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Differential dependence on Beclin 1 for the regulation of pro-survival autophagy by Bcl-2 and Bcl-xL in HCT116 colorectal cancer cells.

Priault M, Hue E, Marhuenda F, Pilet P, Oliver L, Vallette FM - PLoS ONE (2010)

Bottom Line: Still an intact BH3-binding site was required for Bcl-xL to stimulate a fully functional autophagic pathway.This study highlights that, in addition to their well-established anti-death function during apoptosis, Bcl-2 and Bcl-xL have a broader role in cell survival.Should Bcl-2 and Bcl-xL stand at the cross-roads between pro-survival and pro-death autophagy, this study introduces the new concept that the regulation of autophagy by Bcl-2 and Bcl-xL is adjusted according to its survival or death outcome.

View Article: PubMed Central - PubMed

Affiliation: CNRS IBGC UMR 5095, Bordeaux, France. muriel.priault@ibgc.cnrs.fr

ABSTRACT
Autophagy is described to be involved in homeostasis, development and disease, both as a survival and a death process. Its involvement in cell death proceeds from interrelationships with the apoptotic pathway. We focused on survival autophagy and investigated its interplays with the apoptotic machinery. We found that while Mcl-1 remained ineffective, Bcl-2 and Bcl-xL were required for starved cells to display a fully functional autophagic pathway as shown by proteolysis activity and detection of autophagic vesicles. Such pro-autophagic functions of Bcl-2 and Bcl-xL were independent of Bax. However they appeared to operate through non redundant mechanisms as Bcl-xL wielded a tighter control than Bcl-2 over the regulation of autophagy: unlike Bcl-2, Bcl-xL and Atg7 manipulation yielded identical phenotypes suggesting they could be components of the same signalling pathway; Bcl-xL subcellular localisation was modified upon starvation, and importantly Bcl-xL acted independently of Beclin 1. Still an intact BH3-binding site was required for Bcl-xL to stimulate a fully functional autophagic pathway. This study highlights that, in addition to their well-established anti-death function during apoptosis, Bcl-2 and Bcl-xL have a broader role in cell survival. Should Bcl-2 and Bcl-xL stand at the cross-roads between pro-survival and pro-death autophagy, this study introduces the new concept that the regulation of autophagy by Bcl-2 and Bcl-xL is adjusted according to its survival or death outcome.

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Differences between Bcl-2 and Bcl-xL behaviour during autophagy.(A) Co-immunoprecipitation of endogenous Beclin 1 with endogenous Bcl-2 and Bcl-xL in HCT116-Bax KO cells grown in complete medium or starved for 6 hours. (B) Subcellular fractionation of HCT116-Bax KO cells grown in complete medium or starved for 6 hours. Cells were broken with a Dounce homogeniser, post-nuclear supernatants were loaded on top of a continuous 10–55% sucrose gradient for ultracentrifugation overnight. Fractions of 500 µl were collected, and total protein precipitated. The same volume of each fraction was separated on a 14% tris-tricine SDS-PAGE. Western-blots were as described in the methods section. (C) Immunocytochemistry on endogenous Bcl-xL and ATP1 in HCT116-BaxKO cells grown in complete medium or starved for 6 hours.
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pone-0008755-g005: Differences between Bcl-2 and Bcl-xL behaviour during autophagy.(A) Co-immunoprecipitation of endogenous Beclin 1 with endogenous Bcl-2 and Bcl-xL in HCT116-Bax KO cells grown in complete medium or starved for 6 hours. (B) Subcellular fractionation of HCT116-Bax KO cells grown in complete medium or starved for 6 hours. Cells were broken with a Dounce homogeniser, post-nuclear supernatants were loaded on top of a continuous 10–55% sucrose gradient for ultracentrifugation overnight. Fractions of 500 µl were collected, and total protein precipitated. The same volume of each fraction was separated on a 14% tris-tricine SDS-PAGE. Western-blots were as described in the methods section. (C) Immunocytochemistry on endogenous Bcl-xL and ATP1 in HCT116-BaxKO cells grown in complete medium or starved for 6 hours.

Mentions: In our paradigm of cytoprotective autophagy, endogenous Beclin 1 was immunoprecipitated from control or starved cell lysates (Fig. 5A). Bcl-2 was efficiently pulled down but not Bcl-xL; of note, Bcl-2/Beclin 1 association did not vary in starved versus control cells. The converse immunoprecipitation of endogenous Bcl-2 confirmed these observations, while Bcl-xL again failed to pull-down any detectable Beclin 1. These experiments support a differential regulation of pro-survival autophagy by Bcl-2 and Bcl-xL, but further experiments were needed to ascertain that Bcl-xL acted independently of Beclin 1. Subcellular fractionation showed that excepting the mitochondrial portion of Bcl-2, substantial amounts of Bcl-2 and Beclin 1 overlap in light fractions, and in further keeping with IP results, none of these proteins changed localisation in control or starved cells. In contrast, Bcl-xL was detected throughout the whole gradient in control cells and was relocalised to very light fractions in starved cells; given the number of compartments in which Bcl-xL is present, it is reasonable to assume that it may interact with many partners, and this may account for the fact that Beclin 1 is not found as its main interacting partner through IP experiments. Confocal analyses showed that Bcl-xL co-localised with the mitochondrial inner membrane protein Atp1 in control and starved cells (Fig. 5C); hence the light fractions hosting Bcl-xL in starved cells are in the close vicinity of mitochondria.


Differential dependence on Beclin 1 for the regulation of pro-survival autophagy by Bcl-2 and Bcl-xL in HCT116 colorectal cancer cells.

Priault M, Hue E, Marhuenda F, Pilet P, Oliver L, Vallette FM - PLoS ONE (2010)

Differences between Bcl-2 and Bcl-xL behaviour during autophagy.(A) Co-immunoprecipitation of endogenous Beclin 1 with endogenous Bcl-2 and Bcl-xL in HCT116-Bax KO cells grown in complete medium or starved for 6 hours. (B) Subcellular fractionation of HCT116-Bax KO cells grown in complete medium or starved for 6 hours. Cells were broken with a Dounce homogeniser, post-nuclear supernatants were loaded on top of a continuous 10–55% sucrose gradient for ultracentrifugation overnight. Fractions of 500 µl were collected, and total protein precipitated. The same volume of each fraction was separated on a 14% tris-tricine SDS-PAGE. Western-blots were as described in the methods section. (C) Immunocytochemistry on endogenous Bcl-xL and ATP1 in HCT116-BaxKO cells grown in complete medium or starved for 6 hours.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2807451&req=5

pone-0008755-g005: Differences between Bcl-2 and Bcl-xL behaviour during autophagy.(A) Co-immunoprecipitation of endogenous Beclin 1 with endogenous Bcl-2 and Bcl-xL in HCT116-Bax KO cells grown in complete medium or starved for 6 hours. (B) Subcellular fractionation of HCT116-Bax KO cells grown in complete medium or starved for 6 hours. Cells were broken with a Dounce homogeniser, post-nuclear supernatants were loaded on top of a continuous 10–55% sucrose gradient for ultracentrifugation overnight. Fractions of 500 µl were collected, and total protein precipitated. The same volume of each fraction was separated on a 14% tris-tricine SDS-PAGE. Western-blots were as described in the methods section. (C) Immunocytochemistry on endogenous Bcl-xL and ATP1 in HCT116-BaxKO cells grown in complete medium or starved for 6 hours.
Mentions: In our paradigm of cytoprotective autophagy, endogenous Beclin 1 was immunoprecipitated from control or starved cell lysates (Fig. 5A). Bcl-2 was efficiently pulled down but not Bcl-xL; of note, Bcl-2/Beclin 1 association did not vary in starved versus control cells. The converse immunoprecipitation of endogenous Bcl-2 confirmed these observations, while Bcl-xL again failed to pull-down any detectable Beclin 1. These experiments support a differential regulation of pro-survival autophagy by Bcl-2 and Bcl-xL, but further experiments were needed to ascertain that Bcl-xL acted independently of Beclin 1. Subcellular fractionation showed that excepting the mitochondrial portion of Bcl-2, substantial amounts of Bcl-2 and Beclin 1 overlap in light fractions, and in further keeping with IP results, none of these proteins changed localisation in control or starved cells. In contrast, Bcl-xL was detected throughout the whole gradient in control cells and was relocalised to very light fractions in starved cells; given the number of compartments in which Bcl-xL is present, it is reasonable to assume that it may interact with many partners, and this may account for the fact that Beclin 1 is not found as its main interacting partner through IP experiments. Confocal analyses showed that Bcl-xL co-localised with the mitochondrial inner membrane protein Atp1 in control and starved cells (Fig. 5C); hence the light fractions hosting Bcl-xL in starved cells are in the close vicinity of mitochondria.

Bottom Line: Still an intact BH3-binding site was required for Bcl-xL to stimulate a fully functional autophagic pathway.This study highlights that, in addition to their well-established anti-death function during apoptosis, Bcl-2 and Bcl-xL have a broader role in cell survival.Should Bcl-2 and Bcl-xL stand at the cross-roads between pro-survival and pro-death autophagy, this study introduces the new concept that the regulation of autophagy by Bcl-2 and Bcl-xL is adjusted according to its survival or death outcome.

View Article: PubMed Central - PubMed

Affiliation: CNRS IBGC UMR 5095, Bordeaux, France. muriel.priault@ibgc.cnrs.fr

ABSTRACT
Autophagy is described to be involved in homeostasis, development and disease, both as a survival and a death process. Its involvement in cell death proceeds from interrelationships with the apoptotic pathway. We focused on survival autophagy and investigated its interplays with the apoptotic machinery. We found that while Mcl-1 remained ineffective, Bcl-2 and Bcl-xL were required for starved cells to display a fully functional autophagic pathway as shown by proteolysis activity and detection of autophagic vesicles. Such pro-autophagic functions of Bcl-2 and Bcl-xL were independent of Bax. However they appeared to operate through non redundant mechanisms as Bcl-xL wielded a tighter control than Bcl-2 over the regulation of autophagy: unlike Bcl-2, Bcl-xL and Atg7 manipulation yielded identical phenotypes suggesting they could be components of the same signalling pathway; Bcl-xL subcellular localisation was modified upon starvation, and importantly Bcl-xL acted independently of Beclin 1. Still an intact BH3-binding site was required for Bcl-xL to stimulate a fully functional autophagic pathway. This study highlights that, in addition to their well-established anti-death function during apoptosis, Bcl-2 and Bcl-xL have a broader role in cell survival. Should Bcl-2 and Bcl-xL stand at the cross-roads between pro-survival and pro-death autophagy, this study introduces the new concept that the regulation of autophagy by Bcl-2 and Bcl-xL is adjusted according to its survival or death outcome.

Show MeSH
Related in: MedlinePlus