Limits...
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.

Show MeSH

Related in: MedlinePlus

CED-3-dependent translocation of CNT-1 to the plasma membrane blocks AKT-1 membrane recruitment. (a–e) Immunostaining of CNT-1 in C. elegans embryos. FITC (left) and Differential Interference Contrast (DIC, right) images of the stained embryos with the indicated genotype were shown. –HS, no heat-shock treatment. +HS, with heat-shock treatment. (f–j) Microscopy analysis of AKT-1-PH::GFP localization in C. elegans embryos. All strains contained the same ex[Psur-5AKT-1-PH::GFP] transgenic array. Embryos with the indicated genotype were examined with or without heat-shock treatment. GFP (left) and DIC (right) images of transgenic embryos were shown. In all panels (a–j), scale bar represents 10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4256149&req=5

Figure 7: CED-3-dependent translocation of CNT-1 to the plasma membrane blocks AKT-1 membrane recruitment. (a–e) Immunostaining of CNT-1 in C. elegans embryos. FITC (left) and Differential Interference Contrast (DIC, right) images of the stained embryos with the indicated genotype were shown. –HS, no heat-shock treatment. +HS, with heat-shock treatment. (f–j) Microscopy analysis of AKT-1-PH::GFP localization in C. elegans embryos. All strains contained the same ex[Psur-5AKT-1-PH::GFP] transgenic array. Embryos with the indicated genotype were examined with or without heat-shock treatment. GFP (left) and DIC (right) images of transgenic embryos were shown. In all panels (a–j), scale bar represents 10 μm.

Mentions: Several proteins containing the PH domain translocate from cytosol to plasma membrane via a PIP3-mediated mechanism45–47. We analyzed subcellular localization of CNT-1 using an antibody raised against tCNT-1b (Supplementary Fig. 1d). CNT-1 localized in the cytoplasm of all cells in wild type embryos (Fig. 7a) but was not detected in cnt-1(tm2313) embryos (Fig. 7b), demonstrating the specificity of the antibody. In smIs82 (PhspEGL-1) embryos that were induced to undergo global apoptosis and widespread CED-3 activation through heat-shock treatment, CNT-1 staining was observed on the plasma membrane of all cells, in addition to staining in the cytoplasm (Fig. 7d and Supplementary Fig. 7c, d), indicating that a portion of CNT-1 translocated from cytosol to plasma membrane upon apoptosis activation. By contrast, in smIs82 embryos without heat-shock treatment or in smIs82; ced-3(n717) embryos with heat-shock treatment, CNT-1 remained in the cytoplasm (Fig. 7c, e and Supplementary Fig. 7a, b), indicating that CNT-1 translocation from cytosol to plasma membrane is apoptosis- and CED-3-dependent. In cnt-1(tm2313); smIs82 embryos expressing CNT-1a(D1E), the CED-3 uncleavable form of CNT-1a, CNT-1a(D1E) did not translocate to the plasma membrane after heat-shock treatment (Supplementary Fig. 7g–i). By contrast, in cnt-1(tm2313); smIs82 embryos expressing CNT-1a, CNT-1a was observed on plasma membrane after heat-shock treatment (Supplementary Fig. 7j–l). These results confirm that CED-3 cleavage is required for CNT-1 translocation to plasma membrane during apoptosis. Consistently, loss of cnt-1 partially suppressed and expression of tCNT-1a enhanced ectopic cell death induced by smIs82 (Supplementary text and Supplementary Fig. 4c d), providing further support to the finding that cleavage of CNT-1 and activation of tCNT-1 is an important downstream event of CED-3 activation and 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-dependent translocation of CNT-1 to the plasma membrane blocks AKT-1 membrane recruitment. (a–e) Immunostaining of CNT-1 in C. elegans embryos. FITC (left) and Differential Interference Contrast (DIC, right) images of the stained embryos with the indicated genotype were shown. –HS, no heat-shock treatment. +HS, with heat-shock treatment. (f–j) Microscopy analysis of AKT-1-PH::GFP localization in C. elegans embryos. All strains contained the same ex[Psur-5AKT-1-PH::GFP] transgenic array. Embryos with the indicated genotype were examined with or without heat-shock treatment. GFP (left) and DIC (right) images of transgenic embryos were shown. In all panels (a–j), scale bar represents 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4256149&req=5

Figure 7: CED-3-dependent translocation of CNT-1 to the plasma membrane blocks AKT-1 membrane recruitment. (a–e) Immunostaining of CNT-1 in C. elegans embryos. FITC (left) and Differential Interference Contrast (DIC, right) images of the stained embryos with the indicated genotype were shown. –HS, no heat-shock treatment. +HS, with heat-shock treatment. (f–j) Microscopy analysis of AKT-1-PH::GFP localization in C. elegans embryos. All strains contained the same ex[Psur-5AKT-1-PH::GFP] transgenic array. Embryos with the indicated genotype were examined with or without heat-shock treatment. GFP (left) and DIC (right) images of transgenic embryos were shown. In all panels (a–j), scale bar represents 10 μm.
Mentions: Several proteins containing the PH domain translocate from cytosol to plasma membrane via a PIP3-mediated mechanism45–47. We analyzed subcellular localization of CNT-1 using an antibody raised against tCNT-1b (Supplementary Fig. 1d). CNT-1 localized in the cytoplasm of all cells in wild type embryos (Fig. 7a) but was not detected in cnt-1(tm2313) embryos (Fig. 7b), demonstrating the specificity of the antibody. In smIs82 (PhspEGL-1) embryos that were induced to undergo global apoptosis and widespread CED-3 activation through heat-shock treatment, CNT-1 staining was observed on the plasma membrane of all cells, in addition to staining in the cytoplasm (Fig. 7d and Supplementary Fig. 7c, d), indicating that a portion of CNT-1 translocated from cytosol to plasma membrane upon apoptosis activation. By contrast, in smIs82 embryos without heat-shock treatment or in smIs82; ced-3(n717) embryos with heat-shock treatment, CNT-1 remained in the cytoplasm (Fig. 7c, e and Supplementary Fig. 7a, b), indicating that CNT-1 translocation from cytosol to plasma membrane is apoptosis- and CED-3-dependent. In cnt-1(tm2313); smIs82 embryos expressing CNT-1a(D1E), the CED-3 uncleavable form of CNT-1a, CNT-1a(D1E) did not translocate to the plasma membrane after heat-shock treatment (Supplementary Fig. 7g–i). By contrast, in cnt-1(tm2313); smIs82 embryos expressing CNT-1a, CNT-1a was observed on plasma membrane after heat-shock treatment (Supplementary Fig. 7j–l). These results confirm that CED-3 cleavage is required for CNT-1 translocation to plasma membrane during apoptosis. Consistently, loss of cnt-1 partially suppressed and expression of tCNT-1a enhanced ectopic cell death induced by smIs82 (Supplementary text and Supplementary Fig. 4c d), providing further support to the finding that cleavage of CNT-1 and activation of tCNT-1 is an important downstream event of CED-3 activation and 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