Caspase-9 (ICE-LAP6, Mch6) is a member of the CED-3 subfamily and bears similarity to Caspase-3, though there is a main difference in the active-site pentapeptide: Caspase-9 shows the sequence QACGG instead of the more usual QACRG. Compared to Caspase-3, Caspase-9 possesses a longer N-terminal prodomain with high similarity to the prodomains of CED-3 and Caspase-2, which contain CARDs (Duan et al., 1996, J. Biol. Chem., 271:16720-16724, Srinivasula et al., 1996, J. Biol. Chem., 271: 27099-27106). Caspase-9 is ubiquitous expressed, with high levels of mRNA expression in the heart, testis and ovary. Multiple mRNA species are found, due to alternative splicing. Activated Caspase-9 is able to cleave Caspase-3 in vitro (Li et al., 1997, Cell, 91:479-89).
In cytoplasmic extracts of HeLa cells, Caspase-3 is activated in a strictly dATP-dependent manner by the cooperative action of three protein factors, designated apoptotic protease activating factors (Apafs). While Apaf-1 contains a sequence homologous to the C.elegans CED-4, and while Apaf-2 was identified as Cytochrome c, Apaf-3 was shown to be identical with Caspase-9. It also was demonstrated that, in presence of dATP, Caspase-9 is directly activated by Apaf-1 and Cytochrome c. Active Caspase-9 activates Caspase-3 and by this the apoptotic machinery that leads to DNA fragmentation and cell death (Li et al., 1997, Cell, 91:479-89).
Interestingly, it has been reported that Caspase-9 activity is regulated by phosphorylation (Cardone et al., 1998, Science, 282: 1318-1320): the kinase Akt phosphorylates Pro-Caspase-9 at Ser196 and by this inhibits proteolytic processing of pro-Caspase-9. Akt itself is activated by the phosphatidylinositol 3-kinase (PI3K) pathway which is positively controlled by Ras and negatively by the PTEN tumor suppressor.
Two papers reported the knockout of Caspase-9 in mice (Kuida et al., 1998, Cell, 94: 325-337; Hakem et al., 1998, Cell, 9:339-352).
Caspase-9 knockout and embryogenesis
These studies showed, that homozygous Caspase-9 knockout (Casp9 -/-) is in most cases lethal, and that Casp9 -/- newborn mice were consistently smaller than control littermates, and most viable knockout mice died before pnd 3. Casp -/- mice displayed protrusions of the brain tissue from the skull: gross perturbations of brain structure were observed that were most severe within the cortex and the forebrain. No apparent histological abnormalities were noticed in various other tissues of those knockout mice. TUNEL labeling demonstrated substantially less programmed cell death within the developing CNS of Casp9 -/- embryos, but no decrease in apoptosis was observed in brain-associated mesenchymal tissues. Thus, the absence of Caspase-9 results in brain specific malformations. The gross morphological features observed in Casp9 -/- mice are remarkably similar to those observed in mice lacking Caspase-3 (Kuida et al., 1996, Nature, 384: 368-372).
Casp9 -/- Cells and Apoptosis
Hakem et al. (1998, Cell, 9:339-352) propose at least four different apoptotic pathways in mammalian cells:
ES cells from Casp9 -/- knockout mice were resistant to a variety of apoptotic stimuli (e.g. UV, gamma irradiation, etoposide, cisplatinum, adriamycin). Also MEFs from knockout mice were resistant to apoptosis induced by UV and gamma irradiation, adriamycin and etoposide, but still were sensitive to TNF-alpha induced apoptosis (thus, the TNF-induced pathway of apoptosis does not depend on Capase-9). Transfection of Casp9 -/- MEFs with a wildtype human Caspase-9 gene turned them sensitive to adriamycin mediated apoptosis.
Thymocytes and lymphocytes develop normal in Casp9 -/- mice, but are resistant to apoptosis induced by dexamethasone or gamma irradiation (in contrast to Casp3 -/- thymocytes which remain sensitive). Furthermore, Casp9 -/- thymocytes showed resistance to etoposide, but were sensitive to stimuli like anti-Fas, TNF-alpha, UV irradiation, or heat shock. These data indicate that the requirement for Caspase-9 in apoptosis is cell-type and stimulus-specific, indicating the existence of multiple apoptotic pathways.
Caspase-9 and Caspase-3 dependent apoptotic pathway:
This pathway is correlated to the
dATP/Cytochrome c/Apaf-1 induced activation of Caspase-9 and subsequent Caspase-3 activation. UV irradiation of ES cells from Casp9 -/- mice (resistant towards UV), for example, did not induce Caspase-3 processing and PARP cleavage. Thus, UV irradiation preferentially triggers the activation of an apoptotic pathway involving Caspase-9 and Caspase-3. Mutation of Caspase-3 and Caspase-9 produces similar effects on brain apoptosis and development, suggesting thata the pathway dependent on both Caspase-3 and Caspase-9 is physiologically required for PCD in the developing brain.
Caspase-9 and Caspase-3 independent apoptotic pathway:
The requirement for Caspase-9 in apoptosis is cell type- and stimulus specific: unlike ES cells from Casp9 -/- mice, Casp9 -/- thymocytes and splenocytes underwent PCD in response to UV irradiation! Casp3 -/- thymocytes and splenocytes are also sensitive to UV-induced cell death. Thus, it is likely that an apoptotic pathway independent of both Caspase-9 and Caspase-3 exists in vivo.
Caspase-9 independent and Caspase-3 dependent apoptotic pathway:
This pathway focuses on Fas-mediated apoptosis: while recently Caspase-3 deficiency has been shown to protect activated splenocytes from Fas-mediated apoptosis (Woo et al., 1998, Genes Dev. 12: 806-819), Caspase-9 deficiency did not protect activated T cells from Caspase-3 processing and and apoptosis following anti-Fas stimulation. Maybe, other caspases with CARD-prodomains (e.g. Caspase-4 or Caspase-8) substitute for Caspase-9 in this Fas pathway.
Caspase-9 dependent and Caspase-3 independent apoptotic pathway:
Casp3 -/- thymocytes have been shown to be sensitive to all apoptotic stimuli tested, including dexamethasone and gamma-irradiation (Kuida et al., 1996, nature, 384: 368-372). In contrast, Casp9 -/- thymocytes were resistant to PCD induced by dexamethasone or gamma-irradiation!
Kuida et al., 1996, nature, 384: 368-372, Caspase-3 deficiency;
Woo et al., 1998, Genes Dev. 12: 806-819, Caspase-3 deficiency;
Kuida et al., 1998, Cell, 94: 325-337, Caspase-9 deficiency;
Hakem et al., 1998, Cell, 9:339-352, Caspase-9 deficiency;
Li et al., 1997, Cell, 91:479-89, Caspase-9 and Apaf-1 ...;