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First published online 21 September 2004
doi: 10.1242/jcs.01376

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Mechanism of Dronc activation in Drosophila cells

Israel Muro^*, Kristin Monser^* and Rollie J. Clem

Molecular, Cellular, and Developmental Biology Program, Division of Biology, Ackert Hall, Kansas State University, Manhattan, KS 66506, USA

View larger version (31K): [in a new window]   Fig. 1. Dronc is processed at E352 when produced in E. coli. (A) Full-length Dronc protein. Mutations to alanine were made at the indicated residues. (B) Coomassie-blue stain of recombinant proteins separated by SDS-PAGE. Wild-type Dronc and the D113A and D135A mutants were fully processed, whereas E352A Dronc remained mostly full length. The migration of mass markers is indicated to the left of the gel in kDa.

View larger version (41K): [in a new window]   Fig. 2. Full-length Dronc is cleaved by active Dronc at E352 and by active Drice at D135. (A) Full-length in-vitro-translated wild-type (WT) or E352A Dronc was incubated with active recombinant Dronc, Drice or both at 30°C for 1 hour and analysed by SDS-PAGE and autoradiography. (B) Full-length in-vitro-translated D113A, D135A, D113A/D135A or D133A/D135A/E352A Dronc was incubated with active recombinant Dronc, Drice or both at 30°C for 1 hour. Reactions were then separated by SDS-PAGE and examined by autoradiography. (C) The three processed forms of Dronc: FL, full-length Dronc; L, Dronc large subunit; P, Dronc prodomain. The asterisk indicates a Dronc cleavage product resulting from cleavage of full-length Dronc by Drice at D113.

View larger version (11K): [in a new window]   Fig. 3. Processing at E352 is required for Dronc activity in vitro. Recombinant wild-type or E352A Dronc was incubated in caspase buffer A alone (filled bars) or in caspase buffer A plus S2 cell lysate (empty bars) at 30°C for 30 minutes. IETD-afc was then added to both reactions, which were then incubated for another 30 minutes, and activity was determined by comparing fluorescence units. The results shown are representative of three independent experiments.

View larger version (27K): [in a new window]   Fig. 4. Dronc processing is required for apoptosis in S2 cells. (A) S2 cells were transfected with plasmids expressing wild-type Dronc or the indicated Dronc mutants. Wild-type Dronc was also transfected into Dark-RNAi-treated cells. Cell lysates were immunoblotted with anti-Dronc antiserum. The single asterisk represents the Dronc cleavage product resulting from cleavage at D113 and E352, whereas the double asterisk represents a non-specific band detected by the antiserum. L, Dronc large subunit. (B) S2 cells were co-transfected with various Dronc constructs and a plasmid expressing eGFP. At 48 hours after transfection, eGFP-positive cells were counted and this number compared with the number of surviving cells co-transfected with CAT and eGFP (set at 100%).

View larger version (24K): [in a new window]   Fig. 5. Drice is required for apoptosis in S2 cells. (A) Cells were either mock treated or treated with cat dsRNA or drice dsRNA for 20 hours before the addition of diap1 dsRNA. Cell viability was determined at 6 hours, 12 hours and 24 hours after the addition of diap1 dsRNA. (B) An anti-DIAP1 immunoblot of S2 cells pretreated with drice dsRNA and then diap1 dsRNA, and harvested at the times shown after diapl dsRNA addition. (C) An anti-Dronc immunoblot of S2 cells pretreated with drice dsRNA and then diap1 dsRNA, and harvested at the times shown after diapl dsRNA addition. The double asterisk represents a non-specific band detected by the Dronc antiserum.

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