isoform is activated, the availability of its substrate phosphatidylinositol 4,5-bisphosphate (PIP 2 ) is an important determinant of inositol phosphate production (5,18), and it has been reported that PIP 2 levels may be controlled by tyrosine kinase activity (15). However, the kinase involved in this process was not identified, and the effects of lowering PIP 2 levels by tyrosine kinase inhibitors on Ca 2ϩ signaling were not studied.…”

Bacterial protein toxins and lipids: role in toxin targeting and activity

“…Large clostridial toxins, such as Clostridium difficile toxin B and toxin A, also block PLD activity induced by PMA, or GTP [S] in vitro, probably by reducing PI4-P 5-kinase activity and decreasing PIP 2 level (Schmidt et al, 1996a;Rümenapp et al, 1998). Indeed, PLD, which contains a PH (pleckstrin homology) domain and a PX (Phox homology) domain, as well as binding sites for phospholipids, has been reported to anchor to PIP 2 where it is close to its substrates (Frohman and Morris, 1999).…”

LY-294002 [2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one] Affects Calcium Signaling in Airway Smooth Muscle Cells Independently of Phosphoinositide 3-Kinase Inhibition

“…PI(3,4,5)P 3 may affect Ca 2ϩ release by increasing activity of the gamma isoform of PLC (PLC␥) either directly through binding to the pleckstrin homology domain (Falasca et al, 1998) and the Src homology (SH2) domain (Rameh et al, 1998), or indirectly after activating Bruton's kinase Btk (Li et al, 1997). PI(3,4,5)P 3 has been also implicated in the activation of small GTPase proteins from the Rho family, which in turn stimulate production of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ] through activation of PI(4)5-kinase (Smith and Chang, 1989;Hartwig et al, 1995;Tolias et al, 1995;Ren et al, 1996;Carpenter et al, 1997;Rumenapp et al, 1998). Because PI3K uses PI(4,5)P 2 as a substrate, activation of PI3K may potentially have two opposing effects: it may cause a decrease in PI(4,5)P 2 levels (by using it as a substrate) and increase in PI(4,5)P 2 levels by modulating the activity of PI(4)5-kinase.…”