Effects of overexpressing inactive Taspase1 mutants in trans on Taspase1’s processing of various target proteins.

Leukemic (K562) and solid tumor cells were transfected with the indicated amounts of the different indicator plasmids, together with respective control plasmids, or expression plasmids encoding active or inactive Taspase1 mutants, and analyzed 24 h later. The number of cells showing cytoplasmic (C) or nuclear (N) fluorescence was counted in at least 200 indicator protein-expressing cells. Results from one representative experiment are shown. Whereas the number of transfectants displaying cytoplasmic fluorescence, i.e., uncleaved indicator protein, significantly decreased upon co-transfection of 0.1 µg Tasp-BFP expression plasmid (***: pIn transfectants with high (SaOs) or intermediate (SW480) levels of endogenous Taspase1, the A•M_S1/2 indicator protein (0.2 µg expression plasmid) is already fully or partially cleaved in absence of ectopically expressed protease resulting in its predominant nuclear localization. A similar localization was observed upon co-expression of the inactive Taspase1 variants (1 µg expression plasmid), indicating that the activity of endogenous Taspase1 is not inhibited in trans.