Figure 3.

TGF-β signaling induces the phosphorylation of P85 (Y458) and disruption of the PI3K heterodimer. Murine CD4⁺ T cells were isolated by negative selection. Tyrosine phosphorylated proteins were immunoprecipitated from T cells activated for 10 min ± 10 ng/ml TGF-β. Label-free quantitative MS was used to identify proteins in the phospho-tyrosine IPs. A and B, label-free quantitation of the mass spectrometric data are illustrated for (A) PIP4K2α and (B) P85α. Shown are mean ± S.D.; p values were calculated with a two-tailed Student's t test. Murine CD4⁺ T cells isolated by negative selection were in vitro activated through TCR and CD28 costimulation for various time points with or without 10 ng/ml TGF-β. C, immunoblotting was performed on cell lysates for the phosphorylated and total protein abundance for P85. D, densitometry was performed across three independent experiments to quantitate the abundance of tyrosine phosphorylation for P85 (Tyr-458). The abundance of phosphorylated protein was normalized to total protein. Shown are mean ± S.D.; p values were calculated by two-way ANOVA. CD4⁺ T cells isolated by negative selection were in vitro activated through TCR and CD28 costimulation for 10 mins with various doses of TGF-β. E) immunoblotting was performed for p-P85 (Tyr-458) and total P85 on cell lysates. One representative blot from three independent experiments is shown. F, densitometry was performed across three independent experiments to quantitate the abundance of tyrosine P85 (Tyr-458) phosphorylation normalized to total P85 abundance as a function of TGF-β concentration. Shown are mean ± S.D.; p values were calculated by one-way ANOVA with a Tukey multiple comparison test. G, the Thr-458 phosphorylation site on P85 was mapped onto the P85–P110 heterodimer crystal structure (PDB ID, 2RD0). CD4⁺ T cells isolated by negative selection were in vitro activated through TCR and CD28 costimulation for 10 min in the absence or presence of 10 ng/ml TGF-β. P85 was immunoprecipitated from total cell lysates and immunoblotting was performed for P110 and P85. H, one representative blot from three independent experiments is shown. I, densitometry was performed across three independent experiments to quantitate the amount of P110 that coimmunoprecipitated with P85, which was normalized to the abundance of P85 immunoprecipitated. Shown are mean ± S.D.; p values were calculated by one-way ANOVA with a Tukey multiple comparison test. J, mass ELISA assays were utilized to measure PtdIns(3,4,5)P3 in murine CD4⁺ T cells activated for 10 min ± 10 ng/ml TGF-β. Shown are mean ± S.D.; p values were calculated by one-way ANOVA with a Tukey multiple comparison test.