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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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Cleavage of CNT-1 by CED-3 activates its proapoptotic activity. Cell corpses were scored in embryos with the indicated genotypes and at the indicated embryonic stages. The y axis represents the 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.
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Figure 3: Cleavage of CNT-1 by CED-3 activates its proapoptotic activity. Cell corpses were scored in embryos with the indicated genotypes and at the indicated embryonic stages. The y axis represents the 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.

Mentions: We determined whether CNT-1 cleavage by CED-3 is important for apoptosis. Expression of CNT-1a or CNT-1b from a globally active dpy-30 promoter fully rescued the delay-of-cell-death defect in cnt-1(tm2313) animals (Fig. 3a and Supplementary Fig. 2a). However, Pdpy-30CNT-1a(D1E) and Pdpy-30CNT-1b(D1E), expressing CNT-1 mutants resistant to CED-3 cleavage, did not rescue the cnt-1(tm2313) mutant (Fig. 3b and Supplementary Fig. 2b), indicating that cleavage of CNT-1 by CED-3 is critical for its proapoptotic function. Expression of tCNT-1a or tCNT-1b, at 20% of the endogenous CNT-1 level (Supplementary Fig. 2c, d), led to increased cell death in most embryonic stages compared to wild type and cnt-1(tm2313) embryos (Fig. 3c and Supplementary Fig. 2e). The increased cell corpses observed in animals carrying Pdpy-30tCNT-1a or Pdpy-30tCNT-1b were due to ectopic cell death, because some pharyngeal cells that normally live were randomly lost in transgenic larvae (Supplementary Table 3). In comparison, co-expression of the other two CNT-1a CED-3 cleavage fragments, CNT-1a F2 and CNT-1a F3 (Fig. 2a), did not induce ectopic cell death or rescue the cnt-1(tm2313) mutant (Fig. 3d), whereas co-expression of all three CED-3 cleavage products did (Supplementary Fig. 2f). These results indicate that tCNT-1, activated by CED-3 cleavage, is responsible for CNT-1’s proapoptotic activity.


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)

Cleavage of CNT-1 by CED-3 activates its proapoptotic activity. Cell corpses were scored in embryos with the indicated genotypes and at the indicated embryonic stages. The y axis represents the 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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4256149&req=5

Figure 3: Cleavage of CNT-1 by CED-3 activates its proapoptotic activity. Cell corpses were scored in embryos with the indicated genotypes and at the indicated embryonic stages. The y axis represents the 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.
Mentions: We determined whether CNT-1 cleavage by CED-3 is important for apoptosis. Expression of CNT-1a or CNT-1b from a globally active dpy-30 promoter fully rescued the delay-of-cell-death defect in cnt-1(tm2313) animals (Fig. 3a and Supplementary Fig. 2a). However, Pdpy-30CNT-1a(D1E) and Pdpy-30CNT-1b(D1E), expressing CNT-1 mutants resistant to CED-3 cleavage, did not rescue the cnt-1(tm2313) mutant (Fig. 3b and Supplementary Fig. 2b), indicating that cleavage of CNT-1 by CED-3 is critical for its proapoptotic function. Expression of tCNT-1a or tCNT-1b, at 20% of the endogenous CNT-1 level (Supplementary Fig. 2c, d), led to increased cell death in most embryonic stages compared to wild type and cnt-1(tm2313) embryos (Fig. 3c and Supplementary Fig. 2e). The increased cell corpses observed in animals carrying Pdpy-30tCNT-1a or Pdpy-30tCNT-1b were due to ectopic cell death, because some pharyngeal cells that normally live were randomly lost in transgenic larvae (Supplementary Table 3). In comparison, co-expression of the other two CNT-1a CED-3 cleavage fragments, CNT-1a F2 and CNT-1a F3 (Fig. 2a), did not induce ectopic cell death or rescue the cnt-1(tm2313) mutant (Fig. 3d), whereas co-expression of all three CED-3 cleavage products did (Supplementary Fig. 2f). These results indicate that tCNT-1, activated by CED-3 cleavage, is responsible for CNT-1’s proapoptotic activity.

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