Analysis of phosphatidylinositol 3,4,5 trisphosphate 5-phosphatase activity by in vitro and in vivo assays.

Phosphatidylinositol 3,4,5 trisphosphate [PtdIns(3,4,5)P3] is a potent membrane-bound signaling molecule transiently synthesized by phosphoinositide 3-kinase (PI3-kinase) in response to extracellular agonists. PtdIns(3,4,5)P3 signals need to be strictly controlled. PtdIns(3,4,5)P3 recruits and binds effectors that function in oncogenic signaling pathways. PtdIns(3,4,5)P3 activates cell proliferation, growth, and migration as well as regulating insulin signaling. The inositol polyphosphate 5-phosphatase family of enzymes dephosphorylate and thereby modulate PtdIns(3,4,5)P3 levels, attenuating PI3-kinase-dependent signaling. PtdIns(3,4,5)P3 5-phosphatase enzyme activity can be assessed in vitro by analysis of the hydrolysis of radiolabeled or fluorescently labeled PtdIns(3,4,5)P3 and in vivo by visualization of the recruitment and turnover of the PtdIns(3,4,5)P3-specific biosensor GFP-PH/ ARNO or other PtdIns(3,4,5)P3 binding proteins at the plasma membrane.