Fig. 6: PPP metabolites promote AKT activation by inhibiting PHLDA3.

a–c R5P or uridine blocks glucose starvation-induced PHLDA3 upregulation and AKT inhibition. Pten null mES cells were treated with various concentrations of R5P or uridine for 4 h (a), or indicated time periods with (b) with or without (c) 12 h of glucose starvation. Cell lysates were subjected to immunoblotting with the indicated antibodies. d The Pten null mES cells were treated with 6-AN for 24 h, followed by treatment with R5P, uridine and G6P for 2 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. e PHLDA3 WT and knockout HeLa cells were treated with 6-AN in the presence or absence of R5P or uridine. Three days later, colony numbers were counted. f PPP controls PHLDA3 expression by regulating its promoter activity. PC3 cells transfected with the PHLDA3-luciferase reporter plasmid (upper) were treated with 6-AN or incubated with PPP metabolites for 2 h. The cell lysates were harvested for the luciferase activity assay (lower). g PHLDA3 driven by an exogenous promoter fails to respond to 6-AN treatment or R5P and uridine supplements. PC3 cells were transfected without or with a CMV-driven FLAG-PHLDA3 expression plasmid and treated with 6-AN for 24 h or R5P or uridine for 2 h. Cell lysates were subjected to RT-qPCR or immunoblotting with the indicated antibodies. h Truncation analysis narrows down the PHLDA3 promoter region necessary for PPP regulation to p3 (−829 to +431). i, j PPP regulates PHLDA3 and AKT independent of p53 status. Pten null mES cells and LNCAP cells (p53 WT) were treated without or with PFTα (5 μM) or 6-AN (100 nM). The Phlda3 mRNA and protein levels were measured (i). PTEN null LNCAP (p53 WT) and PC (p53 null) cells were treated with 6-AN. Cell lysates were subjected to immunoblotting with the indicated antibodies (j). Data are presented as the mean ± SD and were compared with untreated cells. Each experiment was performed n = 3 (e) and n = 4 (f–i) independent times. *p < 0.05, **p < 0.001, and ***p < 0.001, based on Student’s t test (two-sided ANOVA). See also Supplementary Fig. 5. Source data are provided as a Source Data file.