Bcl-2 and Caspase Inhibition Cooperate to Inhibit Tumor Necrosis Factor-α-induced Cell Death in a Bcl-2 Cleavage-independent Fashion

Abstract

The ability of proteins of the Bcl-2 family to either induce or inhibit apoptosis is dependent on both cell type and the apoptotic stimulus. We have shown in the murine pro-B cell line FL5.12 that Bcl-2 is incapable of inhibiting tumor necrosis factor ␣ (TNF␣)-induced cell death and is cleaved during this process. One potential explanation for this observation is that caspase activation directly or indirectly inhibits Bcl-2 function. It has been suggested that caspase cleavage of Bcl-2 is responsible for its inability to block certain cell deaths. Consistent with Bcl-2 cleavage being a caspase-mediated event, this cleavage is inhibitable by 50 M CBZ-Val-AlaAsp-fluoromethylketone (zVAD-fmk). Furthermore, Bcl-2 can cooperate with the caspase inhibitor zVADfmk in a dose-dependent manner to block TNF␣-induced cell death. Overexpression of Bcl-2 results in a 10-fold decrease in the amount of zVAD-fmk required to inhibit TNF␣-induced apoptosis. However, cleavage-defective mutants (D31A and D34A) show no enhanced viability relative to wild-type Bcl-2 in response to TNF␣-induced cell death and also show the same cooperativity with zVAD-fmk. These results suggest that Bcl-2 cleavage is not important for the inhibition of TNF␣-induced cell death but do not preclude an involvement in a postcommitment phase of apoptosis.

Procaspase-3 regulates fibronectin secretion and influences adhesion, migration and survival independent of catalytic function

“…Cells were harvested, lysed and subjected to western blotting as described previously (Johnson and Boise, 1999). Primary antibody against caspase-3 (Cell Signaling Technology) was used.…”

“…Primary antibody against caspase-3 (Cell Signaling Technology) was used. Actin (Sigma) was used as a loading control and was visualized as described previously (Johnson and Boise, 1999).…”

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“…Studies have demonstrated that cleavage of Bcl2 and Bcl-X L by caspases, the molecular scissors effecting intracellular apoptosis, may affect their antiapoptotic function. [90][91][92] Caspases are cysteine proteases that cleave target proteins specifically at aspartic acid residues. 93 Caspases have emerged as critical players in apoptotic pathways.…”

“…92 The deletion of the BH4 domain and resulting possible 'exposure' of the BH3, pore/channel forming region apparently can turn Bcl2 from an anti-apoptotic to pro-apoptotic molecule, much like a Jekyll and Hyde plot. 91 The putative caspase cleavage site for Bcl-X L occurs within the flexible loop region (at D61). 91 Other mechanisms involving the proteolytic degradation of Bcl2 may also be involved in its regulation.…”

“…91 The putative caspase cleavage site for Bcl-X L occurs within the flexible loop region (at D61). 91 Other mechanisms involving the proteolytic degradation of Bcl2 may also be involved in its regulation. One recent study has found that phosphorylation of Bcl2 blocks TNF␣-induced apoptosis in both HeLa and HUVECs by a mechanism that may involve ubiquitination of Bcl2.…”