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. 2001 Aug;21(16):5346-58.
doi: 10.1128/MCB.21.16.5346-5358.2001.

Antiapoptotic signaling generated by caspase-induced cleavage of RasGAP

J Y Yang  1 , C Widmann
Affiliations

Antiapoptotic signaling generated by caspase-induced cleavage of RasGAP

J Y Yang et al. Mol Cell Biol. 2001 Aug.

Abstract

Activation of caspases 3 and 9 is thought to commit a cell irreversibly to apoptosis. There are, however, several documented situations (e.g., during erythroblast differentiation) in which caspases are activated and caspase substrates are cleaved with no associated apoptotic response. Why the cleavage of caspase substrates leads to cell death in certain cases but not in others is unclear. One possibility is that some caspase substrates generate antiapoptotic signals when cleaved. Here we show that RasGAP is one such protein. Caspases cleave RasGAP into a C-terminal fragment (fragment C) and an N-terminal fragment (fragment N). Fragment C expressed alone induces apoptosis, but this effect could be totally blocked by fragment N. Fragment N could also block apoptosis induced by low levels of caspase 9. As caspase activity increases, fragment N is further cleaved into fragments N1 and N2. Apoptosis induced by high levels of caspase 9 or by cisplatin was strongly potentiated by fragment N1 or N2 but not by fragment N. The present study supports a model in which RasGAP functions as a sensor of caspase activity to determine whether or not a cell should survive. When caspases are mildly activated, the partial cleavage of RasGAP protects cells from apoptosis. When caspase activity reaches levels that allow completion of RasGAP cleavage, the resulting RasGAP fragments turn into potent proapoptotic molecules.

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Figures

FIG. 1
FIG. 1
Characterization of the RasGAP cleavage sites. (A) Jurkat cells were stimulated with anti-Fas antibodies for the indicated periods of time. Cell lysates (200 μg) were analyzed by Western blotting using antibodies specific for either the amino or the carboxy terminus of RasGAP. (B) In vitro-translated RasGAP (wild type [wt] or D455→A mutant, generated from plasmids HA-GAP.dn3 and HA-D455A.dn3, respectively) was incubated or not with 200 ng of purified caspase 3 for 1 h at 37°C. The reaction mixture was subjected to 10% polyacrylamide gel electrophoresis. The gel was then dried and autoradiographed. (C) In vitro-translated RasGAP mutants (D157→A and D160→A, generated from plasmids HA-D157A.dn3 and HA-D160.dn3, respectively) were incubated for 1 h at 37°C with the indicated quantities of purified caspase 3. The samples were then processed as described for panel B. (D) In vitro-translated wild-type RasGAP (produced as described for panel B) was incubated with the indicated amounts of caspase 3 and further processed as described for panel B. (E) Schematic representation of RasGAP cleavage by caspases. SH, Src homology domain; PH, pleckstrin homology domain; PPPP, proline-rich region; C2, calcium-dependent phospholipid binding domain; GAP domain, GTPase-activating domain.
FIG. 2
FIG. 2
The C-terminal and N-terminal fragments of RasGAP differentially regulate apoptosis and cell shape. HeLa cells were transfected with increasing amounts of plasmids encoding HA-tagged forms of wild-type RasGAP (using plasmid HA-GAP.dn3) or of the indicated RasGAP caspase cleavage fragments (using plasmids GFP-GAPC, HA-GAPN.dn3, HA-N1.dn3, and HA-N2.dn3) together with 1 μg of a plasmid encoding GFP (to visualize the transfected cells). (A) Nomarski and fluorescent images of control cells or cells expressing fragment C or fragment N2. Arrows show cells with pycnotic (condensed) nuclei, and arrowheads show cells that are rounding up. Pycnotic nuclei were commonly observed in spread cells expressing fragment C (e.g., the cell labeled with an asterisk). These cells eventually round up (e.g., the cell labeled with a white dot) and detach. Fragment N2 induced cell rounding with no associated nuclear condensation or fragmentation. (B to G) One day after transfection, the number of fluorescent cells that were round and/or that displayed pycnotic nuclei was determined. In panels C to G, these parameters are expressed as a function of transfected protein levels (as determined by quantitative Western blot analysis using an anti-HA-specific antibody). In panel B, cell lysates corresponding to conditions leading to similar protein expression levels (as determined from panels C to G) were analyzed by Western blotting using an antibody that specifically recognizes the active form of caspase 3 or an antibody that recognizes the caspase-generated p85 fragment of PARP.
FIG. 3
FIG. 3
Fragment N2 does not stimulate cell detachment. HeLa cells were transfected with 2 μg of an empty vector (pcDNA3) or with 2 μg of a plasmid encoding the HA-tagged form of fragment N2 (plasmid HA-N2.dn3) together with 2 μg of a plasmid encoding GFP (to monitor transfected cells). The percentage of round cells and the percentage of detached cells among GFP-positive cells were then scored. These results are the pooled means ± standard deviations from four independent experiments.
FIG. 4
FIG. 4
Fragment C induces the appearance of pycnotic nuclei in a caspase-dependent manner. HeLa cells were transfected with plasmids encoding HA-tagged forms of fragment N2 or fragment C (plasmids HA-N2.dn3 and GFP-GAPC, respectively) together with 1 μg of a plasmid encoding GFP (to monitor transfected cells) in the presence or in the absence of 30 μM z-VAD. One day later, the percentage among transfected cells of round cells with a normal nucleus and the percentage of spread cells with a pycnotic nucleus were determined. These results correspond to the means ± standard errors of the means of duplicate values (this experiment was repeated four times with similar results).
FIG. 5
FIG. 5
The N-terminal fragments inhibit fragment C-induced apoptosis. HeLa cells were transfected with 4 μg of the plasmid encoding HA-tagged fragment C with or without increasing quantities (0, 0.125, 0.25, 0.5, and 1 μg) of plasmids encoding the indicated N-terminal fragments (N, N1, N2, and N1 plus N2, all tagged with HA) or a plasmid encoding β-galactosidase as a specificity control. The closed circle in panel B corresponds to cells transfected only with GFP. The extent of apoptosis was then scored (A and B), and the expression levels of the HA-tagged RasGAP fragments were determined by Western blotting using an anti-HA antibody (C). Transfection of HeLa cells with fragment C resulted in the appearance of two closely migrating bands. The asterisk indicates a nonspecific immunoreactive band that migrates close to fragment N2 (arrow). The experiment depicted in panel B is representative of two independent experiments performed in duplicate. The other experiments are each representative of four independent experiments.
FIG. 6
FIG. 6
The N-terminal RasGAP fragments differentially regulate apoptosis in a manner that is dependent on the levels of caspase 9 expression. HeLa cells were transfected with 1 μg of empty vector (pcDNA3), with plasmids encoding fragments N1 and N2 (HA-N1.dn3 and HA-N2.dn3, 1 μg each), or with 1 μg of a plasmid encoding an uncleavable form of fragment N (N-D157A.dn3) in the presence of increasing amounts of a plasmid encoding caspase 9. The number of transfected cells undergoing apoptosis was then scored (mean ± standard deviation from triplicate determinations). This figure is representative of two different experiments. Fragments N, N1, and N2 inhibited apoptosis induced by low levels of caspase 9, but only fragments N1 and N2 potentiated apoptosis induced by high caspase 9 levels.
FIG. 7
FIG. 7
Fragments N1 and N2 sensitize cells towards DNA damage-induced apoptosis. (A) HeLa cells were transfected as described for Fig. 2 with plasmids encoding HA-tagged forms of RasGAP or the indicated HA-tagged RasGAP caspase cleavage fragments. The amounts of plasmid used for the transfection were adapted so as to result in similar protein expression levels. The cells were treated or not with 0.1 μM cisplatin for 16 to 18 h. The number of transfected cells undergoing apoptosis was then scored. The results are expressed as the mean ± standard deviation from the indicated number of experiments. (B) HeLa cells were transfected with 1 μg of a GFP-expressing plasmid with or without plasmids encoding fragment N1 and N2 (1 μg each) and C (4 μg) in the presence of increasing concentrations of cisplatin. The number of transfected cells undergoing apoptosis was then scored and expressed as the mean ± standard error of the mean from duplicate determinations. This figure is a representative example of eight different experiments. (C) HeLa cells were transfected as for panel B. The cells were then incubated with the indicated cisplatin concentrations for 18 h and lysed, and the presence of activated caspase 3 in the cell lysates was detected by Western blot analysis using an antibody specifically recognizing the active form of the caspase. (D) HeLa cells were incubated with the indicated cisplatin concentrations for 18 h. The cells were then lysed, and the presence of RasGAP, fragment N, and fragment N2 was identified by Western blot analysis using an antibody directed at the SH2-SH3-SH2 domains of RasGAP. (E) HeLa cells were incubated with the indicated cisplatin concentrations for 18 h in the absence (control) or in the presence of 30 μM caspase inhibitor z-VAD. The cells were then processed as described for panel D. Inhibition of caspases blocked the appearance of both fragment N and fragment N2. (F) HeLa cells were transfected with 1 μg of a GFP-expressing plasmid with or without the indicated combinations of plasmids (4 μg of the fragment C-encoding plasmid; 1 μg of the others) in the presence of increasing concentrations of cisplatin. The number of transfected cells undergoing apoptosis was then scored. β-gal, β-galactosidase.
FIG. 8
FIG. 8
Role of Ras in the regulation of apoptosis by the RasGAP fragments. (A) HeLa cells were transfected with 1 μg of a GFP-expressing plasmid together with either empty pcDNA3 vector (control), 4 μg of the fragment C-encoding plasmid, 4 μg of the fragment C-encoding plasmid and 1 μg of the plasmid encoding fragment N, or 4 μg of the fragment C-encoding plasmid and 1 μg of plasmids encoding fragments N1 and N2 in the absence (−) or in the presence of 1 μg of a plasmid encoding the constitutively active V12Ras mutant (V12) or the dominant negative N17Ras mutant (N17). The number of transfected cells undergoing apoptosis was then scored and expressed as the mean ± standard deviation from the number of determinations indicated over the bars. The asterisk denotes a significant difference between the indicated conditions (P < 0.001). (B) HeLa cells were transfected with 1 μg of a GFP-expressing plasmid together with either empty pcDNA3 vector (control) or 1 μg of plasmids encoding fragments N1 and N2 in the absence (−) or in the presence of 1 μg of a plasmid encoding the N17Ras mutant. The cells were then stimulated or not with 0.1 μM cisplatin for 18 h. The number of transfected cells undergoing apoptosis was then scored and expressed as the mean ± standard deviation from four independent determinations. The asterisk denotes a significant difference between the indicated conditions (P < 0.001).
FIG. 9
FIG. 9
Model of the roles of RasGAP caspase cleavage fragments in the regulation of apoptosis. See text for details.
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