Interleukin-3 （IL-3） deprivation of the mouse pro-B cell line Ba/F3 induces cell death that is abrogated by B-cell lymphoma 2 （Bcl-2） overexpression, but remains unaffected by the pan-caspase inhibitor carbobenzox...Interleukin-3 （IL-3） deprivation of the mouse pro-B cell line Ba/F3 induces cell death that is abrogated by B-cell lymphoma 2 （Bcl-2） overexpression, but remains unaffected by the pan-caspase inhibitor carbobenzoxy-valyl-analyl- aspartyl-[O-methyl]-fluoromethylketone （zVAD-fmk）. IL-3 withdrawal causes receptor-interacting protein （RIP）I cleavage into C-terminal fragments of 30 and 25 kDa, and only cleavage leading to the former was prevented by zVAD-fmk, siRNA experiments demonstrated that generation of the 25-kDa fragment was due to a Bcl-2-modulated release of the mitochondrial serine protease high temperature requirement protein A2 （HtrA2）/Omi. Accordingly, recombinant HtrA2/Omi efficiently cleaved mouse RIP1 in vitro, generating fragments matching those observed in IL-3-deprived Ba/F3 cells. The HtrA2/Omi cleavage site in mouse RIP1 was mapped to the intermediate domain and the corresponding N- and C-terminal fragments were impaired in their ability to activate nuclear factor-r,B, c-Jun N-terminal kinase and p38 mitogen-activated protein kinase. Interestingly, knockdown of HtrA2/Omi afforded pro- tection against IL-3 withdrawal-induced death in the presence of zVAD-fmk, demonstrating a role for HtrA2/Omi in caspase-independent cell death during growth factor withdrawal by cleaving RIP1.展开更多
文摘Interleukin-3 （IL-3） deprivation of the mouse pro-B cell line Ba/F3 induces cell death that is abrogated by B-cell lymphoma 2 （Bcl-2） overexpression, but remains unaffected by the pan-caspase inhibitor carbobenzoxy-valyl-analyl- aspartyl-[O-methyl]-fluoromethylketone （zVAD-fmk）. IL-3 withdrawal causes receptor-interacting protein （RIP）I cleavage into C-terminal fragments of 30 and 25 kDa, and only cleavage leading to the former was prevented by zVAD-fmk, siRNA experiments demonstrated that generation of the 25-kDa fragment was due to a Bcl-2-modulated release of the mitochondrial serine protease high temperature requirement protein A2 （HtrA2）/Omi. Accordingly, recombinant HtrA2/Omi efficiently cleaved mouse RIP1 in vitro, generating fragments matching those observed in IL-3-deprived Ba/F3 cells. The HtrA2/Omi cleavage site in mouse RIP1 was mapped to the intermediate domain and the corresponding N- and C-terminal fragments were impaired in their ability to activate nuclear factor-r,B, c-Jun N-terminal kinase and p38 mitogen-activated protein kinase. Interestingly, knockdown of HtrA2/Omi afforded pro- tection against IL-3 withdrawal-induced death in the presence of zVAD-fmk, demonstrating a role for HtrA2/Omi in caspase-independent cell death during growth factor withdrawal by cleaving RIP1.
