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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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Sequential inhibition of caspases and proteasome provides collaborative protection of BAG3.A. Proteasome inhibition provides dose-dependent partial protection of BAG3. FL-BAG3 cells were pretreated with 20 µM MG-132 for 1 or 4 hrs prior to exposure to 2 µM STS. Inhibition of proteasomal degradation by pretreatment with MG-132 prior to STS exposure reduced BAG3 degradation. B. BAG3 is ubiquitinated in response to STS treatment. FL-BAG3 cells were treated with 2 µM STS alone or following 4 hr preincubation with 20 µM MG-132. Lysates were immunoprecipitated with anti-BAG3 and the immunoblots were probed for ubiquitin. BAG3 was ubiquitinated and degraded in the absence of proteasome inhibition. C. STS-induced loss of BAG3 is minimized by the combination of caspase and proteasomal inhibition. FL-BAG3 cells were preincubated with 0.05% DMSO vehicle or 20 µM MG-132 for 4 hours with or without 1 additional hour of zVAD preincubation (85 µM). Treatments were followed by a total STS exposure of up to 20 hrs. A progressive retention of BAG3 was seen with inclusion of each inhibitor. Relative levels of BAG3, determined using ImageJ™, are indicated below the respective blots and plotted as percent remaining BAG3 with increasing STS time.
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pone-0005136-g004: Sequential inhibition of caspases and proteasome provides collaborative protection of BAG3.A. Proteasome inhibition provides dose-dependent partial protection of BAG3. FL-BAG3 cells were pretreated with 20 µM MG-132 for 1 or 4 hrs prior to exposure to 2 µM STS. Inhibition of proteasomal degradation by pretreatment with MG-132 prior to STS exposure reduced BAG3 degradation. B. BAG3 is ubiquitinated in response to STS treatment. FL-BAG3 cells were treated with 2 µM STS alone or following 4 hr preincubation with 20 µM MG-132. Lysates were immunoprecipitated with anti-BAG3 and the immunoblots were probed for ubiquitin. BAG3 was ubiquitinated and degraded in the absence of proteasome inhibition. C. STS-induced loss of BAG3 is minimized by the combination of caspase and proteasomal inhibition. FL-BAG3 cells were preincubated with 0.05% DMSO vehicle or 20 µM MG-132 for 4 hours with or without 1 additional hour of zVAD preincubation (85 µM). Treatments were followed by a total STS exposure of up to 20 hrs. A progressive retention of BAG3 was seen with inclusion of each inhibitor. Relative levels of BAG3, determined using ImageJ™, are indicated below the respective blots and plotted as percent remaining BAG3 with increasing STS time.

Mentions: Proteasomal degradation is a major protein regulatory mechanism [16], [17]. Chemical inhibitors of the 20S subunit of the proteasome, such as lactacystin and MG-132, prevent degradation of polyubiquitinated proteins [18]. Neither mutation of the KEVD caspase-recognition site, nor pharmacological inhibition of selected caspases afforded complete protection of BAG3 in the face of intrinsic apoptosis pathway activation, suggesting complementary proteolytic mechanisms. Addition of MG-132 to STS resulted in near complete BAG3 retention (Figure 4A). MG-132 pretreatment in the absence of STS caused accumulation of poly-ubiquitinated BAG3, indicating that some BAG3 ubiquitination occurs basally (Figure 4B, lane 1). Augmented ubiquitination of BAG3 was demonstrated with the combination of STS and MG-132 arguing that BAG3 is targeted to the proteasome under STS stress (Figure 4B, lane 4 v. lane 1). We next investigated the complementarity of pharmacological inhibition of both caspases and the proteasome on BAG3 rescue. Some protection of BAG3 was seen with proteasome inhibition alone (Figure 4C), while there was no BAG3 degradation when zVAD and MG-132 were used in combination. A plot of these data demonstrates progressive loss of BAG3 in the presence of MG-132. This is attenuated with the combination of zVAD and MG-132. These results were recapitulated when lactacystin was used as a proteasomal inhibitor. Disappearance of BAG3 signal caused by STS was prevented by pre-treatment with lactacystin (Figure S3A). The combination zVAD and lactacystin prevented loss of BAG3 in FL and dPXXP cells, while minimally affecting the already resistant dPXXP form (Figure S3B). Taken together these data suggest that both caspase cleavage and proteasomal degradation are required for the degradation of BAG3 in STS-driven apoptosis and that the role of caspase cleavage may be primary.


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)

Sequential inhibition of caspases and proteasome provides collaborative protection of BAG3.A. Proteasome inhibition provides dose-dependent partial protection of BAG3. FL-BAG3 cells were pretreated with 20 µM MG-132 for 1 or 4 hrs prior to exposure to 2 µM STS. Inhibition of proteasomal degradation by pretreatment with MG-132 prior to STS exposure reduced BAG3 degradation. B. BAG3 is ubiquitinated in response to STS treatment. FL-BAG3 cells were treated with 2 µM STS alone or following 4 hr preincubation with 20 µM MG-132. Lysates were immunoprecipitated with anti-BAG3 and the immunoblots were probed for ubiquitin. BAG3 was ubiquitinated and degraded in the absence of proteasome inhibition. C. STS-induced loss of BAG3 is minimized by the combination of caspase and proteasomal inhibition. FL-BAG3 cells were preincubated with 0.05% DMSO vehicle or 20 µM MG-132 for 4 hours with or without 1 additional hour of zVAD preincubation (85 µM). Treatments were followed by a total STS exposure of up to 20 hrs. A progressive retention of BAG3 was seen with inclusion of each inhibitor. Relative levels of BAG3, determined using ImageJ™, are indicated below the respective blots and plotted as percent remaining BAG3 with increasing STS time.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2662420&req=5

pone-0005136-g004: Sequential inhibition of caspases and proteasome provides collaborative protection of BAG3.A. Proteasome inhibition provides dose-dependent partial protection of BAG3. FL-BAG3 cells were pretreated with 20 µM MG-132 for 1 or 4 hrs prior to exposure to 2 µM STS. Inhibition of proteasomal degradation by pretreatment with MG-132 prior to STS exposure reduced BAG3 degradation. B. BAG3 is ubiquitinated in response to STS treatment. FL-BAG3 cells were treated with 2 µM STS alone or following 4 hr preincubation with 20 µM MG-132. Lysates were immunoprecipitated with anti-BAG3 and the immunoblots were probed for ubiquitin. BAG3 was ubiquitinated and degraded in the absence of proteasome inhibition. C. STS-induced loss of BAG3 is minimized by the combination of caspase and proteasomal inhibition. FL-BAG3 cells were preincubated with 0.05% DMSO vehicle or 20 µM MG-132 for 4 hours with or without 1 additional hour of zVAD preincubation (85 µM). Treatments were followed by a total STS exposure of up to 20 hrs. A progressive retention of BAG3 was seen with inclusion of each inhibitor. Relative levels of BAG3, determined using ImageJ™, are indicated below the respective blots and plotted as percent remaining BAG3 with increasing STS time.
Mentions: Proteasomal degradation is a major protein regulatory mechanism [16], [17]. Chemical inhibitors of the 20S subunit of the proteasome, such as lactacystin and MG-132, prevent degradation of polyubiquitinated proteins [18]. Neither mutation of the KEVD caspase-recognition site, nor pharmacological inhibition of selected caspases afforded complete protection of BAG3 in the face of intrinsic apoptosis pathway activation, suggesting complementary proteolytic mechanisms. Addition of MG-132 to STS resulted in near complete BAG3 retention (Figure 4A). MG-132 pretreatment in the absence of STS caused accumulation of poly-ubiquitinated BAG3, indicating that some BAG3 ubiquitination occurs basally (Figure 4B, lane 1). Augmented ubiquitination of BAG3 was demonstrated with the combination of STS and MG-132 arguing that BAG3 is targeted to the proteasome under STS stress (Figure 4B, lane 4 v. lane 1). We next investigated the complementarity of pharmacological inhibition of both caspases and the proteasome on BAG3 rescue. Some protection of BAG3 was seen with proteasome inhibition alone (Figure 4C), while there was no BAG3 degradation when zVAD and MG-132 were used in combination. A plot of these data demonstrates progressive loss of BAG3 in the presence of MG-132. This is attenuated with the combination of zVAD and MG-132. These results were recapitulated when lactacystin was used as a proteasomal inhibitor. Disappearance of BAG3 signal caused by STS was prevented by pre-treatment with lactacystin (Figure S3A). The combination zVAD and lactacystin prevented loss of BAG3 in FL and dPXXP cells, while minimally affecting the already resistant dPXXP form (Figure S3B). Taken together these data suggest that both caspase cleavage and proteasomal degradation are required for the degradation of BAG3 in STS-driven apoptosis and that the role of caspase cleavage may be primary.

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