Figure 2

Increased PI3K activity and downstream signaling in LCLs from FXS patients. (A, B) Immunoprecipitated PI3K catalytic subunit p110β from LCLs shows higher PI3K enzymatic activity in FXS patient cells compared with a healthy control in two different assays. (A) PI3K activity was detected with a radioactive assay using phosphoinositide (PI) and radiolabeled ATP as substrate, followed by thin layer chromatography and autoradiography. Densitometric quantification of autoradiographies showed significantly higher PI3K activity in LCLs from a patient with a full trinucleotide expansion (FXS (■), cell line GM03200) compared with healthy control LCLs (Ctr (□), cell line GM10851) (n = 5, *P = 0.048, paired ttest). An example autoradiography and FMRP-specific Western blot analysis is shown on top. Tubulin served as loading control. Note that residual FMRP expression can be detected in the FXS LCLs. (B) Increased PI3K activity in FXS LCLs was also detected with a competitive ELISA (Echelon Biosciences, Salt Lake City, UT, USA) using PI(4,5)P₂ as substrate (n = 5, *P= 0.005, paired t test). (C) PI3K activity was measured in healthy control LCLs (Ctr-b, cell line J1), LCLs from a patient with a deletion within the FMR1 gene (Fdel, cell line DM316, no FMRP detectable by Western blot, as shown above) and the patient with a full trinucleotide expansion (FXS, same as in (A) and (B), residual FMRP levels detectable) using a competitive ELISA. PI3K activity was plotted against FMRP levels in the same lysates. Correlation analysis shows a significant negative correlation of PI3K activity with FMRP levels (n = 12, 4 samples per cell line, Spearman ρ = −0.68, *P = 0.015, α = 0.05). An example autoradiography of a radioactive PI3K assay for these LCLs is shown in Figure S1. (D, E) Increased activity of PI3K/mTOR downstream signaling is shown by elevated phosphorylation of Akt (D, n = 4, *P = 0.01, paired t test) and S6 (E, n (pS6) = 5, n (S6) = 4, *P = 0.029, paired t test] in patient LCLs. Phospho-specific Western blot signals were normalized to total levels of Akt and S6, respectively. Akt and S6 levels normalized to tubulin were not significantly different in patient cells compared with healthy control cells (^n.s.P(Akt) = 0.50, ^n.s.P (S6) = 0.27, paired t tests]. a.u., Arbitrary units.; norm., normalized; n.s., not significant; ◊, Unaffected control; ▨, Fmr1 CGG repeat expansion;▲, Fmr1 nucleotide deletion.