Assaying Endogenous Phosphatidylinositol-4-Phosphate 5-Kinase (PIP5K) Activities

Phosphoinositides are a family of lipid second messengers interlinked by an extensive and highly regulated network of kinases and phosphatases. The modulation of phosphoinositide profiles can regulate numerous cancer-related pathways, including cell survival, cell proliferation, migration, integrin activation, and transcription. PtdIns(4,5)P 2 is at the heart of phosphoinositide signaling; its levels are controlled by enzymes that synthesize it and those that degrade it. Phosphatidylinositol-4-phosphate 5-kinases (PIP5 K) phosphorylate PtdIns4P on the 5-position and constitute the major pathway for the generation of PtdIns(4,5)P 2 . We will discuss how to suppress the expression of human PIP5 Kβ using RNAi and how to measure the activity and levels of the endogenous enzyme. We describe a method to immunoprecipitate the endogenous PIP5 Kβ and to assay its activity. Western blotting with another panel of antibodies is then used to determine the levels of endogenous PIP5 Kβ in the immunoprecipitates.