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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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CED-3-activated phosphoinositide binding of CNT-1 blocks PIP3 binding by AKT and SGK kinases. (a) Lipid binding assays of CNT-1 in the presence of CED-3. The indicated proteins labeled with 35S-Methionine (*) were incubated with or without CED-3 and then incubated with membrane strips (see METHODS). (b) AKT-1 lipid binding assay in the presence of CNT-1 and CED-3. Unlabeled GST-CNT-1a and GST-CNT-1b were incubated with CED-3 and then added to membrane strips with radiolabeled AKT-1(*) as described in METHODS. (c) AKT-2 and SGK-1 lipid binding assay in the presence of CNT-1 and CED-3. Radiolabeled AKT-2(*) and SGK-1(*) were incubated with membrane strip as described in (b). In all panels, lipids spotted on the strips are: triacylglyceride (TAG), diacylglyceride (DAG), phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylglycerol (PG), cardiolipin (CL), phosphatidylinositol (PI), Phosphatidylinositol (4)-phosphate (PIP), Phosphatidylinositol (4,5)-bisphosphate (PIP2), Phosphatidylinositol (3,4,5)-trisphosphate (PIP3), cholesterol (Chol), sphingomyelin (SM), and 3-sulfogalactosylceramide (SGC).
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Figure 5: CED-3-activated phosphoinositide binding of CNT-1 blocks PIP3 binding by AKT and SGK kinases. (a) Lipid binding assays of CNT-1 in the presence of CED-3. The indicated proteins labeled with 35S-Methionine (*) were incubated with or without CED-3 and then incubated with membrane strips (see METHODS). (b) AKT-1 lipid binding assay in the presence of CNT-1 and CED-3. Unlabeled GST-CNT-1a and GST-CNT-1b were incubated with CED-3 and then added to membrane strips with radiolabeled AKT-1(*) as described in METHODS. (c) AKT-2 and SGK-1 lipid binding assay in the presence of CNT-1 and CED-3. Radiolabeled AKT-2(*) and SGK-1(*) were incubated with membrane strip as described in (b). In all panels, lipids spotted on the strips are: triacylglyceride (TAG), diacylglyceride (DAG), phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylglycerol (PG), cardiolipin (CL), phosphatidylinositol (PI), Phosphatidylinositol (4)-phosphate (PIP), Phosphatidylinositol (4,5)-bisphosphate (PIP2), Phosphatidylinositol (3,4,5)-trisphosphate (PIP3), cholesterol (Chol), sphingomyelin (SM), and 3-sulfogalactosylceramide (SGC).

Mentions: Given that age-1 activates AKT kinases through PIP3 and daf-18 inactivates this pathway through dephosphorylation of PIP3, we hypothesized that tCNT-1, with a PH domain, promotes cell death by interfering with the binding of PIP3 by AKT kinases. We examined whether tCNT-1 binds PIP3 using an in vitro lipid-binding assay. 35S-Met labeled GST-CNT-1a on its own did not bind any lipid, but showed strong binding to PIP, PIP2 and PIP3 and weak binding to phosphatidic acid (PA) and cardiolipin (CL) after pre-treated with CED-3 (Fig. 5a). GST-tCNT-1a displayed an identical lipid-binding pattern to that of CED-3-treated GST-CNT-1a (Fig. 5a). So did GST-CNT-1b pretreated with CED-3 and GST-tCNT-1b (Fig. 5a). When we altered one of the highly conserved, phosphoinositide-binding lysine residues in the PH domain of CNT-1a (K284A)44, neither CNT-1a(K284A) pretreated with CED-3 nor tCNT-1a(K284A) showed any lipid-binding activity (Fig. 5a). Moreover, expression of CNT-1a(K284A) or tCNT-1a(K284A) in cnt-1(tm2313) animals did not rescue the cell death defect or cause ectopic cell death (Fig. 3e, f). Since the K284A mutation did not alter the expression levels of CNT-1a proteins in vitro or in vivo (Supplementary Fig. 5), these results indicate that the PH domain is required for tCNT-1 to acquire phosphoinositide-binding after CED-3 cleavage and to promote apoptosis.


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)

CED-3-activated phosphoinositide binding of CNT-1 blocks PIP3 binding by AKT and SGK kinases. (a) Lipid binding assays of CNT-1 in the presence of CED-3. The indicated proteins labeled with 35S-Methionine (*) were incubated with or without CED-3 and then incubated with membrane strips (see METHODS). (b) AKT-1 lipid binding assay in the presence of CNT-1 and CED-3. Unlabeled GST-CNT-1a and GST-CNT-1b were incubated with CED-3 and then added to membrane strips with radiolabeled AKT-1(*) as described in METHODS. (c) AKT-2 and SGK-1 lipid binding assay in the presence of CNT-1 and CED-3. Radiolabeled AKT-2(*) and SGK-1(*) were incubated with membrane strip as described in (b). In all panels, lipids spotted on the strips are: triacylglyceride (TAG), diacylglyceride (DAG), phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylglycerol (PG), cardiolipin (CL), phosphatidylinositol (PI), Phosphatidylinositol (4)-phosphate (PIP), Phosphatidylinositol (4,5)-bisphosphate (PIP2), Phosphatidylinositol (3,4,5)-trisphosphate (PIP3), cholesterol (Chol), sphingomyelin (SM), and 3-sulfogalactosylceramide (SGC).
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Figure 5: CED-3-activated phosphoinositide binding of CNT-1 blocks PIP3 binding by AKT and SGK kinases. (a) Lipid binding assays of CNT-1 in the presence of CED-3. The indicated proteins labeled with 35S-Methionine (*) were incubated with or without CED-3 and then incubated with membrane strips (see METHODS). (b) AKT-1 lipid binding assay in the presence of CNT-1 and CED-3. Unlabeled GST-CNT-1a and GST-CNT-1b were incubated with CED-3 and then added to membrane strips with radiolabeled AKT-1(*) as described in METHODS. (c) AKT-2 and SGK-1 lipid binding assay in the presence of CNT-1 and CED-3. Radiolabeled AKT-2(*) and SGK-1(*) were incubated with membrane strip as described in (b). In all panels, lipids spotted on the strips are: triacylglyceride (TAG), diacylglyceride (DAG), phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylglycerol (PG), cardiolipin (CL), phosphatidylinositol (PI), Phosphatidylinositol (4)-phosphate (PIP), Phosphatidylinositol (4,5)-bisphosphate (PIP2), Phosphatidylinositol (3,4,5)-trisphosphate (PIP3), cholesterol (Chol), sphingomyelin (SM), and 3-sulfogalactosylceramide (SGC).
Mentions: Given that age-1 activates AKT kinases through PIP3 and daf-18 inactivates this pathway through dephosphorylation of PIP3, we hypothesized that tCNT-1, with a PH domain, promotes cell death by interfering with the binding of PIP3 by AKT kinases. We examined whether tCNT-1 binds PIP3 using an in vitro lipid-binding assay. 35S-Met labeled GST-CNT-1a on its own did not bind any lipid, but showed strong binding to PIP, PIP2 and PIP3 and weak binding to phosphatidic acid (PA) and cardiolipin (CL) after pre-treated with CED-3 (Fig. 5a). GST-tCNT-1a displayed an identical lipid-binding pattern to that of CED-3-treated GST-CNT-1a (Fig. 5a). So did GST-CNT-1b pretreated with CED-3 and GST-tCNT-1b (Fig. 5a). When we altered one of the highly conserved, phosphoinositide-binding lysine residues in the PH domain of CNT-1a (K284A)44, neither CNT-1a(K284A) pretreated with CED-3 nor tCNT-1a(K284A) showed any lipid-binding activity (Fig. 5a). Moreover, expression of CNT-1a(K284A) or tCNT-1a(K284A) in cnt-1(tm2313) animals did not rescue the cell death defect or cause ectopic cell death (Fig. 3e, f). Since the K284A mutation did not alter the expression levels of CNT-1a proteins in vitro or in vivo (Supplementary Fig. 5), these results indicate that the PH domain is required for tCNT-1 to acquire phosphoinositide-binding after CED-3 cleavage and to promote apoptosis.

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