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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 in vitro and in vivo. (a) CED-3 cleavage assay of GST-CNT-1a. Left, 35S-Methionine labeled GST-CNT-1a and its mutant derivatives were incubated with or without CED-3 and resolved by 15% SDS polyacrylamide gel (PAGE). CED-3 cleavage products are indicated by arrows. Right, a schematic diagram of GST-CNT-1a with the sizes of the CED-3 cleavage products indicated (tCNT-1a, CNT-1a F2, and CNT-1a F3). Three predicted CED-3 cleavage sites are indicated with arrows. The black box at the N-terminus represents the region that is different from CNT-1b. The Lysine residue in the PH domain (K284) critical for lipid binding is indicated with an asterisk. (b) CNT-1b cleavage by CED-3. Left, GST-CNT-1b and its mutant derivatives were synthesized, labeled, digested with CED-3, and resolved on SDS-PAGE as described in a. Right, a schematic diagram of GST-CNT-1b with the sizes of the CED-3 cleavage products indicated. The gray box at the N-terminus represents the region that is different from CNT-1a. (c) Immunoblotting analysis of C. elegans embryos to show CED-3-mediated cleavage of CNT-1 in vivo. Left, embryos with the indicated genotype were treated with or without heat-shock, lysed and resolved on SDS-PAGE (see METHODS). Antibodies used were anti-CNT-1 antibody (upper panel) or an anti-alpha-tubulin antibody (lower panel)(Uncropped image in Supplementary Fig. 1e). Right, a schematic diagram of CNT-1a, tCNT-1a, CNT-1b, and tCNT-1b and their sizes.
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Figure 2: Cleavage of CNT-1 by CED-3 in vitro and in vivo. (a) CED-3 cleavage assay of GST-CNT-1a. Left, 35S-Methionine labeled GST-CNT-1a and its mutant derivatives were incubated with or without CED-3 and resolved by 15% SDS polyacrylamide gel (PAGE). CED-3 cleavage products are indicated by arrows. Right, a schematic diagram of GST-CNT-1a with the sizes of the CED-3 cleavage products indicated (tCNT-1a, CNT-1a F2, and CNT-1a F3). Three predicted CED-3 cleavage sites are indicated with arrows. The black box at the N-terminus represents the region that is different from CNT-1b. The Lysine residue in the PH domain (K284) critical for lipid binding is indicated with an asterisk. (b) CNT-1b cleavage by CED-3. Left, GST-CNT-1b and its mutant derivatives were synthesized, labeled, digested with CED-3, and resolved on SDS-PAGE as described in a. Right, a schematic diagram of GST-CNT-1b with the sizes of the CED-3 cleavage products indicated. The gray box at the N-terminus represents the region that is different from CNT-1a. (c) Immunoblotting analysis of C. elegans embryos to show CED-3-mediated cleavage of CNT-1 in vivo. Left, embryos with the indicated genotype were treated with or without heat-shock, lysed and resolved on SDS-PAGE (see METHODS). Antibodies used were anti-CNT-1 antibody (upper panel) or an anti-alpha-tubulin antibody (lower panel)(Uncropped image in Supplementary Fig. 1e). Right, a schematic diagram of CNT-1a, tCNT-1a, CNT-1b, and tCNT-1b and their sizes.

Mentions: Given that cnt-1 acts downstream of ced-3, we tested whether CNT-1 is a substrate of CED-3 protease. We incubated 35S-Methionine labeled glutathione S-transferase (GST) fusion with the longer CNT-1 isoform, GST-CNT-1a, with CED-3 and found that it was cleaved by CED-3, yielding two species of approximately 68 and 24 kilodalton (kDa) (Fig. 2a). Since GST-CNT-1a is 116 kDa, this cleavage pattern suggests that the 24 kDa species might contain two cleavage products of similar sizes and that CNT-1a is cleaved at two different sites. Based on the consensus CED-3 cleavage sites (DXXD; X can be any amino acid)40, we identified three potential CED-3 cleavage sites, Asp382 (DSVD), Asp508 (DSID), and Asp609 (DVQD). We then generated Aspartic acid to Glutamic acid substitutions in each potential cleavage site, which is named D1, D2 and D3, respectively, and tested CED-3 cleavage of the mutant proteins. The D1E mutation completely abolished cleavage of CNT-1a by CED-3 (Fig. 2a), suggesting that D1 is required for CED-3 cleavage. The D2E mutation did not alter the cleavage pattern (Fig. 2a), indicating that D2 is not a CED-3 cleavage site in vitro. The D3E mutation abolished the 24 kDa species, and instead, produced a new 47 kDa band along with the 68 kDa band (Fig. 2a), confirming that CED-3 cleavage at D3 generates two cleavage products of similar sizes (23 and 24 kDa). Because D1E also blocked cleavage at the D3 site, CED-3 cleavage at the D1 site is required for cleavage at the D3 site. In CNT-1b, a shorter isoform of CNT-1 that has the same 564 amino acid C-terminus, including a pleckstrin homology (PH) domain, but has a different 177-residue N-terminal sequence (Fig. 2b), three identical CED-3 cleavage sites were found (Asp298, Asp424, and Asp525). We observed similar CED-3 cleavage patterns with GST-CNT-1b and GST-CNT-1b mutants carrying D1E, D2E and D3E, respectively (Fig. 2b). These data indicate that D1 is the primary CED-3 cleavage site in CNT-1 and is required for subsequent CED-3 cleavage at D3.


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 in vitro and in vivo. (a) CED-3 cleavage assay of GST-CNT-1a. Left, 35S-Methionine labeled GST-CNT-1a and its mutant derivatives were incubated with or without CED-3 and resolved by 15% SDS polyacrylamide gel (PAGE). CED-3 cleavage products are indicated by arrows. Right, a schematic diagram of GST-CNT-1a with the sizes of the CED-3 cleavage products indicated (tCNT-1a, CNT-1a F2, and CNT-1a F3). Three predicted CED-3 cleavage sites are indicated with arrows. The black box at the N-terminus represents the region that is different from CNT-1b. The Lysine residue in the PH domain (K284) critical for lipid binding is indicated with an asterisk. (b) CNT-1b cleavage by CED-3. Left, GST-CNT-1b and its mutant derivatives were synthesized, labeled, digested with CED-3, and resolved on SDS-PAGE as described in a. Right, a schematic diagram of GST-CNT-1b with the sizes of the CED-3 cleavage products indicated. The gray box at the N-terminus represents the region that is different from CNT-1a. (c) Immunoblotting analysis of C. elegans embryos to show CED-3-mediated cleavage of CNT-1 in vivo. Left, embryos with the indicated genotype were treated with or without heat-shock, lysed and resolved on SDS-PAGE (see METHODS). Antibodies used were anti-CNT-1 antibody (upper panel) or an anti-alpha-tubulin antibody (lower panel)(Uncropped image in Supplementary Fig. 1e). Right, a schematic diagram of CNT-1a, tCNT-1a, CNT-1b, and tCNT-1b and their sizes.
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Figure 2: Cleavage of CNT-1 by CED-3 in vitro and in vivo. (a) CED-3 cleavage assay of GST-CNT-1a. Left, 35S-Methionine labeled GST-CNT-1a and its mutant derivatives were incubated with or without CED-3 and resolved by 15% SDS polyacrylamide gel (PAGE). CED-3 cleavage products are indicated by arrows. Right, a schematic diagram of GST-CNT-1a with the sizes of the CED-3 cleavage products indicated (tCNT-1a, CNT-1a F2, and CNT-1a F3). Three predicted CED-3 cleavage sites are indicated with arrows. The black box at the N-terminus represents the region that is different from CNT-1b. The Lysine residue in the PH domain (K284) critical for lipid binding is indicated with an asterisk. (b) CNT-1b cleavage by CED-3. Left, GST-CNT-1b and its mutant derivatives were synthesized, labeled, digested with CED-3, and resolved on SDS-PAGE as described in a. Right, a schematic diagram of GST-CNT-1b with the sizes of the CED-3 cleavage products indicated. The gray box at the N-terminus represents the region that is different from CNT-1a. (c) Immunoblotting analysis of C. elegans embryos to show CED-3-mediated cleavage of CNT-1 in vivo. Left, embryos with the indicated genotype were treated with or without heat-shock, lysed and resolved on SDS-PAGE (see METHODS). Antibodies used were anti-CNT-1 antibody (upper panel) or an anti-alpha-tubulin antibody (lower panel)(Uncropped image in Supplementary Fig. 1e). Right, a schematic diagram of CNT-1a, tCNT-1a, CNT-1b, and tCNT-1b and their sizes.
Mentions: Given that cnt-1 acts downstream of ced-3, we tested whether CNT-1 is a substrate of CED-3 protease. We incubated 35S-Methionine labeled glutathione S-transferase (GST) fusion with the longer CNT-1 isoform, GST-CNT-1a, with CED-3 and found that it was cleaved by CED-3, yielding two species of approximately 68 and 24 kilodalton (kDa) (Fig. 2a). Since GST-CNT-1a is 116 kDa, this cleavage pattern suggests that the 24 kDa species might contain two cleavage products of similar sizes and that CNT-1a is cleaved at two different sites. Based on the consensus CED-3 cleavage sites (DXXD; X can be any amino acid)40, we identified three potential CED-3 cleavage sites, Asp382 (DSVD), Asp508 (DSID), and Asp609 (DVQD). We then generated Aspartic acid to Glutamic acid substitutions in each potential cleavage site, which is named D1, D2 and D3, respectively, and tested CED-3 cleavage of the mutant proteins. The D1E mutation completely abolished cleavage of CNT-1a by CED-3 (Fig. 2a), suggesting that D1 is required for CED-3 cleavage. The D2E mutation did not alter the cleavage pattern (Fig. 2a), indicating that D2 is not a CED-3 cleavage site in vitro. The D3E mutation abolished the 24 kDa species, and instead, produced a new 47 kDa band along with the 68 kDa band (Fig. 2a), confirming that CED-3 cleavage at D3 generates two cleavage products of similar sizes (23 and 24 kDa). Because D1E also blocked cleavage at the D3 site, CED-3 cleavage at the D1 site is required for cleavage at the D3 site. In CNT-1b, a shorter isoform of CNT-1 that has the same 564 amino acid C-terminus, including a pleckstrin homology (PH) domain, but has a different 177-residue N-terminal sequence (Fig. 2b), three identical CED-3 cleavage sites were found (Asp298, Asp424, and Asp525). We observed similar CED-3 cleavage patterns with GST-CNT-1b and GST-CNT-1b mutants carrying D1E, D2E and D3E, respectively (Fig. 2b). These data indicate that D1 is the primary CED-3 cleavage site in CNT-1 and is required for subsequent CED-3 cleavage at D3.

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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Related in: MedlinePlus