Attenuation of focal adhesion kinase signaling following depletion of agonist-sensitive pools of phosphatidylinositol 4,5-bisphosphate

The effect of phosphoinositide depletion on focal adhesion kinase (FAK) sig naling was investigated in two neuronal cell lines. Treatment of either SH- SY5Y neuroblastoma cells or PC12 cells with wortmannin, at a concentration that inhibits phosphatidylinositol 4-kinase activity, led to a selective de pletion of phosphatidylinositol 4-phosphate without significantly altering phosphatidylinositol 4,5-bisphosphate (PIP2) content. An enhanced tyrosine phosphorylation of FAK elicited by agonist occupancy of phospholipase C-cou pled receptors (muscarinic cholinergic in SH-SY5Y neuroblastoma or bradykin in in PC12 cells) was blocked completely by wortmannin. Under the above con ditions, phosphoinositide resynthesis was prevented, and as a consequence, receptor stimulation led to a marked depletion of PIP2. In contrast, the in creased tyrosine phosphorylation of FAK elicited by agents that do not acti vate phospholipase C (phenylarsine oxide, lysophosphatidic acid, or phorbol ester) persisted in the presence of wortmannin. However, the ability of th ese agents to elicit an increase in FAK phosphorylation was also prevented if PIP2 was depleted by activation of a phospholipase C-coupled receptor in the presence of wortmannin. The results suggest that agonist-sensitive poo ls of PIP2 must be maintained far FAK signaling to occur in response to a m echanistically diverse range of stimuli.