Overexpression of the Catalytically Impaired Tas1T234V or Taspase1D233A Variants Does Not Have a Dominant Negative Effect in T(4;11) Leukemia Cells
Methodology/Findings: The consequences of overexpressing the catalytically dead Taspase1 mutant, Taspase1T234V, or the highly attenuated variant, Taspase1D233A, on Taspase1’s processing of AF4NMLL and of other Taspase1 targets was analyzed in living cancer cells employing an optimized cell-based assay. Notably, even a nine-fold overexpression of the respective Taspase1 mutants neither inhibited Taspase1’s cis- nor trans-cleavage activity in vivo. Likewise, enforced expression of the a- or b-subunits showed no trans-dominant effect against the ectopically or endogenously expressed enzyme. Notably, co- expression of the individual a- and b-subunits did not result in their assembly into an enzymatically active protease complex. Probing Taspase1 multimerization in living cells by a translocation-based protein interaction assay as well as by biochemical methods indicated that the inactive Taspase1 failed to assemble into stable heterocomplexes with the wild type enzyme.
Conclusions: Collectively, our results demonstrate that inefficient heterodimerization appears to be the mechanism by which inactive Taspase1 variants fail to inhibit wild type Taspase1’s activity in trans. Our work favours strategies targeting Taspase1’s catalytic activity rather than attempts to block the formation of active Taspase1 dimers to interfere with the pathobiological function of AF4NMLL.
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