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Michael H. Cardone,*^ Natalie Roy,* Henning R. Stennicke, Guy S. Salvesen, Thomas F. Franke, Eric Stanbridge, Steven Frisch, John C. Reed^^
Caspases are intracellular proteases that function as initiators and effectors of apoptosis. The kinase Akt and p21-Ras, an Akt activator, induced phosphorylation of pro-caspase-9 (pro-Casp9) in cells. Cytochrome c-induced proteolytic processing of pro-Casp9 was defective in cytosolic extracts from cells expressing either active Ras or Akt. Akt phosphorylated recombinant Casp9 in vitro on serine-196 and inhibited its protease activity. Mutant pro-Casp9(Ser196Ala) was resistant to Akt-mediated phosphorylation and inhibition in vitro and in cells, resulting in Akt-resistant induction of apoptosis. Thus, caspases can be directly regulated by protein phosphorylation.
Many apoptotic stimuli induce release of cytochrome c (cyto c) from mitochondria into the cytosol, where it binds to the CED-4 homolog Apaf-1, inducing binding to proCasp9 and resulting in proteolytic processing and activation of pro-Casp9. Active Casp9 then directly cleaves to and activates proCasp3, initiating a cascade of additional caspase activation that culminates in apoptosis. Cyto c induces caspase activation when added to cytosolic extracts in vitro with deoxyadenosine triphosphate (dATP) (1). We noticed that epithelial cell lines 267 (prostate) and MCDK (kidney) transfected with transforming Ki-Ras(Val12) or Ha-Ras(Val12) displayed resistance to apoptotic stimuli that are known to cause cyto c release (2), such as staurosporine and etoposide (VP16) (Fig. 1, A and B), and that cytosolic extracts derived from these cells were resistant to cyto c-induced caspase activation, as measured by cleavage of Ac-DEVD-AFC (3) (Fig.1 C) (4). Reduced caspase activity was not due to lower concentrations of pro-Casp3 but correlated with inhibition of proteolytic processing of pro-Casp3 (Fig. lB) (4), implying a defect at or upstream of this caspase. Ras extracts, however, were not resistant to caspase activation induced by granzyme B (GraB) (5), which implies that other routes of protease activation were intact (Fig. 1D).
A farnesyl transferase inhibitor (FTI) reversed the resistance of Ras cytosolic extracts to cyto c (Fig. 1E), which suggests that the phenomenon is not due to secondary genetic changes in these cells. Moreover, resistance to cyto c-mediated activation of caspases was not an artifact of over-expressing oncogenic Ras, because extracts from DLD- colon cancer cells, which contain an endogenous activated Ki-Ras gene, displayed similar resistance...