Classical phosphoinositide (PI) metabolism leading to the well-known second messengers diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (InsP3), was elucidated more than 10 years ago (Fig. 4-1A). Many mitogenic signals stimulate PI turnover and transformed cells have constitutively activated PI turnover. It was work on this classical pathway that eventually led to the discovery of the novel PI pathway. Let us first look briefly at the classical pathway, in which phosphatidylinositol (Ptdlns) is phosphorylated by PtdIns 4-kinase to PtdIns-4-P, which is subsequently phosphorylated by PtdIns-4-P 5-kinase to form PtdIns-4,5-P2. Much of the PtdIns-4,5-P2 in the cell is found on the inner leaflet of the plasma membrane. This lipid can serve as a substrate for PI-specific phospholipase C (PLC), liberating DAG and IP3. IP3 is a water-soluble molecule which, when released into the cytosol, acts to liberate intracellular stores of Ca2+, increasing the intracellular concentration of Ca2+ from the resting level of ~110 nM to 400–1000 nM, which in turn can activate a number of Ca2+-sensitive enzymes and channels. The DAG released from Ptdlns-4,5-P2 remains in the membrane and serves as a cofactor in activating many of the protein kinase C (PKC) isotypes.