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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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STS induced apoptosis and degradation of BAG3.A. STS induces cellular injury in a dose and time-dependent fashion. DAPI-stained STS-treated MDA435 cells show chromatin condensation consistent with apoptotic injury. Apoptotic bodies were observed with 2 and 4 µM STS at 4 hr (arrows) and there was a net loss of cells at 16 hrs. B. STS-mediated degradation of BAG3 occurs concomitant to activation of caspases 3, 7 and 9. Cleavage of caspases 3 and 9 were observed as early as 4 hrs into STS treatment. In comparison activation of caspases 8 and 10 was delayed, occurring after BAG protein loss was initiated. C, D. STS induces apoptosis in HeLa cells. Apoptotic bodies were observed in DAPI stained cells. Data points are the mean and SEM of five independent fields (n = 2). E, F. BAGs 3, 4, and 6 are lost progressively with STS treatment. Floating and adherent MDA435 cells were collected, lysed, and subjected to immunoblot. BAGs 3 and 4 (E), and 6 (F) were lost with STS exposures of 3–16 hrs. No reduction in the p50, p46, p34, or p29 BAG1 isoforms was observed (E). G. STS-mediated BAG3 degradation is neither cell line nor construct-specific. EGFP-BAG3 or EGFP-C1 empty vector were stably expressed in HeLa cells. Cells were exposed to 2 µM STS and both endogenous BAG3 and EGFP-BAG3 fusion proteins were lost over time, as early as 6 h.
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pone-0005136-g001: STS induced apoptosis and degradation of BAG3.A. STS induces cellular injury in a dose and time-dependent fashion. DAPI-stained STS-treated MDA435 cells show chromatin condensation consistent with apoptotic injury. Apoptotic bodies were observed with 2 and 4 µM STS at 4 hr (arrows) and there was a net loss of cells at 16 hrs. B. STS-mediated degradation of BAG3 occurs concomitant to activation of caspases 3, 7 and 9. Cleavage of caspases 3 and 9 were observed as early as 4 hrs into STS treatment. In comparison activation of caspases 8 and 10 was delayed, occurring after BAG protein loss was initiated. C, D. STS induces apoptosis in HeLa cells. Apoptotic bodies were observed in DAPI stained cells. Data points are the mean and SEM of five independent fields (n = 2). E, F. BAGs 3, 4, and 6 are lost progressively with STS treatment. Floating and adherent MDA435 cells were collected, lysed, and subjected to immunoblot. BAGs 3 and 4 (E), and 6 (F) were lost with STS exposures of 3–16 hrs. No reduction in the p50, p46, p34, or p29 BAG1 isoforms was observed (E). G. STS-mediated BAG3 degradation is neither cell line nor construct-specific. EGFP-BAG3 or EGFP-C1 empty vector were stably expressed in HeLa cells. Cells were exposed to 2 µM STS and both endogenous BAG3 and EGFP-BAG3 fusion proteins were lost over time, as early as 6 h.

Mentions: STS caused dose- and time-dependent apoptosis in MDA435 human breast cancer cells (Figure 1A). Concomitant with nuclear condensation and cell death due to STS was progressive activation of caspases 3, 7, 8, 9, and 10 (Figure 1B). Caspases 3, 9 and 7 were cleaved earlier and at lower STS doses than caspases 8 or 10, confirming the expected predominant activation of the intrinsic apoptotic pathway. A similar effect was observed in HeLa cells. Apoptosis, demonstrated by the presence of apoptotic bodies, occurred earlier, at 4 and 8 hours (Figure 1C, D and Figure S2B, DMSO control). Cells lacked normal nuclear morphology at later time points, consistent with progressive injury (Fig 1C, arrow head). In BAG3 overexpressing HeLa and MDA435 cells, BAG3 colocalized with active mitochondria early in STS-mediated injury (Figure S1, arrows). Higher concentrations of, or longer exposure to, STS resulted in a generalized uptake of Mito-Tracker into the nucleus, indicating the lack of mitochondrial membrane integrity seen in apoptosis (Figure S1). The dose and time course of activation of caspases 3, 9 and 7 (Figure 1B) paralleled the progressive loss of BAG3 (Figure 1E). Family members BAGs 4, and 6 were similarly lost with STS treatment (Figure 1E, F) as were the commonly used ‘housekeeping proteins’ GAPDH and β-tubulin (Figure S5), while the four isotypes of BAG1, p50BAG1L, p46BAG1M, p34BAG1, and p29BAG1S, were unaffected, arguing against a global toxic effect of STS. Both endogenous and forced BAG3 were susceptible to this proteotoxicity. HeLa cells stably expressing EGFP-BAG3 displayed time-dependent loss of the fusion protein, as well as endogenous BAG3 (Figure 1G). The parallel between caspase activation and loss of BAGs 3, 4, and 6 suggested that intrinsic pathway caspases might be involved in BAG3 degradation.


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)

STS induced apoptosis and degradation of BAG3.A. STS induces cellular injury in a dose and time-dependent fashion. DAPI-stained STS-treated MDA435 cells show chromatin condensation consistent with apoptotic injury. Apoptotic bodies were observed with 2 and 4 µM STS at 4 hr (arrows) and there was a net loss of cells at 16 hrs. B. STS-mediated degradation of BAG3 occurs concomitant to activation of caspases 3, 7 and 9. Cleavage of caspases 3 and 9 were observed as early as 4 hrs into STS treatment. In comparison activation of caspases 8 and 10 was delayed, occurring after BAG protein loss was initiated. C, D. STS induces apoptosis in HeLa cells. Apoptotic bodies were observed in DAPI stained cells. Data points are the mean and SEM of five independent fields (n = 2). E, F. BAGs 3, 4, and 6 are lost progressively with STS treatment. Floating and adherent MDA435 cells were collected, lysed, and subjected to immunoblot. BAGs 3 and 4 (E), and 6 (F) were lost with STS exposures of 3–16 hrs. No reduction in the p50, p46, p34, or p29 BAG1 isoforms was observed (E). G. STS-mediated BAG3 degradation is neither cell line nor construct-specific. EGFP-BAG3 or EGFP-C1 empty vector were stably expressed in HeLa cells. Cells were exposed to 2 µM STS and both endogenous BAG3 and EGFP-BAG3 fusion proteins were lost over time, as early as 6 h.
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pone-0005136-g001: STS induced apoptosis and degradation of BAG3.A. STS induces cellular injury in a dose and time-dependent fashion. DAPI-stained STS-treated MDA435 cells show chromatin condensation consistent with apoptotic injury. Apoptotic bodies were observed with 2 and 4 µM STS at 4 hr (arrows) and there was a net loss of cells at 16 hrs. B. STS-mediated degradation of BAG3 occurs concomitant to activation of caspases 3, 7 and 9. Cleavage of caspases 3 and 9 were observed as early as 4 hrs into STS treatment. In comparison activation of caspases 8 and 10 was delayed, occurring after BAG protein loss was initiated. C, D. STS induces apoptosis in HeLa cells. Apoptotic bodies were observed in DAPI stained cells. Data points are the mean and SEM of five independent fields (n = 2). E, F. BAGs 3, 4, and 6 are lost progressively with STS treatment. Floating and adherent MDA435 cells were collected, lysed, and subjected to immunoblot. BAGs 3 and 4 (E), and 6 (F) were lost with STS exposures of 3–16 hrs. No reduction in the p50, p46, p34, or p29 BAG1 isoforms was observed (E). G. STS-mediated BAG3 degradation is neither cell line nor construct-specific. EGFP-BAG3 or EGFP-C1 empty vector were stably expressed in HeLa cells. Cells were exposed to 2 µM STS and both endogenous BAG3 and EGFP-BAG3 fusion proteins were lost over time, as early as 6 h.
Mentions: STS caused dose- and time-dependent apoptosis in MDA435 human breast cancer cells (Figure 1A). Concomitant with nuclear condensation and cell death due to STS was progressive activation of caspases 3, 7, 8, 9, and 10 (Figure 1B). Caspases 3, 9 and 7 were cleaved earlier and at lower STS doses than caspases 8 or 10, confirming the expected predominant activation of the intrinsic apoptotic pathway. A similar effect was observed in HeLa cells. Apoptosis, demonstrated by the presence of apoptotic bodies, occurred earlier, at 4 and 8 hours (Figure 1C, D and Figure S2B, DMSO control). Cells lacked normal nuclear morphology at later time points, consistent with progressive injury (Fig 1C, arrow head). In BAG3 overexpressing HeLa and MDA435 cells, BAG3 colocalized with active mitochondria early in STS-mediated injury (Figure S1, arrows). Higher concentrations of, or longer exposure to, STS resulted in a generalized uptake of Mito-Tracker into the nucleus, indicating the lack of mitochondrial membrane integrity seen in apoptosis (Figure S1). The dose and time course of activation of caspases 3, 9 and 7 (Figure 1B) paralleled the progressive loss of BAG3 (Figure 1E). Family members BAGs 4, and 6 were similarly lost with STS treatment (Figure 1E, F) as were the commonly used ‘housekeeping proteins’ GAPDH and β-tubulin (Figure S5), while the four isotypes of BAG1, p50BAG1L, p46BAG1M, p34BAG1, and p29BAG1S, were unaffected, arguing against a global toxic effect of STS. Both endogenous and forced BAG3 were susceptible to this proteotoxicity. HeLa cells stably expressing EGFP-BAG3 displayed time-dependent loss of the fusion protein, as well as endogenous BAG3 (Figure 1G). The parallel between caspase activation and loss of BAGs 3, 4, and 6 suggested that intrinsic pathway caspases might be involved in BAG3 degradation.

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