Detection of Phosphatidylinositol-4-Phosphate 5-Kinase Activity Using Thin-Layer Chromatography

Phosphatidylinositol-4-phosphate 5-kinases (PIP5Ks; EC 2.7.1.68) synthesize phosphatidylinositol 4,5-bisphosphate (PI 4,5-P₂ ) from phosphatidylinositol 4-phosphate (PI 4-P). The hydrolysis of PI 4,5-P₂ by phosphoinositide-specific phospholipase C generates the second messengers 1,2-diacylglycerol, which activates several protein kinase C isoforms, and inositol 1,4,5-trisphosphate, which stimulates the release of calcium from intracellular stores (1 ). PI 4,5-P₂ can also be phosphorylated by the PI 3-kinase, generating phosphatidylinositol 3,4,5-trisphosphate (PI 3,4,5-P₃ ), a second messenger of generally unknown function; however, some isoforms of protein kinase C may be targets (2 ) as well as proteins that contain pleckstrin homology (PH) domains (3 ). Furthermore, PI 4,5-P₂ regulates multiple enzymes and several actin-binding proteins (4 ), is bound by PH domains found in some signaling proteins (5 ), and appears to play a role in the secretory vesicle cycle (6 ,7 ).