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

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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.


In vitro cleavage of raptor by recombinant caspase-1 and -6. (a) Jurkat T-cell lysates were incubated with two units of recombinant caspase-1 (C1), caspase-2 (C2), caspase-3 (C3), caspase-6 (C6), caspase-7 (C7), caspase-8 (C8) or caspase-9 (C9) and raptor cleavage was monitored and compared with a STS-treated Jurkat T-cell lysate. (b) Time-dependant in vitro cleavage of raptor by caspase-1, -3 or -6 in Jurkat T-cell lysates using two units of each recombinant proteins.
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fig3: In vitro cleavage of raptor by recombinant caspase-1 and -6. (a) Jurkat T-cell lysates were incubated with two units of recombinant caspase-1 (C1), caspase-2 (C2), caspase-3 (C3), caspase-6 (C6), caspase-7 (C7), caspase-8 (C8) or caspase-9 (C9) and raptor cleavage was monitored and compared with a STS-treated Jurkat T-cell lysate. (b) Time-dependant in vitro cleavage of raptor by caspase-1, -3 or -6 in Jurkat T-cell lysates using two units of each recombinant proteins.

Mentions: To determine more precisely which caspase(s) could be implicated in raptor cleavage, recombinant caspase(s)-1, -2, -3, -6, -7, -8 and -9 were incubated with Jurkat T-cell lysates (Figure 3a). In comparison with cells treated with STS, used a positive control, caspase-6 was the only caspase that induced processing of raptor into the expected 100 kDa band, whereas caspase-1 was able to cleave raptor into two smaller fragments of roughly 70 and 80 kDa. Although recombinant caspase-1 and -6 induced an in vitro time-dependant cleavage of raptor in Jurkat T-cell lysates (Figure 3b), activation of the inflammatory caspase-1 in bone marrow-derived macrophages (BMDMø) did not highlighted processing of raptor, suggesting that caspase-1 most likely did not account for physiological raptor cleavage (Supplementary Figure S1).41


Caspase-mediated cleavage of raptor participates in the inactivation of mTORC1 during cell death
In vitro cleavage of raptor by recombinant caspase-1 and -6. (a) Jurkat T-cell lysates were incubated with two units of recombinant caspase-1 (C1), caspase-2 (C2), caspase-3 (C3), caspase-6 (C6), caspase-7 (C7), caspase-8 (C8) or caspase-9 (C9) and raptor cleavage was monitored and compared with a STS-treated Jurkat T-cell lysate. (b) Time-dependant in vitro cleavage of raptor by caspase-1, -3 or -6 in Jurkat T-cell lysates using two units of each recombinant proteins.
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Related In: Results  -  Collection

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fig3: In vitro cleavage of raptor by recombinant caspase-1 and -6. (a) Jurkat T-cell lysates were incubated with two units of recombinant caspase-1 (C1), caspase-2 (C2), caspase-3 (C3), caspase-6 (C6), caspase-7 (C7), caspase-8 (C8) or caspase-9 (C9) and raptor cleavage was monitored and compared with a STS-treated Jurkat T-cell lysate. (b) Time-dependant in vitro cleavage of raptor by caspase-1, -3 or -6 in Jurkat T-cell lysates using two units of each recombinant proteins.
Mentions: To determine more precisely which caspase(s) could be implicated in raptor cleavage, recombinant caspase(s)-1, -2, -3, -6, -7, -8 and -9 were incubated with Jurkat T-cell lysates (Figure 3a). In comparison with cells treated with STS, used a positive control, caspase-6 was the only caspase that induced processing of raptor into the expected 100 kDa band, whereas caspase-1 was able to cleave raptor into two smaller fragments of roughly 70 and 80 kDa. Although recombinant caspase-1 and -6 induced an in vitro time-dependant cleavage of raptor in Jurkat T-cell lysates (Figure 3b), activation of the inflammatory caspase-1 in bone marrow-derived macrophages (BMDMø) did not highlighted processing of raptor, suggesting that caspase-1 most likely did not account for physiological raptor cleavage (Supplementary Figure S1).41

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.