FIGURE 5.

Identification of the H₂O₂/Syk-dependent PIP5Kβ tyrosine phosphorylation site(s). A, map of potential tyrosine phosphorylation sites in PIP5Kβ. PIP5Kβ (human isoform designation used here; equivalent to mouse PIP5Kα) has 540 residues and its kinase core spans residues 26 to 398. Potential tyrosine phosphorylation sites predicted by the NetPhos program are indicated. Tyr-105 (bold) is identified as the bona fide phosphorylation site in this paper. B, tyrosine phosphorylation of PIP5Kβ mutants in COS cells overexpressing Syk. Point or truncated mutants were coexpressed with Syk in COS cells and immunoprecipitated. The immunoprecipitates were blotted with α-Tyr(P) and α-Myc. The images shown were assembled from a single Western blot in which 4 lanes were removed. Data are representative of at least three independent experiments. C, response of PIP5Kβ mutants to H₂O₂, in the absence of overexpressed Syk. PIP5Kβ mutants were immunoprecipitated from COS cells and immunoblotted. Top, Western blot of a representative experiment. Bottom, relative extent of tyrosine phosphorylation. The intensity of the α-Tyr(P) band was normalized against that of the α-Myc band. The response of WT PIP5Kβ (extent of Tyr(P)) is defined as 1. Data from three independent experiments (mean ± S.E.) are shown in the lower panel. Asterisks denote statistically significant, with p < 0.05. Error bars indicate S.E.M.