Hydrolysis of phosphatidylinositol 3,4-bisphosphate by a 4-phosphatase associated with the p85/p110 phosphoinositide 3-kinase

The phosphoinositide 3-kinase (PI 3-kinase) associates with activated tyrosine kinase receptors resulting in the production of phosphatidylinositol 3-phosphate (PtdIns(3)P), phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2) and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3). The time course for formation of these 3-phosphoinositides suggests PtdIns(3,4)P2 is dephosphorj'lated by a 4-phosphatase forming PtdIns(3)P. We report here the identification of a PtdIns(3,4)P2 4-phosphatase associated with the p85/l 10 form of the PI 3-kinase. NW 3T3 cells were stimulated with platelet-derived growth factor (PDGF) (30 ng/ml) for 030 minutes and the p85 subunit of the PI 3-kinase, or the PDGF receptor immunoprecipitated using specific polyclonal antibodies. PI 3-kinase activity in the immunoprecipitates was determined using either Ptdlns or PtdIns(4)P as the substrate. Within 10 minutes of PDGF-treatment, using Ptdlns as a substrate, the formation of PtdIns(3)P was stimulated 1.6-fold in p85, and 4-fold in PDGF receptor immunoprecipitates. When PtdIns(4)P was used as a substrate in p85, or PDGF receptor immunoprecipitates 60% of the product the reaction was PtdIns(3,4)P₂ and the remainder PtdIns(3)P, suggesting the presence of PtdIns(3,4)P₃ 4-phosphatase. A 3.7-fold increase in total 3-phosphorylated phosphoinositides formed was observed in p85 immunoprecipitates following PDGF-treatment of NIH 3T3 cells, however the hydrolysis of PtdIns(3,4)P₂ to PtdIns(3)P was not enhanced. The immunoprecipitated 4-phosphatase hydrolysed PtdIns(³²P-3,4)P2 forming PtdIns(³²P-3)P, but did not hydrolyse inositol 1,3,4-trisphosphate. These studies indicate the presence of a PtdIns(3,4)P₂ 4-phosphatase associated with the p85 subunit of the PI 3-kinase.