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. 2008 Feb;20(2):347-58.
doi: 10.1016/j.cellsig.2007.10.032. Epub 2007 Nov 17.

HSP105 interacts with GRP78 and GSK3 and promotes ER stress-induced caspase-3 activation

Gordon P Meares  1 , Anna A Zmijewska, Richard S Jope
Affiliations

HSP105 interacts with GRP78 and GSK3 and promotes ER stress-induced caspase-3 activation

Gordon P Meares et al. Cell Signal. 2008 Feb.

Abstract

Stress of the endoplasmic reticulum (ER stress) is caused by the accumulation of misfolded proteins, which occurs in many neurodegenerative diseases. ER stress can lead to adaptive responses or apoptosis, both of which follow activation of the unfolded protein response (UPR). Heat shock proteins (HSP) support the folding and function of many proteins, and are important components of the ER stress response, but little is known about the role of one of the major large HSPs, HSP105. We identified several new partners of HSP105, including glycogen synthase kinase-3 (GSK3), a promoter of ER stress-induced apoptosis, and GRP78, a key component of the UPR. Knockdown of HSP105 did not alter UPR signaling after ER stress, but blocked caspase-3 activation after ER stress. In contrast, caspase-3 activation induced by genotoxic stress was unaffected by knockdown of HSP105, suggesting ER stress-specificity in the apoptotic action of HSP105. However, knockdown of HSP105 did not alter cell survival after ER stress, but instead diverted signaling to a caspase-3-independent cell death pathway, indicating that HSP105 is necessary for apoptotic signaling after UPR activation by ER stress. Thus, HSP105 appears to chaperone the responses to ER stress through its interactions with GRP78 and GSK3, and without HSP105 cell death following ER stress proceeds by a non-caspase-3-dependent process.

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Figures

Figure 1
Figure 1. HSP105 interacting proteins
A. GSK3β was immunoprecipitated (IP) from cerebral cortical homogenates of two C57BL/6 mice followed by immunoblotting for HSP105 and GSK3β. B. SH-SY5Y cells stably expressing HSP105-V5 were generated by lentiviral transduction, and the modestly increased expression level of HSP105 was demonstrated by immunoblotting for HSP105. HSP105-V5 was immunoprecipitated using an anti-V5 antibody from non-transduced (-) or HSP105-V5 transduced (+) cells, followed by immunoblotting for GSK3β and HSP105. C. HEK293 cells were transiently transfected with HSP105-V5. HSP105-V5 was then immunoprecipitated from 3 mg of cell lysate, followed by SDS-PAGE and coomassie staining. Several bands were extracted from the gel and the proteins listed were identified by MALDI-TOF. D. GRP78 was immunoprecipitated (IP) from cerebral cortical homogenates of two C57BL/6 mice using an anti-KDEL antibody followed by immunoblotting for HSP105 and GRP78. E. HSP105-V5 was immunoprecipitated with a V5 antibody from HEK293 cells transiently transfected with HSP105-V5 (+) or nontransfected cells (-), followed by immunoblotting for GRP78.
Figure 2
Figure 2. Characterization of the interaction of HSP105 with GSK3 and shRNAi knockdown of HSP105
A. SH-SY5Y cells were subjected to heat shock (HS) by incubation in a 45°C water bath for 0-120 min, followed by immunoprecipitation (IP) of GSK3β and immunoblotting for HSP105 and GSK3β. B. SH-SY5Y cells were placed in 45°C water bath for 0-30 min, cytosolic and nuclear fractions were prepared and immunoblotted for HSP105. C. SH-SY5Y cells were placed in a 45°C water bath for 0-30 min, cytosolic and nuclear fractions were prepared, followed by immunoprecipitation of GSK3β and immunoblotting for HSP105 and GSK3β. The levels of nuclear co-immunoprecipitated HSP105 and immunoprecipitated GSK3β were quantified; Means ± SEM; n = 3; *p<0.05 compared with cells not subjected to heat shock. D. SH-SY5Y cells were placed in a 45°C water bath for 0-30 min, cytosolic and nuclear fractions were prepared, followed by immunoprecipitation of GSK3α and immunoblotting for HSP105 and GSK3α. The levels of nuclear co-immunoprecipitated HSP105 and immunoprecipitated GSK3α were quantified; Means ± SEM; n = 3; *p<0.05 compared with cells not subjected to heat shock. E. SH-SY5Y cells were treated with 100 nM wortmannin (Wort) for 1 hr or 20 mM lithium (Li) for 2 hr followed by immunoprecipitation of GSK3β and immunoblotting for HSP105 and GSK3β (right panel). Cell lysates were immunoblotted for phospho-ser9-GSK3β and total levels of GSK3β, HSP105 and β-actin (left panel). F. Stable SH-SY5Y cells were generated by lentiviral transduction using the empty shRNAi vector that still expresses GFP, or with one of two shRNAi directed to HSP105. The RNAi were designated HSP105 RNAi #2 and HSP105 RNAi #4. The efficiency of knockdown of HSP105 and the lack of effects on HSP90 and HSP70 levels were measured in immunoblots, and quantitative values (means ± SEM) were obtained from three independent samples. G. Vector or HSP105 RNAi #2 expressing cells were treated with 50 ng/ml IGF-1 for 0-30 min followed by immunoblotting for phospho-Ser473-Akt, phospho-Thr308-Akt, phospho-Ser9-GSK3β, and β-actin. H. Vector or HSP105 RNAi #2 expressing cells were maintained in serum (S) or placed in serum-free (SF) media for 2 h, then incubated with or without 50 ng/ml IGF-1 for 30 min. The nuclei were isolated and the level of GSK3α/β measured by immunoblot.
Figure 2
Figure 2. Characterization of the interaction of HSP105 with GSK3 and shRNAi knockdown of HSP105
A. SH-SY5Y cells were subjected to heat shock (HS) by incubation in a 45°C water bath for 0-120 min, followed by immunoprecipitation (IP) of GSK3β and immunoblotting for HSP105 and GSK3β. B. SH-SY5Y cells were placed in 45°C water bath for 0-30 min, cytosolic and nuclear fractions were prepared and immunoblotted for HSP105. C. SH-SY5Y cells were placed in a 45°C water bath for 0-30 min, cytosolic and nuclear fractions were prepared, followed by immunoprecipitation of GSK3β and immunoblotting for HSP105 and GSK3β. The levels of nuclear co-immunoprecipitated HSP105 and immunoprecipitated GSK3β were quantified; Means ± SEM; n = 3; *p<0.05 compared with cells not subjected to heat shock. D. SH-SY5Y cells were placed in a 45°C water bath for 0-30 min, cytosolic and nuclear fractions were prepared, followed by immunoprecipitation of GSK3α and immunoblotting for HSP105 and GSK3α. The levels of nuclear co-immunoprecipitated HSP105 and immunoprecipitated GSK3α were quantified; Means ± SEM; n = 3; *p<0.05 compared with cells not subjected to heat shock. E. SH-SY5Y cells were treated with 100 nM wortmannin (Wort) for 1 hr or 20 mM lithium (Li) for 2 hr followed by immunoprecipitation of GSK3β and immunoblotting for HSP105 and GSK3β (right panel). Cell lysates were immunoblotted for phospho-ser9-GSK3β and total levels of GSK3β, HSP105 and β-actin (left panel). F. Stable SH-SY5Y cells were generated by lentiviral transduction using the empty shRNAi vector that still expresses GFP, or with one of two shRNAi directed to HSP105. The RNAi were designated HSP105 RNAi #2 and HSP105 RNAi #4. The efficiency of knockdown of HSP105 and the lack of effects on HSP90 and HSP70 levels were measured in immunoblots, and quantitative values (means ± SEM) were obtained from three independent samples. G. Vector or HSP105 RNAi #2 expressing cells were treated with 50 ng/ml IGF-1 for 0-30 min followed by immunoblotting for phospho-Ser473-Akt, phospho-Thr308-Akt, phospho-Ser9-GSK3β, and β-actin. H. Vector or HSP105 RNAi #2 expressing cells were maintained in serum (S) or placed in serum-free (SF) media for 2 h, then incubated with or without 50 ng/ml IGF-1 for 30 min. The nuclei were isolated and the level of GSK3α/β measured by immunoblot.
Figure 3
Figure 3. HSP105 and UPR-induced signaling
A. SH-SY5Y cells were treated with 4 μg/ml tunicamycin for 0 to 6 h followed by immunoblotting for phospho-eIF2α, CHOP, cleaved caspase-3, cleaved PARP, and β-actin. B. SH-SY5Y cells were treated with 2 μM thapsigargin for 0 to 6 h followed by immunoblotting for phospho-eIF2α, CHOP, cleaved caspase-3, cleaved PARP, and β-actin. C. To examine XBP-1 splicing, vector or HSP105 RNAi expressing cells were treated with 2 μM thapsigargin for 2 or 4 h. The cellular RNA was isolated and spliced XBP-1 examined by RT-PCR. D. Vector or HSP105 RNAi #2 expressing cells were treated with 4 μg/ml tunicamycin for 0 to 90 min, then immunoblotted for phospho-eIF2α or total eIF2α. E. Vector or HSP105 RNAi #2 cells were treated with 4 μg/ml tunicamycin for 0 to 4 h, then immunoblotted for CHOP. F. Untransduced SH-SY5Y cells, vector cells, or HSP105 RNAi #2 expressing cells were treated with 2 μM thapsigargin for 2 or 3 h, then immunoblotted for cleaved caspase-3 and HSP105. G. Vector or HSP105 RNAi#2 expressing cells were treated with 4 μg/ml tunicamycin, 2 μM thapsigargin, or 1 μM camptothecin, for 3 h, then stained for active caspase-3 or DAPI and examined by fluorescence microscopy. Scale bar: 100 μm
Figure 4
Figure 4. HSP105 knockdown attenuates ER stress-induced caspase-3 activation
A. Vector cells or HSP105 RNAi #2 expressing cells were treated with 4 μg/ml tunicamycin for 0 to 4 h and active caspase-3 examined by immunocytochemistry. Quantitative values are means ± SEM, n=3, * p<0.05. Cleaved PARP, HSP105, and β-actin were examined by immunoblot. Vector cells and HSP105 RNAi #4 expressing cells were treated with 4 μg/ml tunicamycin (Tunic) for 0 to 4 h and immunoblotted for cleaved PARP and HSP105. B. Vector cells or HSP105 RNAi #2 expressing cells were treated with 2 μM thapsigargin for 0 to 4 h and active caspase-3 examined by immunocytochemistry. Quantitative values are means ± SEM, n=3, * p<0.05. Cleaved PARP, HSP105, and β-actin were examined by immunoblot. C. Vector cells or HSP105 RNAi #2 expressing cells were treated with 1 μM camptothecin for 0 to 4 h and active caspase-3 examined by immunocytochemistry. Quantitative values are means ± SEM, n=3, * p<0.05. Cleaved PARP, HSP105, and β-actin were examined by immunoblot. D. Total levels of caspase-3, caspase-9, Bcl-XL, Bcl-2, Bad cIAP-1, cIAP22, and xIAP were measured by immunoblot in vector control cells and HSP105 RNAi #2 expressing cells.
Figure 5
Figure 5. Knockdown of HSP105 does not promote cell survival
A. Vector or HSP105 RNAi #2 expressing cells were treated with 4 μg/ml tunicamycin for 3 or 4 h, and immunoblotted for MCL1 and β-actin. B. To examine Bax activation, vector or HSP105 RNAi #2 expressing cells were treated with 4 μg/ml tunicamycin for 0 to 4 h. The active conformation specific Bax antibody 6A7 was used to immunoprecipitate active Bax followed by immunoblotting for total Bax in the immunoprecipitate and the cell lysate. C. To examine nuclear morphology, vector or HSP105 RNAi #2 expressing cells were treated with 4 μg/ml tunicamycin for 4 or 8 h, then fixed and stained with Hoechst 33342 and examined by fluorescence microscopy. Scale bar: 100 μm D. Vector or HSP105 RNAi #2 expressing cells were treated with 4 μg/ml tunicamycin or 2 μM thapsigargin for 0 to 16 h, and cell death was assessed by measuring the release of LDH. Quantitative values are means ± SEM, n=3.
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