FIGURE 2.

H₂O₂ induces PIP5Kβ tyrosine phosphorylation and decreases its lipid kinase activity. A, PV and H₂O₂ have similar effects on phosphoinositide homeostasis. HeLa cells labeled with [³²P]orthophosphate were treated with either 1 mm H₂O₂ or 10 μm PV for 15 min. Lipids were extracted and resolved by TLC. Left, fluorogram; right, quantitation. The amount of PIP₂ or PIP in the control sample was set as 1. B, PIP5Kβ tyrosine phosphorylation. COS cells transfected with Myc-PIP5Kβ were exposed to 0.5 or 1 mm H₂O₂ for 20 min. Myc-PIP5Kβ was immunoprecipitated and Western blotted with α-Myc and α-Tyr(P). Top, Western blots; bottom, quantitation of tyrosine phosphorylation. The ratio of α-Tyr(P) to α-Myc PIP5Kβ intensity in the absence of H₂O₂ is set as 1. Values shown are mean ± S.E., n = 3. Asterisks denote statistically significant compared with control, with p < 0.05, in this and all other panels in this figure. C, H₂O₂ induces tyrosine phosphorylation of PIP5Kβ and γ87, but not PIP5Kα. Cells were incubated with 1 mm H₂O₂ for 15 min. D, H₂O₂ has differential effects on the lipid kinase activity of the three PIP5K isoforms. COS cells transiently transfected with Myc-PIP5K isoforms were exposed to either 1 mm H₂O₂ or 10 μm PV for 15 min. Immunoprecipitated Myc-PIP5Ks were used in an in vitro lipid kinase assay and their activity in the linear range was normalized against the amount of immunoprecipitated kinase. The specific activity (mean ± S.E., n = 3) of each control untreated PIP5K isoform was set as 1. E, H₂O₂ also induces PIP5Kβ Ser/Thr dephosphorylation. COS cells expressing HA-PIP5Kβ were labeled with ³²P and exposed to 1 mm H₂O₂. Immunoprecipitated HA-PIP5Kβ was subjected to SDS-PAGE. ³²P-Labeled proteins were detected by PhosphorImager and total HA-PIP5Kβ was detected by Western blotting. Data shown are representative of those from two independent experiments. F, effects of PIP5Kβ depletion by RNAi on the H₂O₂-induced decrease in PIP₂. HeLa cells were transfected with small interfering RNA oligonucleotides targeting PIP5Kβ or firefly luciferase (negative control). Cells were stimulated with H₂O₂ (0.5 mm, 15 min), and ³²P incorporation into PIP and PIP₂ was quantitated after TLC. The amount of [³²P]phosphoinositide was expressed as a percentage of total labeled phospholipids. Data shown are mean ± S.E., n = 7 from three independent experiments. Error bars indicate S.E.M.