Figure 3
Identification of a novel and conserved caspase cleavage motif in p27 by mass spectrometry. (a) Recombinant purified p27 was incubated in presence + or absence − of a caspase-3 preparation. Proteins were separated by SDS–PAGE and stained with Coomassie brilliant blue. *, full-length p27; Δ, cleaved p27. (b) Analysis of the reaction in presence (bottom) or absence (top) of caspase-3 as shown in a by mass spectrometry (MALDI-TOF). The mass of peak fragments in Da is shown above the corresponding peaks. Upon caspase processing, the peak of 21.9 kDa declines and a novel peak of 19.6 kDa arises. *, peak for full-length p27; Δ, novel peak after incubation with caspase-3. (c) Table with p27 sequences that fit the mass of 19.6 kDa. A sequence starting at the N-terminus of the protein is highlighted in red. This sequence carries the aspartate residue 176 at the very C-terminus. (d) Alignment of the C-terminal sequences of p27 orthologs from different mammalian species. Caspase consensus sequences are shown in boxes. (e) Primary thymocytes were cultured with or without the addition of dexamethasone (10−7 M). Extracts from MEFs transiently overexpressing full-length and truncated forms of murine p27 were loaded for size comparison. The levels of transiently expressed p27 mutants were matched for western blot analysis. Extracts were blotted for p27, PARP1 and GAPDH. (f) Recombinant human and murine p27 were incubated with recombinant caspase-3 (C3). Samples were taken at the indicated time points (min). Proteins were separated by SDS–PAGE and stained with Coomassie brilliant blue. The caspase cleavage products are indicated by arrows. Human p27 was untagged and murine p27 carries an N-terminal 6xHis tag. −, purified p27 incubated in the absence of caspase-3. (g) Schematic representation of p27. The nuclear localization signal (NLS) and two C-terminal phosphorylation sites (T187 and T198) that can regulate p27 stability are indicated. A previously proposed and the newly identified caspase cleavage sites are shown.
