Calpain-Mediated Bid Cleavage and Calpain-Independent Bak Modulation: Two Separate Pathways in Cisplatin-Induced Apoptosis

Abstract

Calpain is a ubiquitous protease with potential involvement in apoptosis. We report that in human melanoma cells, cisplatin-induced calpain activation occurs early in apoptosis. Calpain activation and subsequent apoptosis were inhibited by calpeptin and PD150606, two calpain inhibitors with different modes of action. Furthermore, cisplatin induced cleavage of the BH3-only protein Bid, yielding a 14-kDa fragment similar to proapoptotic, caspase-cleaved Bid. However, Bid cleavage was inhibited by inhibitors of calpain, but not by inhibitors of caspases or of cathepsin L. Recombinant Bid was cleaved in vitro by both recombinant calpain and by lysates of cisplatin-treated cells. Cleavage was calpeptin sensitive, and the cleavage site was mapped between Gly70 and Arg71. Calpain-cleaved Bid induced cytochrome c release from isolated mitochondria. While calpeptin did not affect cisplatin-induced modulation of Bak to its proapoptotic conformation, a dominant-negative mutant of MEKK1 (dnMEKK) inhibited Bak modulation. dnMEKK did not, however, block Bid cleavage. The combination of dnMEKK and calpeptin had an additive inhibitory effect on apoptosis. In summary, calpain-mediated Bid cleavage is important in drug-induced apoptosis, and cisplatin induces at least two separate apoptotic signaling pathways resulting in Bid cleavage and Bak modulation, respectively.

Constitutive proteasome-mediated turnover of Bfl-1/A1 and its processing in response to TNF receptor activation in FL5.12 pro-B cells convert it into a prodeath factor

“…Calpains have been shown to cleave several proteins involved in apoptosis including members of the Bcl-2 family like proapoptotic members Bid and Bax, and cleavage of the prosurvival factor Bcl-xL leads to the emergence of a proapoptotic fragment. 11,[36][37][38] Pretreatment with the cellpermeable calpain inhibitor I ALLN significantly decreased the 'enhanced death' phenotype of GFP-Bfl-1 in TNFa/CHXtreated FL5.12 cells (Figure 6a), similar to proteasome inhibitor MG132 that incidentally can also suppress the activity of calpains 39 (Figure 2a; R Mellgren, personal communication). However, since ALLN is a competitive Figure 5 Mutations that interfere with efficient ubiquitination of GFP-Bfl-1 suppress its prodeath phenotype in FL5.12 cells treated with TNFa/CHX.…”

“…Radiolabeled GFP-Bfl-1, GFP-Bfl-1DC, GFP-Bfl-1/-xL, GFP-Bcl-xL, GFP or untagged Bfl-1 proteins (5 ml) or Flag-Bax control (1 ml) were incubated with purified porcine m-calpain (Calbiochem; 10 ml) in 30 ml of m-calpain reaction buffer (30 mM Tris-HCl pH 7.5, 750 mM CaCl 2 , 1.5 mM DTT), 68 or with recombinant rat m-calpain (10 ml; Calbiochem) in 30 ml of m-calpain reaction buffer (20 mM HEPES pH 7.5, 50 mM KCl, 2 mM MgCl 2 , 5 mM CaCl 2 , 1 mM DTT). 36 Control reactions included buffer lacking CaCl 2 and supplemented with calcium chelator EGTA (1.5 mM), or containing calpain inhibitor I ALLN (Calbiochem) or calpain inhibitor II ALLM (Calbiochem) at 10 mM each. Reactions were incubated at 301C for 2 h for m-calpain or at 371C for 2 h for m-calpain, and terminated on ice by addition of 2 Â SDS loading buffer.…”

Multistep and multitask Bax activation

“…This truncated Bax is highly active, possibly because a negative regulation signal has been removed (Toyota et al, 2003). In addition, calpain were also shown to cleave Bid to a cleavage site distinct from caspase 8 (Chen et al, 2001); this calpain-dependent t-Bid shares similar pro-apoptotic activity with caspase 8-cleaved t-Bid (Mandic et al, 2002), including Bax recruitment. Thus Ca 2+ alterations may elicit at least two proapoptotic signals via calpain activation (Fig.…”

Role of the pro-survival molecule Bfl-1 in melanoma

“…Noxa has been shown to be upregulated in melanoma in response to proteasome inhibition resulting in cells becoming re-sensitized to apoptosis 301 , although again there is no evidence that loss of Noxa causes carcinogenesis. Interestingly, Bid appears to play an important role in cisplatin induced apoptosis in melanoma 302 , however in general it appears that Bid expression does not correlate with sensitivity to chemotherapy in many cancers 303 .…”