FLCN phosphomimetic mutations inactivate the effect of WT FLCN on cell cycle.

Abstract

<p><i>(A) A FLCN phosphomimetic mutant (S62/73E) results in more rapid progression through the cell cycle compared to the FLCN WT, while the FLCN phosphoinactivating mutant (S62/73A) retains the ability to delay cell cycle progression.</i> Representative cell cycle profiles from UOK257 cells and the isogenic UOK257 cells expressing FLCN WT, or the phosphomutants S62/73E or S62/73A, collected 12 hours after release from thymidine. UOK257 cells expressing FLCN WT or S62/73A have more cells in G2/M and fewer cells entering G1 compared to the FLCN null (vector only) and the S62/73E cells as measured by PI staining. (B) UOK257 (black line) and their isogenic counterparts reconstituted with WT FLCN (red line) or FLCN S62/73E mutant (blue line) were synchronized by double thymidine block. Cell cycle progression after release from thymidine was analyzed by FACS. Vertical axis indicates the percent of all cells registering in the specific phase of cell cycle. Horizontal axis indicates hours post thymidine release. The average of three experiments is presented (n = 3) in each panel; bars correspond to standard error of the mean (SEM). (C) Same as in (B), except that the reconstituted phosphomutant is FLCN S62/73A (purple line). For both (B) and (C), * indicates a statistically significant difference between UOK257+ vector only compared to UOK257+ FLCN WT at the corresponding time point; # indicates a difference between UOK257 FLCN WT and the UOK257+ phosphomutant (S62/73E or S62/73A); $ indicates a difference between UOK257+ vector only and UOK257+ phosphomutant (S62/73E or S62/73A) (One-way ANOVA, Tukey's post-test, p<0.05).</p>