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Caspase-mediated cleavage of raptor participates in the inactivation of mTORC1 during cell death

View Article: PubMed Central - PubMed

ABSTRACT

The mammalian target of rapamycin complex 1 (mTORC1) is a highly conserved protein complex regulating key pathways in cell growth. Hyperactivation of mTORC1 is implicated in numerous cancers, thus making it a potential broad-spectrum chemotherapeutic target. Here, we characterized how mTORC1 responds to cell death induced by various anticancer drugs such rapamycin, etoposide, cisplatin, curcumin, staurosporine and Fas ligand. All treatments induced cleavage in the mTORC1 component, raptor, resulting in decreased raptor–mTOR interaction and subsequent inhibition of the mTORC1-mediated phosphorylation of downstream substrates (S6K and 4E-BP1). The cleavage was primarily mediated by caspase-6 and occurred at two sites. Mutagenesis at one of these sites, conferred resistance to cell death, indicating that raptor cleavage is important in chemotherapeutic apoptosis.

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Raptor cleavage by caspase-6 and other caspases. (a) Jurkat T-cell lysates were incubated with recombinant caspases-3, -6, -7 for 2 h. Cleavage of raptor and physiological substrates of caspases (PARP and lamin A/C) were monitored and compared with the positive control sample (Jurkat T cells treated with STS for 2 h). (b) The effect of z-VAD-fmk on the recombinant caspase-6-mediated cleavage of raptor in Jurkat T-cell lysate. (c) Cartoon of recombinant mTORC1 (composed of mLST8, mTOR (1362-end) and recombinant raptor). FAT, focal adhesion targeting; FRB, FKBP12-rapamycin binding; FATC, C-term focal adhesion targeting. (d) Cleavage of the full-length recombinant raptor (inside recombinant mTORC1) by recombinant caspase-6, -3 and -7 in the presence (or not) of z-VAD-fmk. (e) WT versus caspase-6 K.O. KBM-7 cells were treated with STS or FasL for 4 h and then cell lysates were analyzed by SDS-PAGE and western blot.
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fig4: Raptor cleavage by caspase-6 and other caspases. (a) Jurkat T-cell lysates were incubated with recombinant caspases-3, -6, -7 for 2 h. Cleavage of raptor and physiological substrates of caspases (PARP and lamin A/C) were monitored and compared with the positive control sample (Jurkat T cells treated with STS for 2 h). (b) The effect of z-VAD-fmk on the recombinant caspase-6-mediated cleavage of raptor in Jurkat T-cell lysate. (c) Cartoon of recombinant mTORC1 (composed of mLST8, mTOR (1362-end) and recombinant raptor). FAT, focal adhesion targeting; FRB, FKBP12-rapamycin binding; FATC, C-term focal adhesion targeting. (d) Cleavage of the full-length recombinant raptor (inside recombinant mTORC1) by recombinant caspase-6, -3 and -7 in the presence (or not) of z-VAD-fmk. (e) WT versus caspase-6 K.O. KBM-7 cells were treated with STS or FasL for 4 h and then cell lysates were analyzed by SDS-PAGE and western blot.

Mentions: As recombinant caspase-6 generated similar processing of raptor than treatment with pro-apoptotic drugs, we decided to investigate this processing in more detail. In Figure 4a, the cleavage of the poly (ADP-ribose) polymerase (PARP) by caspase-3 and -7,42 and the cleavage of lamin A/C by caspase-643,44 revealed the specificity of these active recombinant caspases in Jurkat T-cell lysate. As shown before, caspase-6 was the only executioner caspase able to cleave raptor in cell lysates and addition of z-VAD-fmk abolished processing of raptor, confirming that the cleavage was depending on the catalytic activity of recombinant caspase-6 (Figures 4a and b).


Caspase-mediated cleavage of raptor participates in the inactivation of mTORC1 during cell death
Raptor cleavage by caspase-6 and other caspases. (a) Jurkat T-cell lysates were incubated with recombinant caspases-3, -6, -7 for 2 h. Cleavage of raptor and physiological substrates of caspases (PARP and lamin A/C) were monitored and compared with the positive control sample (Jurkat T cells treated with STS for 2 h). (b) The effect of z-VAD-fmk on the recombinant caspase-6-mediated cleavage of raptor in Jurkat T-cell lysate. (c) Cartoon of recombinant mTORC1 (composed of mLST8, mTOR (1362-end) and recombinant raptor). FAT, focal adhesion targeting; FRB, FKBP12-rapamycin binding; FATC, C-term focal adhesion targeting. (d) Cleavage of the full-length recombinant raptor (inside recombinant mTORC1) by recombinant caspase-6, -3 and -7 in the presence (or not) of z-VAD-fmk. (e) WT versus caspase-6 K.O. KBM-7 cells were treated with STS or FasL for 4 h and then cell lysates were analyzed by SDS-PAGE and western blot.
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Related In: Results  -  Collection

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fig4: Raptor cleavage by caspase-6 and other caspases. (a) Jurkat T-cell lysates were incubated with recombinant caspases-3, -6, -7 for 2 h. Cleavage of raptor and physiological substrates of caspases (PARP and lamin A/C) were monitored and compared with the positive control sample (Jurkat T cells treated with STS for 2 h). (b) The effect of z-VAD-fmk on the recombinant caspase-6-mediated cleavage of raptor in Jurkat T-cell lysate. (c) Cartoon of recombinant mTORC1 (composed of mLST8, mTOR (1362-end) and recombinant raptor). FAT, focal adhesion targeting; FRB, FKBP12-rapamycin binding; FATC, C-term focal adhesion targeting. (d) Cleavage of the full-length recombinant raptor (inside recombinant mTORC1) by recombinant caspase-6, -3 and -7 in the presence (or not) of z-VAD-fmk. (e) WT versus caspase-6 K.O. KBM-7 cells were treated with STS or FasL for 4 h and then cell lysates were analyzed by SDS-PAGE and western blot.
Mentions: As recombinant caspase-6 generated similar processing of raptor than treatment with pro-apoptotic drugs, we decided to investigate this processing in more detail. In Figure 4a, the cleavage of the poly (ADP-ribose) polymerase (PARP) by caspase-3 and -7,42 and the cleavage of lamin A/C by caspase-643,44 revealed the specificity of these active recombinant caspases in Jurkat T-cell lysate. As shown before, caspase-6 was the only executioner caspase able to cleave raptor in cell lysates and addition of z-VAD-fmk abolished processing of raptor, confirming that the cleavage was depending on the catalytic activity of recombinant caspase-6 (Figures 4a and b).

View Article: PubMed Central - PubMed

ABSTRACT

The mammalian target of rapamycin complex 1 (mTORC1) is a highly conserved protein complex regulating key pathways in cell growth. Hyperactivation of mTORC1 is implicated in numerous cancers, thus making it a potential broad-spectrum chemotherapeutic target. Here, we characterized how mTORC1 responds to cell death induced by various anticancer drugs such rapamycin, etoposide, cisplatin, curcumin, staurosporine and Fas ligand. All treatments induced cleavage in the mTORC1 component, raptor, resulting in decreased raptor–mTOR interaction and subsequent inhibition of the mTORC1-mediated phosphorylation of downstream substrates (S6K and 4E-BP1). The cleavage was primarily mediated by caspase-6 and occurred at two sites. Mutagenesis at one of these sites, conferred resistance to cell death, indicating that raptor cleavage is important in chemotherapeutic apoptosis.

No MeSH data available.


Related in: MedlinePlus