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

Nature Structural & Molecular Biology volume 21, pages 1082–1090 (2014)Cite this article

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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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Figure 1: Cloning and characterization of cps-2.
Figure 2: Cleavage of CNT-1 by CED-3 in vitro and in vivo.
Figure 3: Cleavage of CNT-1 by CED-3 activates its proapoptotic activity.
Figure 4: CNT-1 acts downstream of AGE-1 but upstream of AKT-1, AKT-2 and SGK-1 to promote apoptosis.
Figure 5: CED-3–activated PI binding of CNT-1 blocks PIP3 binding by AKT and SGK kinases.
Figure 6: PIP3 binding activity of tCNT-1a is two orders of magnitude greater than that of AKT-1.
Figure 7: CED-3–dependent translocation of CNT-1 to the plasma membrane blocks AKT-1 membrane recruitment.
Figure 8: A model of CED-3–activated suppression of AKT signaling by CNT-1 in C. elegans.

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Acknowledgements

We thank B. Derry (Hospital for Sick Children) for anti–AKT-1 antibodies, M. Han (University of Colorado) for some RNAi clones, Y. Kohara (Japan National Institute of Genetics) for akt-1 cDNA, M. Valencia and Y. Shi for making some of the constructs, S. Mitani (Tokyo Women's Medical University) and G. Ruvkun (Massachusetts General Hospital) for strains, R.R. Skeen-Gaar for assistance in generating transgenic strains and B.L. Harry, T. Blumenthal, B. Olwin, and J.M. Espinosa for comments on the manuscript. Some of the worm strains used in this study were kindly provided by the Caenorhabditis Genetics Center, which is funded by the US National Institutes of Health. This work was supported by US National Institutes of Health (grants R01 GM59083, R01 GM79097 and R01 GM088241 to D.X.).

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

    Akihisa Nakagawa, Kelly D Sullivan & Ding Xue

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  1. Akihisa Nakagawa
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  2. Kelly D Sullivan
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Contributions

A.N. and D.X. conceived the research and designed experiments. A.N. carried out and analyzed experiments. K.D.S. assisted in some experiments. A.N. and D.X. wrote the paper.

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Correspondence to Ding Xue.

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Integrated supplementary information

Supplementary Figure 1 Analyses of the cps-2(sm8) mutant and anti–CNT-1 antibodies.

(a) Embryonic cell corpses were scored in the indicated animals. The y axis represents the average number of cell corpses and error bars are S.D. “ex” indicates an extrachromosomal array carrying the indicated construct. Statistical significance values were determined by two-way ANOVA, followed by Bonferroni comparison (n = 15 embryos). *, P < 0.001. All other points had P > 0.05. (b) RT-PCR analysis of N2 and cps-2(sm8) animals. Primer sets used for PCR are indicated with arrowheads in the cartoon. Left panel shows cnt-1 and right panel shows rpl-26 as a control. (c) Immunoblotting of N2 and cps-2(sm8) animals. Left panel is probed with anti-CNT-1 antibody and right panel is probed with anti-α-tubulin antibody. (d) Immunoblotting of recombinant GST-tCNT-1a and GST-tCNT-1b. Left panel is probed with anti-CNT-1 antibody and right panel is probed with anti-GST antibody. (e) The uncropped image of α-tubulin blot shown in Fig. 2c.

Supplementary Figure 2 CNT-1 cleavage by CED-3 is required for its proapoptotic function.

Supplementary Figure 3 Further analysis of the roles of AKT kinase genes, age-1 and daf-16 in C. elegans cell death.

Cell corpses were scored in the indicated strains. The y axis represents average number of cell corpses scored and error bars represent S.D. Statistical significance values were determined by two-way ANOVA, followed by Bonferroni comparison (n = 15 embryos for each stage). *, P < 0.001; **, P < 0.05.

Supplementary Figure 4 Analyses of the importance of AKT-1 and AKT-2 kinase activities and CNT-1 in cell death in C. elegans.

Cell corpses were scored in the indicated strains. The y axis represents average number of cell corpses scored and error bars represent S.D. Statistical significance values were determined by two-way ANOVA, followed by Bonferroni comparison (n = 15 embryos for each stage). *, P < 0.001.

Supplementary Figure 5 Expression levels of CNT-1a(K284A) and tCNT-1a(K284A) are comparable to those of wild-type proteins both in vitro and in vivo.

(a) Autoradiogram of GST-CNT-1a, GST-CNT-1a(K284A), GST-tCNT-1a, and GST-tCNT-1a(K284A) synthesized in rabbit reticulocyte lysate and labeled with 35S-Methionine (*). (b) Immunoblotting of worm lysates using an anti-CNT-1 antibody. Ten transgenic animals of the indicated genotype for each lane were subjected to immunoblotting.

Supplementary Figure 6 The expression level of CNT-1 in C. elegans is higher than that of AKT-1.

(a) Left, immunoblotting of 100 N2 embryos at 1.5-fold stage and 1 pmol of GST-CNT-1a purified from bacteria using an anti-CNT-1 antibody. Right, quantification of the relative CNT-1 amount, which was determined from 3 independent experiments, including the one shown in the left panel, as described in Supplementary Fig. 2d. The data are presented as relative CNT-1 amount (CNT-1a+CNT-1b) and S.D. (b) Left, immunoblotting of 100 N2 embryos at 1.5-fold stage and 1 pmol of AKT-1-His6 purified from bacteria using an anti-AKT-1 antibody. Right, the relative AKT-1 amount was determined as in a. Please see Online Methods for detail. Statistical significance values were determined by two-tailed Student’s t-test (n = 3 independent experiments). *, P < 0.001 (a). P > 0.05 (b).

Supplementary Figure 7 Analyses of CNT-1 protein localization in various embryos.

(a-f) The CNT-1 protein is expressed in all cells during embryogenesis. Embryos with the indicated genotype and developmental stage were stained with an anti-CNT-1 antibody. FITC (left) and DIC (right) images are shown. (g-l) CED-3 uncleavable form of CNT-1a does not translocate to the plasma membrane after activation of CED-3 and apoptosis. Transgenic or non-transgenic embryos with the indicated genotype were stained with an anti-CNT-1 antibody. In all panels, -HS, no heat-shock treatment. +HS, with heat-shock treatment. Scale bars represent 10 mm.

Supplementary Figure 8 The cnt-1(tm2313) deletion does not affect life span or thermotolerance.

Animals with the indicated genotype were subjected to either life span analysis (a) or heat stress resistance analysis (b) (see Online Methods).

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Nakagawa, A., Sullivan, K. & Xue, D. Caspase-activated phosphoinositide binding by CNT-1 promotes apoptosis by inhibiting the AKT pathway. Nat Struct Mol Biol 21, 1082–1090 (2014). https://doi.org/10.1038/nsmb.2915

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