Identification of FLCN phosphorylation sites at the G2/M boundary.

(A) U2OS cells were cultured in the presence (lane 2) or absence (lane 1) of nocodazole overnight and immunoprecipitated FLCN protein was detected by Coomassie Blue staining of SDS-PAGE gels. (B) LC-MS/MS analysis of FLCN peptides. Extracted ion chromatograms for FLCN peptide AHSPAEGASVESSSPGPK with no phosphorylation (a), one phosphorylation (b) and 2 phosphorylation sites (c). Intensity corresponds to the absolute intensity of the highest peak in the chromatogram. No normalization was performed. (C) FLCN deficient UOK257 cells were reconstituted with FLCN WT, FLCN double phosphoinactivating mutant S62/73A or FLCN double phosphomimetic mutant S62/73E. Exogenous WT FLCN and the phosphomutants (S62/73A and S62/73E) are expressed in both the nuclear and the cytoplasmic cellular fractions. (D) FLCN deficient UOK257 cells and the isogenic cell lines reconstituted with either FLCN WT, a tumor-associated mutant (FLCN ?F157) or the phosphomutants (S62/73A and S62/73E) were cultured overnight in the absence or presence of nocodazole. (E) UOK257 FLCN WT cells growing asynchronously (Asyn) or arrested during G2/M with nocodazole were treated with either DMSO or cycloheximide (10 μg/ml). Protein stability was determined by Western blot at various time points over the course of 8 hours (V – UOK257 vector only cells; WT – UOK257 FLCN WT cells). Lysates were resolved in SDS-PAGE and FLCN protein detected with anti-FLCN antibody. Actin serves as a loading control.