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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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Cloning and characterization of cps-2. (a) Embryonic cell corpses were counted in animals with the indicated genotype. (b) Mapping and cloning of cps-2. The top bar represents the genetic map with two genetic markers (dpy-10 and rol-1) and three SNPs (pkP2069, CE2-196, and Y43F11A[1]) used for mapping cps-2(sm8). Numbers below are the fractions of the 105 recombinant events that occur between two genetic markers and three SNPs, respectively. The bottom panel shows the cnt-1 coding region, the sm8 mutation, the tm2313 deletion, and rescuing results of the cps-2(sm8) mutant by the Pcnt-1CNT-1a::3xFlag and the Pcnt-1(sm8)CNT-1a::3xFlag constructs. Three independent transgenic arrays were examined for each rescue experiment. (c) Embryonic cell corpses were counted in animals with the indicated genotype. In a and c, the y axis represents average number of cell corpses and error bars represent standard deviation (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 1: Cloning and characterization of cps-2. (a) Embryonic cell corpses were counted in animals with the indicated genotype. (b) Mapping and cloning of cps-2. The top bar represents the genetic map with two genetic markers (dpy-10 and rol-1) and three SNPs (pkP2069, CE2-196, and Y43F11A[1]) used for mapping cps-2(sm8). Numbers below are the fractions of the 105 recombinant events that occur between two genetic markers and three SNPs, respectively. The bottom panel shows the cnt-1 coding region, the sm8 mutation, the tm2313 deletion, and rescuing results of the cps-2(sm8) mutant by the Pcnt-1CNT-1a::3xFlag and the Pcnt-1(sm8)CNT-1a::3xFlag constructs. Three independent transgenic arrays were examined for each rescue experiment. (c) Embryonic cell corpses were counted in animals with the indicated genotype. In a and c, the y axis represents average number of cell corpses and error bars represent standard deviation (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: We investigated whether cps-2 affects apoptosis by examining embryonic cell death in cps-2(sm8) animals. Compared with wild type N2 embryos, cps-2(sm8) embryos had fewer apoptotic cell corpses in early stages of embryogenesis (comma and 1.5-fold stages) and more cell corpses in later stages (Fig. 1a), displaying a characteristic delay-of-cell-death phenotype observed in mutants defective in genes acting downstream of ced-37,35–37. Three-point mapping and single-nucleotide polymorphism (SNP) mapping placed cps-2(sm8) at a position of 12,103,628 base pair (bp) on LGII (Fig. 1b). Since there are no available fosmid or cosmid clones in this region for transformation rescue experiments, we performed an RNA interference (RNAi) screen on nine candidate genes in this region and found that RNAi treatment of cnt-1 caused a similar delay-of-cell-death defect (Supplementary Table 1). We then introduced into cps-2(sm8) animals a cnt-1 minigene, which contains a full-length cnt-1 cDNA fused to 1944 bp of the cnt-1 promoter, and found that it rescued the cps-2(sm8) defect (Fig. 1b and Supplementary Fig. 1a). Moreover, an existing cnt-1 deletion mutation (tm2313) caused a similar delay-of-cell-death defect and failed to complement cps-2(sm8) (Fig. 1a), indicating that tm2313 and sm8 are allelic. Sequencing analysis of cps-2(sm8) animals revealed a nucleotide deletion at 927 bp upstream from the cnt-1 translational start, but no mutation in the coding region of cnt-1 and 1031 bp 3′ untranslated region. Since a cnt-1 minigene carrying this nucleotide deletion failed to rescue the cell death defect in cps-2(sm8) animals (Fig. 1b and Supplementary Fig. 1a) and since neither cnt-1 mRNA nor CNT-1 protein was detected in cps-2(sm8) animals (Supplementary Fig. 1b, c), sm8 likely disrupts a cis-element important for cnt-1 expression. These results indicate that cps-2 is cnt-1. cnt-1(tm2313) was thus used in all subsequent experiments.


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)

Cloning and characterization of cps-2. (a) Embryonic cell corpses were counted in animals with the indicated genotype. (b) Mapping and cloning of cps-2. The top bar represents the genetic map with two genetic markers (dpy-10 and rol-1) and three SNPs (pkP2069, CE2-196, and Y43F11A[1]) used for mapping cps-2(sm8). Numbers below are the fractions of the 105 recombinant events that occur between two genetic markers and three SNPs, respectively. The bottom panel shows the cnt-1 coding region, the sm8 mutation, the tm2313 deletion, and rescuing results of the cps-2(sm8) mutant by the Pcnt-1CNT-1a::3xFlag and the Pcnt-1(sm8)CNT-1a::3xFlag constructs. Three independent transgenic arrays were examined for each rescue experiment. (c) Embryonic cell corpses were counted in animals with the indicated genotype. In a and c, the y axis represents average number of cell corpses and error bars represent standard deviation (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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4256149&req=5

Figure 1: Cloning and characterization of cps-2. (a) Embryonic cell corpses were counted in animals with the indicated genotype. (b) Mapping and cloning of cps-2. The top bar represents the genetic map with two genetic markers (dpy-10 and rol-1) and three SNPs (pkP2069, CE2-196, and Y43F11A[1]) used for mapping cps-2(sm8). Numbers below are the fractions of the 105 recombinant events that occur between two genetic markers and three SNPs, respectively. The bottom panel shows the cnt-1 coding region, the sm8 mutation, the tm2313 deletion, and rescuing results of the cps-2(sm8) mutant by the Pcnt-1CNT-1a::3xFlag and the Pcnt-1(sm8)CNT-1a::3xFlag constructs. Three independent transgenic arrays were examined for each rescue experiment. (c) Embryonic cell corpses were counted in animals with the indicated genotype. In a and c, the y axis represents average number of cell corpses and error bars represent standard deviation (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: We investigated whether cps-2 affects apoptosis by examining embryonic cell death in cps-2(sm8) animals. Compared with wild type N2 embryos, cps-2(sm8) embryos had fewer apoptotic cell corpses in early stages of embryogenesis (comma and 1.5-fold stages) and more cell corpses in later stages (Fig. 1a), displaying a characteristic delay-of-cell-death phenotype observed in mutants defective in genes acting downstream of ced-37,35–37. Three-point mapping and single-nucleotide polymorphism (SNP) mapping placed cps-2(sm8) at a position of 12,103,628 base pair (bp) on LGII (Fig. 1b). Since there are no available fosmid or cosmid clones in this region for transformation rescue experiments, we performed an RNA interference (RNAi) screen on nine candidate genes in this region and found that RNAi treatment of cnt-1 caused a similar delay-of-cell-death defect (Supplementary Table 1). We then introduced into cps-2(sm8) animals a cnt-1 minigene, which contains a full-length cnt-1 cDNA fused to 1944 bp of the cnt-1 promoter, and found that it rescued the cps-2(sm8) defect (Fig. 1b and Supplementary Fig. 1a). Moreover, an existing cnt-1 deletion mutation (tm2313) caused a similar delay-of-cell-death defect and failed to complement cps-2(sm8) (Fig. 1a), indicating that tm2313 and sm8 are allelic. Sequencing analysis of cps-2(sm8) animals revealed a nucleotide deletion at 927 bp upstream from the cnt-1 translational start, but no mutation in the coding region of cnt-1 and 1031 bp 3′ untranslated region. Since a cnt-1 minigene carrying this nucleotide deletion failed to rescue the cell death defect in cps-2(sm8) animals (Fig. 1b and Supplementary Fig. 1a) and since neither cnt-1 mRNA nor CNT-1 protein was detected in cps-2(sm8) animals (Supplementary Fig. 1b, c), sm8 likely disrupts a cis-element important for cnt-1 expression. These results indicate that cps-2 is cnt-1. cnt-1(tm2313) was thus used in all subsequent experiments.

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