Cytosolic expression of dimerization defective mutants of mature Smac potentiate induction of apoptosis without interacting with inhibitor of apoptosis proteins (IAPs)

Abstract

Mature Smac, a homodimeric mitochondrial protein lacking the first 55 amino acids, exits mitochondria and promotes apoptosis by directly antagonizing IAPs. We used the ubiquitin fusion method to express mutant and wild type mature Smac in the cytosol of the 911 line of human embryonic retinoblasts. Mutations V78E, V81D, and F88D in the dimeric interface of Smac each prevented homodimerization, as determined by pull down of transiently expressed mutant Smac‐V5 by transiently expressed mutant Smac‐GST. Each of the Smac mutants also failed to heterodimerize with wild type Smac. We induced apoptosis in 911 cells that stably expressed cytosolic Smac by a 24 h treatment with zIEALal, a peptidyl aldehyde inhibitor of the proteasome. Apoptosis was measured by FACS analysis with annexin V PE and 7‐AAD. Each of the monomeric Smac mutants potentiated the induction of apoptosis by zIEALal at least as well as wild type Smac. Caspase activation in 911 cells that stably expressed cytosolic Smac was demonstrated by western analysis of cleaved PARP after a 10 h zIEALal treatment. Cells that expressed the F88D‐Smac mutant accumulated significantly more cleaved PARP than those that expressed wild type Smac. Interestingly, stable expression of wild type Smac in the cytosol of 911 cells markedly decreased its ability to interact with IAPs, which may be due to an as yet unidentified post‐translational modification. Following transient expression in 911 cells, monomeric F88D‐Smac failed to co‐immunoprecipitate with any of the 6 IAPs tested (cIAP1, cIAP2, XIAP, Survivin, Livin, or Apollon). These findings suggest that mature Smac can exert a proapoptotic action that is independent of a direct interaction with IAPs. Supported by grant GM60383 from NIH.