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Caspase-activated phosphoinositide binding by CNT-1 promotes apoptosis by inhibiting the AKT pathway.

Nakagawa A, Sullivan KD, Xue D - Nat. Struct. Mol. Biol. (2014)

Bottom Line: How this pathway is suppressed to promote apoptosis is poorly understood.Here we report the identification of a CED-3 caspase substrate in Caenorhabditis elegans, CNT-1, that is cleaved during apoptosis to generate an N-terminal phosphoinositide-binding fragment (tCNT-1). tCNT-1 translocates from the cytoplasm to the plasma membrane and blocks AKT binding to phosphatidylinositol (3,4,5)-trisphosphate, thereby disabling AKT activation and its prosurvival activity.Our findings reveal a new mechanism that negatively regulates AKT cell signaling to promote apoptosis and that may restrict cell growth and proliferation in normal cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Boulder, Colorado, USA.

ABSTRACT
Inactivation of cell-survival factors is a crucial step in apoptosis. The phosphoinositide 3-kinase (PI3K)-AKT signaling pathway promotes cell growth, proliferation and survival, and its deregulation causes cancer. How this pathway is suppressed to promote apoptosis is poorly understood. Here we report the identification of a CED-3 caspase substrate in Caenorhabditis elegans, CNT-1, that is cleaved during apoptosis to generate an N-terminal phosphoinositide-binding fragment (tCNT-1). tCNT-1 translocates from the cytoplasm to the plasma membrane and blocks AKT binding to phosphatidylinositol (3,4,5)-trisphosphate, thereby disabling AKT activation and its prosurvival activity. Our findings reveal a new mechanism that negatively regulates AKT cell signaling to promote apoptosis and that may restrict cell growth and proliferation in normal cells.

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cnt-1 acts downstream of age-1 but upstream of akt-1, akt-2, and sgk-1 to promote apoptosis. Cell corpses were scored in the indicated strains as in Fig. 3. The y axis represents average number of cell corpses and error bars represent s.d. Statistical significance values were determined by two-way ANOVA, followed by Bonferroni comparison (n = 15 embryos at each stage). * P < 0.001; ** P < 0.05.
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Figure 4: cnt-1 acts downstream of age-1 but upstream of akt-1, akt-2, and sgk-1 to promote apoptosis. Cell corpses were scored in the indicated strains as in Fig. 3. The y axis represents average number of cell corpses and error bars represent s.d. Statistical significance values were determined by two-way ANOVA, followed by Bonferroni comparison (n = 15 embryos at each stage). * P < 0.001; ** P < 0.05.

Mentions: Because the only recognizable motif in tCNT-1 is the PH domain (Fig. 2), a potential phosphoinositide (PI) binding domain41, and because one of the PI species, PIP3, activates AKT kinases (also containing a PH domain) to promote cell survival8,9, we asked whether C. elegans AKT kinases participate in regulating apoptosis and genetically interact with cnt-1. An activating mutation in akt-1, akt-1(mg144gf)22, caused a delay of cell death defect similar to that of the cnt-1(tm2313) mutant (Fig. 4a). Combination of cnt-1(tm2313) and akt-1(mg144gf) did not enhance this phenotype, suggesting that akt-1 and cnt-1 function in the same pathway. Since the ced-3(n2438); akt-1(mg144gf) double mutant had more extra undead cells than ced-3(n2438) or akt-1(mg144gf) single mutants (Supplementary Table 4), this suggests that akt-1 inhibits cell death. Epistasis analysis using animals carrying a Pdpy-30tCNT-1a transgene showed that akt-1(mg144gf) completely blocked ectopic cell death induced by Pdpy-30tCNT-1a (Fig. 4b), indicating that akt-1 likely acts downstream of cnt-1 to inhibit cell death. Consistently, akt-1(mg144gf) suppressed ectopic cell death induced by smIs111, a transgene expressing acCED-3, to the same extent as cnt-1(tm2313)(Fig. 1c), indicating that akt-1 can act downstream of CED-3 to promote cell survival.


Caspase-activated phosphoinositide binding by CNT-1 promotes apoptosis by inhibiting the AKT pathway.

Nakagawa A, Sullivan KD, Xue D - Nat. Struct. Mol. Biol. (2014)

cnt-1 acts downstream of age-1 but upstream of akt-1, akt-2, and sgk-1 to promote apoptosis. Cell corpses were scored in the indicated strains as in Fig. 3. The y axis represents average number of cell corpses and error bars represent s.d. Statistical significance values were determined by two-way ANOVA, followed by Bonferroni comparison (n = 15 embryos at each stage). * P < 0.001; ** P < 0.05.
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Figure 4: cnt-1 acts downstream of age-1 but upstream of akt-1, akt-2, and sgk-1 to promote apoptosis. Cell corpses were scored in the indicated strains as in Fig. 3. The y axis represents average number of cell corpses and error bars represent s.d. Statistical significance values were determined by two-way ANOVA, followed by Bonferroni comparison (n = 15 embryos at each stage). * P < 0.001; ** P < 0.05.
Mentions: Because the only recognizable motif in tCNT-1 is the PH domain (Fig. 2), a potential phosphoinositide (PI) binding domain41, and because one of the PI species, PIP3, activates AKT kinases (also containing a PH domain) to promote cell survival8,9, we asked whether C. elegans AKT kinases participate in regulating apoptosis and genetically interact with cnt-1. An activating mutation in akt-1, akt-1(mg144gf)22, caused a delay of cell death defect similar to that of the cnt-1(tm2313) mutant (Fig. 4a). Combination of cnt-1(tm2313) and akt-1(mg144gf) did not enhance this phenotype, suggesting that akt-1 and cnt-1 function in the same pathway. Since the ced-3(n2438); akt-1(mg144gf) double mutant had more extra undead cells than ced-3(n2438) or akt-1(mg144gf) single mutants (Supplementary Table 4), this suggests that akt-1 inhibits cell death. Epistasis analysis using animals carrying a Pdpy-30tCNT-1a transgene showed that akt-1(mg144gf) completely blocked ectopic cell death induced by Pdpy-30tCNT-1a (Fig. 4b), indicating that akt-1 likely acts downstream of cnt-1 to inhibit cell death. Consistently, akt-1(mg144gf) suppressed ectopic cell death induced by smIs111, a transgene expressing acCED-3, to the same extent as cnt-1(tm2313)(Fig. 1c), indicating that akt-1 can act downstream of CED-3 to promote cell survival.

Bottom Line: How this pathway is suppressed to promote apoptosis is poorly understood.Here we report the identification of a CED-3 caspase substrate in Caenorhabditis elegans, CNT-1, that is cleaved during apoptosis to generate an N-terminal phosphoinositide-binding fragment (tCNT-1). tCNT-1 translocates from the cytoplasm to the plasma membrane and blocks AKT binding to phosphatidylinositol (3,4,5)-trisphosphate, thereby disabling AKT activation and its prosurvival activity.Our findings reveal a new mechanism that negatively regulates AKT cell signaling to promote apoptosis and that may restrict cell growth and proliferation in normal cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Boulder, Colorado, USA.

ABSTRACT
Inactivation of cell-survival factors is a crucial step in apoptosis. The phosphoinositide 3-kinase (PI3K)-AKT signaling pathway promotes cell growth, proliferation and survival, and its deregulation causes cancer. How this pathway is suppressed to promote apoptosis is poorly understood. Here we report the identification of a CED-3 caspase substrate in Caenorhabditis elegans, CNT-1, that is cleaved during apoptosis to generate an N-terminal phosphoinositide-binding fragment (tCNT-1). tCNT-1 translocates from the cytoplasm to the plasma membrane and blocks AKT binding to phosphatidylinositol (3,4,5)-trisphosphate, thereby disabling AKT activation and its prosurvival activity. Our findings reveal a new mechanism that negatively regulates AKT cell signaling to promote apoptosis and that may restrict cell growth and proliferation in normal cells.

Show MeSH
Related in: MedlinePlus