Distinct OGT-Binding Sites Promote HCF-1 Cleavage
Fig 2
HCF-1PRO-repeat cleavage enhancement by a sequence nearby the HCF-1PRO repeat 1.
(A) HCF-1PRO-repeat cleavage is context dependent in vivo. (Top) Schematic of the HCF-1rep1 precursor subdivided into Region I (25 residues, blue), Region II (58 residues, pink), and Region III (60 residues, gray). (Bottom) HEK 293 cells were transfected with expression vectors encoding HCF-1rep1 FL or deletion constructs, either containing or lacking Regions I, II or III. Proteins were immunoprecipitated by an N-terminal HA-tag and assayed for cleavage by visualization by α-HA-tag immunoblot. *, C-terminal precursor truncations. (B) Region II enhances HCF-1PRO-repeat cleavage in vitro. Cleavage efficiency during an in vitro cleavage assay time course of selected HCF-1rep1 constructs. HCF-1rep1 constructs were incubated with OGT for 0 to 8 h and precursor and resulting N-terminal cleavage products were analyzed for cleavage by α-GST-immunoblot. Uncleaved and cleaved products were quantified and cleavage efficiencies determined as cleaved products over total. Shown are the means and standard deviations of three independent experiments. (C) Region II cleavage-enhancement activity is sequence specific. In vitro cleavage assay of HCF-1rep1 FL and Region II constructs containing a scrambled Region II sequence (+II_scrambled) or an inactive HCF-1PRO repeat (+II_T17–22A). Resulting precursor and N-terminal cleavage products were analyzed for cleavage with the indicated antibodies. (D) Region II activates the inactive POUrep2 construct for cleavage. (Left) Schematic of the GST-fusion construct POUrep2 containing HCF-1PRO repeat 2 (rep2), embedded in between the POU-specific (POUS) and POU-homeo domains (POUH) of Oct-1. Region II or Region III were inserted N-terminal of rep2, respectively. (Right) In vivo cleavage activities in HEK 293 cells, transiently transfected with transfection medium (mock) or POUrep2 encoding plasmids. Precursors and cleaved fragments were purified via immunoprecipitation of an N-terminal HA-tag and cleavage assayed using the indicated antibody. In (A), (C) and (D), prominent (●) and faint (⭕) cleavage products are indicated.