Vol. results suggest that p70S6K is a novel substrate for caspase-3 and that the proteolytic cleavage of p70S6K is important for cisplatin-induced apoptosis. cleavage assay with human recombinant caspase-3 demonstrated that caspase-3 is capable of cleaving p70S6K directly. Furthermore, treatment with as high as 50 M cisplatin had no effect on the cleavage of p70S6K in MCF-7 cells that lack functional caspase-3 but overexpression of caspase-3 in MCF-7 cells resulted in proteolytic cleavage of p70S6K in response to cisplatin. Cleavage of p70S6K was observed in several different cell lines, including A549, H69, H358 and HeLa cells and in response to several different apoptotic stimuli, including cisplatin, doxorubicin, TNF and TRAIL (data not shown), suggesting that proteolytic cleavage of p70S6K during apoptosis is Rabbit Polyclonal to HS1 a general phenomenon. Cleavage of substrates by caspases may result in their activation or inactivation but there are also proteins that are cleaved with the cleavage having no effect on their functions (23, 30-32). These caspase substrates have been described as innocent bystanders (24, 30). Thus, to examine the functional significance of caspase-3-mediated p70S6K cleavage, we first wanted to determine the site at which p70S6K is cleaved. Active caspases cleave key proteins by recognizing a set of four neighboring amino acids in their substrate termed P4-P3-P2-P1 and have a stringent requirement for aspartic acid at the P1 position (13, 25, 33, 34). Although p70S6K contained Asp-Ser-Pro-Asp, which had weak resemblance to the caspase-3 cleavage recognition motif Asp-Glu-Xaa-Asp, the mutation of Asp at 396 to Ala had no effect on caspase-3-mediated cleavage of p70S6K. Using an antibody that recognizes the N-terminal domain of p70S6K, we have demonstrated that the cleavage of p70S6K generates a fragment of an Firsocostat approximate molecular mass of 45-kDa. We have shown that treatment of translated p70S6K with human recombinant caspase-3 generated two fragments of approximate molecular mass 45-kDa and 20-kDa, representing the cleavage of full-length protein into two fragments. Therefore, we mutated several Asp residues Firsocostat that may serve as recognition sites for caspase-3, and identified Thr-Pro-Val-Asp-Ser as the cleavage site for caspase-3. The discovery of substrate cleavage by caspase at non-canonical sites is now becoming increasingly common (35-38). It has been reported that caspase-3 is more tolerant to variations of the cleavage site and the presence of Asp at the P-4 position is not absolutely necessary (36). We have, however, detected a minor cleavage fragment above the major N-terminal cleavage product when in vitro translated EE-p70S6K was incubated with human recombinant caspase-3 (Figure 3). In addition, we could detect a faint band corresponding to the cleavage fragment of p70S6K when HeLa cells expressing D393A mutant p70S6K were treated with cisplatin (Figure 6b). It is conceivable that cleavage of p70S6K at D393 or mutation of Asp-393 to Ala exposes other caspase cleavage sites as we have seen previously during caspase-7-mediated cleavage of PKC (38). Since p70S6K is cleaved by caspase-3, the cleavage of p70S6K is a part of the apoptotic process. Our results suggest that the proteolytic cleavage of p70S6K can also contribute to cisplatin-induced apoptosis in several cell lines. However, it remains to be seen if this is a general phenomenon or if it is cell type-dependent. The mutation of Asp 393 residue at the caspase cleavage site to Ala attenuated cisplatin-induced apoptosis. Since the N-terminal fragment of p70S6K was the major cleavage product, we also generated the N-terminal domain by deleting the amino acid residues after caspase-3 cleavage site at 393 to directly demonstrate the importance of proteolytic cleavage of p70S6K on apoptosis. Introduction of the N-terminal domain alone was sufficient to induce cell death and it further enhanced cisplatin-induced cell death. p70S6K regulates multiple cellular functions, including cell proliferation, protein translation and autophagy. Future studies should determine if the ability of the cleaved p70S6K to enhance apoptosis is due to its ability to influence autophagy. Since.The EMBO journal. recombinant caspase-3. Cisplatin failed to induce cleavage of p70S6K in MCF-7 cells that lack functional caspase-3 but ectopic expression of caspase-3 in MCF-7 cells resulted in the cleavage of p70S6K. p70S6K was primarily cleaved at a noncanonical recognition site Thr-Pro-Val-Asp, after Asp-393. Site-directed mutagenesis of Asp-393 to Ala resulted in protection against cisplatin-mediated apoptosis whereas introduction of the N-terminal cleaved fragment resulted in potentiation of cisplatin-induced apoptosis. These results suggest that p70S6K is a novel substrate for caspase-3 and that the proteolytic cleavage of p70S6K is important for cisplatin-induced apoptosis. cleavage assay with human recombinant caspase-3 demonstrated that caspase-3 is capable of cleaving p70S6K directly. Furthermore, treatment with as high as 50 M cisplatin had no effect on the cleavage of p70S6K in MCF-7 cells that lack practical caspase-3 but overexpression of caspase-3 in MCF-7 cells resulted in proteolytic cleavage of p70S6K in response to cisplatin. Cleavage of p70S6K was observed in several different cell lines, including A549, H69, H358 and HeLa cells and in response to several different apoptotic stimuli, including cisplatin, doxorubicin, TNF and TRAIL (data not demonstrated), suggesting that proteolytic cleavage of p70S6K during apoptosis is definitely a general trend. Cleavage of substrates by caspases may result in their activation or inactivation but there are also proteins that are cleaved with the cleavage having no effect on their functions (23, 30-32). These caspase substrates have been described as innocent bystanders (24, 30). Therefore, to examine the practical significance of caspase-3-mediated p70S6K cleavage, we 1st wanted to determine the site at which p70S6K is definitely cleaved. Active caspases cleave important proteins by realizing a set of four neighboring amino acids in their substrate termed P4-P3-P2-P1 and have a stringent requirement for aspartic acid in the P1 position (13, 25, 33, 34). Although p70S6K contained Asp-Ser-Pro-Asp, which experienced weak resemblance to the caspase-3 cleavage acknowledgement motif Asp-Glu-Xaa-Asp, the mutation of Asp at 396 to Ala experienced no effect on caspase-3-mediated cleavage of p70S6K. Using an antibody that recognizes the N-terminal website of p70S6K, we have demonstrated the cleavage of p70S6K produces a fragment of an approximate molecular mass of 45-kDa. We have demonstrated that treatment of translated p70S6K with human being recombinant caspase-3 generated two fragments of approximate molecular mass 45-kDa and 20-kDa, representing the cleavage of full-length protein into two fragments. Consequently, we mutated several Asp residues that may serve as acknowledgement sites for caspase-3, and recognized Thr-Pro-Val-Asp-Ser as the cleavage site for caspase-3. The finding of substrate cleavage by caspase at non-canonical sites is now becoming increasingly common (35-38). It has been reported that caspase-3 is definitely more tolerant to variations of the cleavage site and the presence of Asp in the P-4 position is not absolutely necessary (36). We have, however, detected a minor cleavage fragment above the major N-terminal cleavage product when in vitro translated EE-p70S6K was incubated with human being recombinant caspase-3 (Number 3). In addition, we could detect a faint band corresponding to the cleavage fragment of p70S6K when HeLa cells expressing D393A mutant p70S6K were treated with cisplatin (Number 6b). It is conceivable that cleavage of p70S6K at D393 or mutation of Asp-393 to Ala exposes additional caspase cleavage sites as we have seen previously during caspase-7-mediated cleavage of PKC (38). Since p70S6K is definitely cleaved by caspase-3, the cleavage of p70S6K is definitely a part of the apoptotic process. Our results suggest that the proteolytic cleavage of p70S6K can also contribute to cisplatin-induced apoptosis in several cell lines. However, it remains to be seen if this is a general phenomenon or if it Firsocostat is cell type-dependent. The mutation of Asp 393 residue in the caspase cleavage site to Ala attenuated cisplatin-induced apoptosis. Since the N-terminal fragment of p70S6K was the major cleavage product, we also generated the N-terminal website by deleting the amino acid residues after caspase-3 cleavage site at 393 to directly demonstrate the importance of proteolytic cleavage of p70S6K on apoptosis. Intro of the N-terminal website alone was adequate to induce cell death and it further enhanced cisplatin-induced cell death. p70S6K regulates multiple cellular functions, including cell proliferation, protein translation and autophagy. Long term studies should determine if the ability of the cleaved p70S6K to enhance apoptosis is due.
