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The anti-apoptotic activity of BAG3 is restricted by caspases and the proteasome.

Virador VM, Davidson B, Czechowicz J, Mai A, Kassis J, Kohn EC - PLoS ONE (2009)

Bottom Line: Caspase and proteasome inhibition resulted in partial and independent protection of BAG3 whereas inhibitors of both blocked BAG3 degradation.STS-induced apoptosis was increased when BAG3 was silenced, and retention of BAG3 was associated with cytoprotection.The need for dual regulation of BAG3 in apoptosis suggests a key role for BAG3 in cancer cell resistance to apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Molecular Signaling Section, Medical Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, United States of America. vvirador@helix.nih.gov

ABSTRACT

Background: Caspase-mediated cleavage and proteasomal degradation of ubiquitinated proteins are two independent mechanisms for the regulation of protein stability and cellular function. We previously reported BAG3 overexpression protected ubiquitinated clients, such as AKT, from proteasomal degradation and conferred cytoprotection against heat shock. We hypothesized that the BAG3 protein is regulated by proteolysis.

Methodology/principal findings: Staurosporine (STS) was used as a tool to test for caspase involvement in BAG3 degradation. MDA435 and HeLa human cancer cell lines exposed to STS underwent apoptosis with a concomitant time and dose-dependent loss of BAG3, suggesting the survival role of BAG3 was subject to STS regulation. zVAD-fmk or caspase 3 and 9 inhibitors provided a strong but incomplete protection of both cells and BAG3 protein. Two putative caspase cleavage sites were tested: KEVD (BAG3(E345A/D347A)) within the proline-rich center of BAG3 (PXXP) and the C-terminal LEAD site (BAG3(E516A/D518A)). PXXP deletion mutant and BAG3(E345A/D347A), or BAG3(E516A/D518A) respectively slowed or stalled STS-mediated BAG3 loss. BAG3, ubiquitinated under basal growth conditions, underwent augmented ubiquitination upon STS treatment, while there was no increase in ubiquitination of the BAG3(E516A/D518A) caspase-resistant mutant. Caspase and proteasome inhibition resulted in partial and independent protection of BAG3 whereas inhibitors of both blocked BAG3 degradation. STS-induced apoptosis was increased when BAG3 was silenced, and retention of BAG3 was associated with cytoprotection.

Conclusions/significance: BAG3 is tightly controlled by selective degradation during STS exposure. Loss of BAG3 under STS injury required sequential caspase cleavage followed by polyubiquitination and proteasomal degradation. The need for dual regulation of BAG3 in apoptosis suggests a key role for BAG3 in cancer cell resistance to apoptosis.

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Loss or silencing of BAG3 sensitizes cells to apoptosis.A. Cells with intact nuclear morphology retain BAG3 signal. In HeLa with basal expression of BAG3, the appearance of apoptotic bodies (arrows) correlates with the loss of BAG3 green signal (arrowhead) in individual cells. In MDA_FL with forced expression of BAG3 the same phenomenon occurs although at higher STS dose and exposure time. B. PARP cleavage is augmented with BAG3 silencing and progressive STS dose. Adhered and floating MDA435 cells were stained with FITC-conjugated anti-cleaved PARP and anti BAG3 antibodies. Top graph shows BAG3 silencing by the leftward shift of the BAG3 positive population (dark blue line). Bottom graph shows the PARP+ population with BAG3 silencing and 18 h STS exposure (si 18S, dark blue line), compared with 18 h STS exposure but no silencing (NS 18STS, light blue line) along with controls (NS DMSO, si DMSO). C. Quantitation of percent apoptosis after 3.5 or 18 h of STS exposure from the cleaved PARP population (median FL-1) in response to BAG3 silencing combined with STS exposure (3.5 h and 18 h); data represent mean and SEM (n = 3).
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pone-0005136-g002: Loss or silencing of BAG3 sensitizes cells to apoptosis.A. Cells with intact nuclear morphology retain BAG3 signal. In HeLa with basal expression of BAG3, the appearance of apoptotic bodies (arrows) correlates with the loss of BAG3 green signal (arrowhead) in individual cells. In MDA_FL with forced expression of BAG3 the same phenomenon occurs although at higher STS dose and exposure time. B. PARP cleavage is augmented with BAG3 silencing and progressive STS dose. Adhered and floating MDA435 cells were stained with FITC-conjugated anti-cleaved PARP and anti BAG3 antibodies. Top graph shows BAG3 silencing by the leftward shift of the BAG3 positive population (dark blue line). Bottom graph shows the PARP+ population with BAG3 silencing and 18 h STS exposure (si 18S, dark blue line), compared with 18 h STS exposure but no silencing (NS 18STS, light blue line) along with controls (NS DMSO, si DMSO). C. Quantitation of percent apoptosis after 3.5 or 18 h of STS exposure from the cleaved PARP population (median FL-1) in response to BAG3 silencing combined with STS exposure (3.5 h and 18 h); data represent mean and SEM (n = 3).

Mentions: Cells with greater expression of BAG3 were less likely to succumb to STS apoptosis. BAG3 signal was lost first in cells that had nuclear evidence of apoptosis (Figure 2A arrows) yet remained detectable in their morphologically normal neighbors (Figure 2A arrow head). This suggested that loss of BAG3 might be required for STS-mediated cell death. The green BAG3 cytoplasmic signal disappeared at higher STS dose or with longer treatment time in MDA435 cells with forced expression of BAG3 than in wild type HeLa cells with less endogenous BAG3. Direct fluorescence comparison between apoptototic and non-apoptotic cells is difficult due to their different cytosolic volume, thus we followed on this observation by investigating whether silencing BAG3 had an effect on apoptosis. Silencing BAG3 (Fig 2B, top panel and Figure S4) sensitized cells to STS-induced apoptosis, as demonstrated by progressive PARP cleavage under increasing STS exposure and silenced BAG3 (Figure 2B, bottom panel and quantitation in Fig 2C). We observed a consistent but transient induction of BAG3 around 4 hours into STS exposure (Figure S2), suggesting an attempt by the cells to resist injury. However, these levels, even with the brief induction, are not sufficient to overcome cellular commitment to apoptosis. These data indicate loss of BAG3 is necessary but may not be sufficient for STS induced apoptosis.


The anti-apoptotic activity of BAG3 is restricted by caspases and the proteasome.

Virador VM, Davidson B, Czechowicz J, Mai A, Kassis J, Kohn EC - PLoS ONE (2009)

Loss or silencing of BAG3 sensitizes cells to apoptosis.A. Cells with intact nuclear morphology retain BAG3 signal. In HeLa with basal expression of BAG3, the appearance of apoptotic bodies (arrows) correlates with the loss of BAG3 green signal (arrowhead) in individual cells. In MDA_FL with forced expression of BAG3 the same phenomenon occurs although at higher STS dose and exposure time. B. PARP cleavage is augmented with BAG3 silencing and progressive STS dose. Adhered and floating MDA435 cells were stained with FITC-conjugated anti-cleaved PARP and anti BAG3 antibodies. Top graph shows BAG3 silencing by the leftward shift of the BAG3 positive population (dark blue line). Bottom graph shows the PARP+ population with BAG3 silencing and 18 h STS exposure (si 18S, dark blue line), compared with 18 h STS exposure but no silencing (NS 18STS, light blue line) along with controls (NS DMSO, si DMSO). C. Quantitation of percent apoptosis after 3.5 or 18 h of STS exposure from the cleaved PARP population (median FL-1) in response to BAG3 silencing combined with STS exposure (3.5 h and 18 h); data represent mean and SEM (n = 3).
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Related In: Results  -  Collection

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pone-0005136-g002: Loss or silencing of BAG3 sensitizes cells to apoptosis.A. Cells with intact nuclear morphology retain BAG3 signal. In HeLa with basal expression of BAG3, the appearance of apoptotic bodies (arrows) correlates with the loss of BAG3 green signal (arrowhead) in individual cells. In MDA_FL with forced expression of BAG3 the same phenomenon occurs although at higher STS dose and exposure time. B. PARP cleavage is augmented with BAG3 silencing and progressive STS dose. Adhered and floating MDA435 cells were stained with FITC-conjugated anti-cleaved PARP and anti BAG3 antibodies. Top graph shows BAG3 silencing by the leftward shift of the BAG3 positive population (dark blue line). Bottom graph shows the PARP+ population with BAG3 silencing and 18 h STS exposure (si 18S, dark blue line), compared with 18 h STS exposure but no silencing (NS 18STS, light blue line) along with controls (NS DMSO, si DMSO). C. Quantitation of percent apoptosis after 3.5 or 18 h of STS exposure from the cleaved PARP population (median FL-1) in response to BAG3 silencing combined with STS exposure (3.5 h and 18 h); data represent mean and SEM (n = 3).
Mentions: Cells with greater expression of BAG3 were less likely to succumb to STS apoptosis. BAG3 signal was lost first in cells that had nuclear evidence of apoptosis (Figure 2A arrows) yet remained detectable in their morphologically normal neighbors (Figure 2A arrow head). This suggested that loss of BAG3 might be required for STS-mediated cell death. The green BAG3 cytoplasmic signal disappeared at higher STS dose or with longer treatment time in MDA435 cells with forced expression of BAG3 than in wild type HeLa cells with less endogenous BAG3. Direct fluorescence comparison between apoptototic and non-apoptotic cells is difficult due to their different cytosolic volume, thus we followed on this observation by investigating whether silencing BAG3 had an effect on apoptosis. Silencing BAG3 (Fig 2B, top panel and Figure S4) sensitized cells to STS-induced apoptosis, as demonstrated by progressive PARP cleavage under increasing STS exposure and silenced BAG3 (Figure 2B, bottom panel and quantitation in Fig 2C). We observed a consistent but transient induction of BAG3 around 4 hours into STS exposure (Figure S2), suggesting an attempt by the cells to resist injury. However, these levels, even with the brief induction, are not sufficient to overcome cellular commitment to apoptosis. These data indicate loss of BAG3 is necessary but may not be sufficient for STS induced apoptosis.

Bottom Line: Caspase and proteasome inhibition resulted in partial and independent protection of BAG3 whereas inhibitors of both blocked BAG3 degradation.STS-induced apoptosis was increased when BAG3 was silenced, and retention of BAG3 was associated with cytoprotection.The need for dual regulation of BAG3 in apoptosis suggests a key role for BAG3 in cancer cell resistance to apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Molecular Signaling Section, Medical Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, United States of America. vvirador@helix.nih.gov

ABSTRACT

Background: Caspase-mediated cleavage and proteasomal degradation of ubiquitinated proteins are two independent mechanisms for the regulation of protein stability and cellular function. We previously reported BAG3 overexpression protected ubiquitinated clients, such as AKT, from proteasomal degradation and conferred cytoprotection against heat shock. We hypothesized that the BAG3 protein is regulated by proteolysis.

Methodology/principal findings: Staurosporine (STS) was used as a tool to test for caspase involvement in BAG3 degradation. MDA435 and HeLa human cancer cell lines exposed to STS underwent apoptosis with a concomitant time and dose-dependent loss of BAG3, suggesting the survival role of BAG3 was subject to STS regulation. zVAD-fmk or caspase 3 and 9 inhibitors provided a strong but incomplete protection of both cells and BAG3 protein. Two putative caspase cleavage sites were tested: KEVD (BAG3(E345A/D347A)) within the proline-rich center of BAG3 (PXXP) and the C-terminal LEAD site (BAG3(E516A/D518A)). PXXP deletion mutant and BAG3(E345A/D347A), or BAG3(E516A/D518A) respectively slowed or stalled STS-mediated BAG3 loss. BAG3, ubiquitinated under basal growth conditions, underwent augmented ubiquitination upon STS treatment, while there was no increase in ubiquitination of the BAG3(E516A/D518A) caspase-resistant mutant. Caspase and proteasome inhibition resulted in partial and independent protection of BAG3 whereas inhibitors of both blocked BAG3 degradation. STS-induced apoptosis was increased when BAG3 was silenced, and retention of BAG3 was associated with cytoprotection.

Conclusions/significance: BAG3 is tightly controlled by selective degradation during STS exposure. Loss of BAG3 under STS injury required sequential caspase cleavage followed by polyubiquitination and proteasomal degradation. The need for dual regulation of BAG3 in apoptosis suggests a key role for BAG3 in cancer cell resistance to apoptosis.

Show MeSH
Related in: MedlinePlus