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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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PIP3 binding activity of tCNT-1a is stronger than that of AKT-1 by two orders of magnitude. (a) Immunoblotting of 5.1 μl of GST-tCNT-1a and 2.9 μl of AKT-1-His6 proteins synthesized and radiolabeled in rabbit reticulocyte lysate (*RRL) and 1 pmol of GST-tCNT-1a or AKT-1-His6 proteins purified from bacteria (Bact) using an anti-GST or an anti-His6 antibody (see METHODS). (b) Autoradiogram of 1 pmol of 35S-Met-labeled GST-tCNT-1a and AKT-1-His6 proteins made in RRL shows comparable radioactive signal intensity. (c) Lipid binding assays of GST-tCNT-1a or AKT-1-His6. Radiolabeled (*) GST-tCNT-1a or AKT-1-His6 with the indicated concentrations were incubated with lipid membrane strips as described in Fig. 5.
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Figure 6: PIP3 binding activity of tCNT-1a is stronger than that of AKT-1 by two orders of magnitude. (a) Immunoblotting of 5.1 μl of GST-tCNT-1a and 2.9 μl of AKT-1-His6 proteins synthesized and radiolabeled in rabbit reticulocyte lysate (*RRL) and 1 pmol of GST-tCNT-1a or AKT-1-His6 proteins purified from bacteria (Bact) using an anti-GST or an anti-His6 antibody (see METHODS). (b) Autoradiogram of 1 pmol of 35S-Met-labeled GST-tCNT-1a and AKT-1-His6 proteins made in RRL shows comparable radioactive signal intensity. (c) Lipid binding assays of GST-tCNT-1a or AKT-1-His6. Radiolabeled (*) GST-tCNT-1a or AKT-1-His6 with the indicated concentrations were incubated with lipid membrane strips as described in Fig. 5.

Mentions: We tested whether tCNT-1 interferes with binding of AKT and SGK kinases to PIP321–23. 35S-Met-labeled AKT-1 alone bound strongly to PIP3 and weakly to cardiolipin and this binding was not affected by addition of unlabeled GST-CNT-1a or GST-CNT-1b (Fig. 5b). Addition of GST-CNT-1a or GST-CNT-1b pre-treated with CED-3 completely blocked PIP3 binding by AKT-1 (Fig. 5b), whereas incubation of AKT-1 with CED-3 did not alter AKT-1 lipid binding (Fig. 5b). Likewise, both AKT-2 and SGK-1 bound strongly to PIP3 and this binding was blocked by GST-CNT-1a pre-treated with CED-3 (Fig. 5c). To probe how tCNT-1 inhibits PIP3 binding by AKT-1, we compared their PIP3 binding affinity. We first determined the concentrations of 35S-Met-labeled GST-tCNT-1a and AKT-1 synthesized in rabbit reticulocyte lysate (RRL) by comparing them with recombinant GST-tCNT-1a and AKT-1 proteins with known concentrations, which were purified from bacteria (Bact) but showed no lipid binding activity (Fig. 6a). Labeled GST-tCNT-1a and AKT-1-His6 displayed comparable radioactive signal intensity at the same concentrations (Fig. 6b). GST-tCNT-1a displayed strong PIP3 binding at 0.04 nM, weak binding at 0.004 nM, and no binding at 0.0004 nM (Fig. 6c). By contrast, AKT-1 showed strong PIP3 binding at 4 nM, weak binding at 0.4 nM, and no binding at 0.04 nM (Fig. 6c), indicating that tCNT-1a binds PIP3 with two order of magnitude higher affinity than AKT-1. Because the expression level of CNT-1 in C. elegans is approximately 70% higher than that of AKT-1 (Supplementary Fig. 6), the combination of higher CNT-1 concentrations and much higher binding affinity to PIP3 than those of AKT-1 allows tCNT-1 to block PIP3 binding by AKT kinases and thus their activation.


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)

PIP3 binding activity of tCNT-1a is stronger than that of AKT-1 by two orders of magnitude. (a) Immunoblotting of 5.1 μl of GST-tCNT-1a and 2.9 μl of AKT-1-His6 proteins synthesized and radiolabeled in rabbit reticulocyte lysate (*RRL) and 1 pmol of GST-tCNT-1a or AKT-1-His6 proteins purified from bacteria (Bact) using an anti-GST or an anti-His6 antibody (see METHODS). (b) Autoradiogram of 1 pmol of 35S-Met-labeled GST-tCNT-1a and AKT-1-His6 proteins made in RRL shows comparable radioactive signal intensity. (c) Lipid binding assays of GST-tCNT-1a or AKT-1-His6. Radiolabeled (*) GST-tCNT-1a or AKT-1-His6 with the indicated concentrations were incubated with lipid membrane strips as described in Fig. 5.
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Figure 6: PIP3 binding activity of tCNT-1a is stronger than that of AKT-1 by two orders of magnitude. (a) Immunoblotting of 5.1 μl of GST-tCNT-1a and 2.9 μl of AKT-1-His6 proteins synthesized and radiolabeled in rabbit reticulocyte lysate (*RRL) and 1 pmol of GST-tCNT-1a or AKT-1-His6 proteins purified from bacteria (Bact) using an anti-GST or an anti-His6 antibody (see METHODS). (b) Autoradiogram of 1 pmol of 35S-Met-labeled GST-tCNT-1a and AKT-1-His6 proteins made in RRL shows comparable radioactive signal intensity. (c) Lipid binding assays of GST-tCNT-1a or AKT-1-His6. Radiolabeled (*) GST-tCNT-1a or AKT-1-His6 with the indicated concentrations were incubated with lipid membrane strips as described in Fig. 5.
Mentions: We tested whether tCNT-1 interferes with binding of AKT and SGK kinases to PIP321–23. 35S-Met-labeled AKT-1 alone bound strongly to PIP3 and weakly to cardiolipin and this binding was not affected by addition of unlabeled GST-CNT-1a or GST-CNT-1b (Fig. 5b). Addition of GST-CNT-1a or GST-CNT-1b pre-treated with CED-3 completely blocked PIP3 binding by AKT-1 (Fig. 5b), whereas incubation of AKT-1 with CED-3 did not alter AKT-1 lipid binding (Fig. 5b). Likewise, both AKT-2 and SGK-1 bound strongly to PIP3 and this binding was blocked by GST-CNT-1a pre-treated with CED-3 (Fig. 5c). To probe how tCNT-1 inhibits PIP3 binding by AKT-1, we compared their PIP3 binding affinity. We first determined the concentrations of 35S-Met-labeled GST-tCNT-1a and AKT-1 synthesized in rabbit reticulocyte lysate (RRL) by comparing them with recombinant GST-tCNT-1a and AKT-1 proteins with known concentrations, which were purified from bacteria (Bact) but showed no lipid binding activity (Fig. 6a). Labeled GST-tCNT-1a and AKT-1-His6 displayed comparable radioactive signal intensity at the same concentrations (Fig. 6b). GST-tCNT-1a displayed strong PIP3 binding at 0.04 nM, weak binding at 0.004 nM, and no binding at 0.0004 nM (Fig. 6c). By contrast, AKT-1 showed strong PIP3 binding at 4 nM, weak binding at 0.4 nM, and no binding at 0.04 nM (Fig. 6c), indicating that tCNT-1a binds PIP3 with two order of magnitude higher affinity than AKT-1. Because the expression level of CNT-1 in C. elegans is approximately 70% higher than that of AKT-1 (Supplementary Fig. 6), the combination of higher CNT-1 concentrations and much higher binding affinity to PIP3 than those of AKT-1 allows tCNT-1 to block PIP3 binding by AKT kinases and thus their activation.

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