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. 2016 Jan 18;212(2):181–198. doi: 10.1083/jcb.201506081

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© 2016 Liu et al.

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Figure 10. — WDR91 and WDR81 inhibit PI3K complex activity. (A) Loss of WDR91 or WDR81 enhances activity of the PI3K complex. Beclin1 was immunoprecipitated from cell lysates of control (Ctrl), KO-91, and KO-81 HeLa cells. Precipitated proteins were detected with indicated antibodies (bottom panel). Equal amounts of precipitated proteins from each genotype were examined for PI3K activity by measuring relative light unit of luminescence (RLU) of ADP converted from ATP. Data representing mean ± SEM are from three independent experiments and are normalized to PI3K activity in Ctrl cells (top panel). ***, P < 0.001. (B) Reinforced expression of Flag-WDR91 or/and Myc-WDR81 inhibits PI3K complex activity. HeLa cells were transfected with Flag-WDR91 or Myc-WDR81 expression vectors, or both. 48 h later, cell lysates were subjected to IP of Beclin1. Precipitates were examined with antibodies of indicated proteins (bottom panel) then analyzed for PI3K activity as in A. Data representing mean ± SEM are from three independent experiments and are normalized to PI3K activity in Ctrl cells (top panel). ***, P < 0.001. (C) Coimmunostaining of PtdIns3P with EEA1 in Ctrl, KO-91, and KO-81 HeLa cells treated without or with wortmannin (200 nM, 1 h). Insets show enlarged (2.5×) views of boxes in merged images. Bars, 10 µm. Right panel: PtdIns3P and EEA1 intensity was quantified in ≥100 cells. ***, P < 0.001. (D) Negative regulation of endosomal PtdIns3P levels by SORF-1/WDR91-SORF-2/WDR81 and Rab switch factors. Solid green arrows, activation; dashed green arrow, potential activation effect; black and red lines, inhibitory effects; dashed black arrows, termination of activation. EE, early endosome; LE, late endosome. See also Discussion.