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Human Molecular Genetics Advance Access originally published online on January 4, 2008
Human Molecular Genetics 2008 17(8):1109-1119; doi:10.1093/hmg/ddm383
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© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Polycystin-2 is regulated by endoplasmic reticulum-associated degradation

Genqing Liang1, Qiang Li1, Yan Tang1, Koichi Kokame2, Tadashi Kikuchi2, Guanqing Wu3 and Xing-Zhen Chen1,*

1 Membrane Protein Research Group, Department of Physiology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7 2 National Cardiovascular Centre Research Institute, Suita, Osaka 565-8565, Japan 3 Department of Medicine, Vanderbilt University, Nashville, TN 32232-0275, USA

* To whom correspondence should be addressed. Tel: +1 780 492 2294; Fax: +1 780 492 8915; Email: xzchen{at}ualberta.ca

Received November 23, 2007; Accepted December 26, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND MATHODS
 FUNDING
 REFERENCES
 
Endoplasmic reticulum(ER)-associated degradation (ERAD) is an essential process for cell homeostasis and remains not well understood. During ERAD, misfolded proteins are recognized, ubiquitinated on ER and subsequently retro-translocated/dislocated from ER to the 26S proteasome in the cytosol for proteolytic elimination. Polycystin-2 (PC2), a member of the transient receptor potential superfamily of cation channels, is a Ca channel mainly located on ER and primary cilium membranes of cells. Mutations in PC2 are associated with autosomal dominant polycystic kidney disease (ADPKD). The cellular and molecular mechanisms underlying the PC2-associated pathogenesis remain unclear. Here we show that PC2 degradation is regulated by the ERAD pathway through the ubiquitin–proteasome system. PC2 interacted with ATPase p97, a well-known ERAD component extracting substrates from ER, and immobilized it in perinuclear regions. PC2 also interacted with Herp, an ubiquitin-like protein implicated in regulation of ERAD. We found that Herp is required for and promotes PC2 degradation. ER stress accelerates the retro-translocation of PC2 for cytosolic degradation, at least in part through increasing the Herp expression. Thus, PC2 is a novel ERAD substrate. Herp also promoted, to varied degrees, the degradation of PC2 truncation mutants, including two pathogenic mutants R872X and E837X, as long as they interact with Herp. In contrast, Herp did not interact with, and has no effect on the degradation of, PC2 mutant missing both the N- and C-termini. The ERAD machinery may thus be important for ADPKD pathogenesis because the regulation of PC2 expression by the ERAD pathway is altered by mutations in PC2.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND MATHODS
 FUNDING
 REFERENCES
 
The endoplasmic reticulum (ER) plays a crucial role in the protein folding and quality control (1). Conditions disrupting the ER homeostasis can cause unfolded protein accumulation, which triggers ER stress and unfolded protein response (UPR) (2). In a process termed ER-associated degradation (ERAD), unfolded proteins are delivered into the cytosol where they are ultimately destroyed by the 26S ubiquitin–proteasome system (1,3,4). Several steps are involved in protein degradation by ERAD, including the recognition of a target substrate on the ER, retro-translocation of the substrate to the cytosol, and transferring of the substrate to the 26S proteasome for final destruction. Efforts to elucidate the pathway have identified some factors involved in ERAD in mammals. E3 ubiquitin ligases, such as HRD1 and gp78, have been shown to mediate the ubiquitination of ERAD substrates (5,6). A protein complex formed by AAA ATPase p97 and its cofactors Ufd1 and Npl4 retro-translocates ubiquitinated substrates from the ER to the cytosol (7–9). Recently, membrane proteins Derlin-1, VIMP, Herp and signal peptide peptidase were demonstrated to mediate the extraction of some substrates, including class I major histocompatibility complex (MHC), CD3-delta and cystic fibrosis transmembrane conductance regulator (10–14). Identification and characterization of new substrates and components of the ERAD pathway will bring novel insights into the molecular mechanism underlying ERAD, which remains largely unclear.

Polycystin-2 (PC2) with a molecular mass of ~110 kDa is a non-selective cation channel permeable to Ca. It is mainly localized on the ER membrane as a Ca release channel (15,16), on epithelial primary cilia membrane of renal tubular and embryonic nodal cells as part of flow sensor (17,18), and may also be present on the plasma membrane for Ca entry (19). The channel is involved in multiple signal transduction pathways such as Wnt, cAMP and Ras/MAPK (20). Mutations in PC2 are associated with autosomal dominant polycystic kidney disease (ADPKD), abnormalities in vascular structure/function and organ left-right asymmetry development (20). At the cellular level, ADPKD is associated with altered cell proliferation, adhesion and differentiation. However, the cellular regulation of PC2 remains largely unknown. In this study, we examined how PC2 and its mutants interact with components of ERAD, in particular, Herp, and how their degradation is regulated by the ERAD pathway.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND MATHODS
 FUNDING
 REFERENCES
 
PC2 is poly-ubiquitinated
To test whether PC2 is a potential substrate of ERAD, we first examined the possible in vivo ubiquitination of the ER Ca channel protein. For this end, mouse inner medullary collecting duct (IMCD) cells were treated with proteasome inhibitor MG-132. Immunoprecipitates of the cell extracts with an anti-PC2 antibody (21,22) were immunoblotted by an anti-ubiquitin antibody. Indeed, ubiquitinated PC2 could be precipitated and the amount of the ubiquitinated fraction was significantly increased in cells treated with MG-132 (Fig. 1A, left panel). Reciprocally, MG-132-induced accumulation of ubiquitinated PC2 was also observed in the immunoprecipitate obtained using the anti-ubiquitin antibody (Fig. 1A, right panel). These data indicate that PC2 is ubiquitinated in vivo and that the ubiquitinated PC2 proteins accumulate when proteasome-dependent degradation is inhibited. We also examined ubiquitination of the over-expressed PC2 in HeLa cells and obtained similar results (Fig. 1B). We then performed similar experiments using HeLa cells transiently expressing GFP-ubiquitin or GFP to examine the effect of ubiquitin on PC2. We found that, similar to the effect of MG-132, over-expression of ubiquitin increased the proportion of ubiquitinated PC2 and also leads to a decreased PC2 level (Fig. 1C), suggesting that PC2 degradation is promoted by ubiquitin. Thus, our data indicate that PC2 is ubiquitinated, consistent with a recent report that PC2 is likely ubiquitinated (23).


Figure 1
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  		Figure 1. PC2 is poly-ubiquitinated in vivo. (A) Ubiquitination of polycystin-2 (PC2). Inner medullary collecting duct (IMCD) cells were treated with 10 µM MG-132 for 4 h, followed by protein preparation and immunoprecipitation (IP) with an anti PC2 antibody (1A11) or ubiquitin (Ub) antibody. Precipitated PC2 and an input (10%) of poly-ubiquitinated PC2 were detected by immunoblotting with the PC2 or ubiquitin antibody. (B) Ubiquitination of GFP-PC2. Extracts of HeLa cells transfected with pEGFP-PC2 or pEGFP (5 µg in 100-mm plates) for 40 h, followed by the MG-132 (10 µM) treatment for 4 h, were subjected to IP with a GFP (gaot) antibody and immunoblotting with the ubiquitin or a GFP (rabbit) antibody. Cell extracts (Input, 30%) was used for immunoblotting with the ubiquitin or GFP (goat) antibody. (C) Effect of over-expression of ubiquitin on PC2 ubiquitination. Extracts of HeLa cells transfected with pEGFP-ubiquitin or vector pEGFP (5 µg in 100-mm plates) for 40 h, followed by the MG-132 (10 µM) treatment for 4 h, were subjected to IP with the PC2 antibody and immunoblotting using the ubiquitin or PC2 antibody. Cell extracts (Input, 10%) were utilized for detecting cellular ubiquitination, GFP-ubiquitin and PC2 expression by immunoblotting with the ubiquitin, GFP (goat) and PC2 antibodies, respectively. β-actin was used as a loading control.

 
PC2 degradation is proteasome-dependent
We next explored further documentations for the involvement of the 26S proteasome in PC2 degradation. For this we inhibited protein synthesis by cycloheximide (CHX) in native HeLa cells and Madin-Darby canine kidney (MDCK) stably expressing GFP-tagged human PC2. Eight hours after treatment with CHX, PC2 protein was decreased by 62 ± 8% (N = 3) (Fig. 2A) and 68 ± 7% (N = 3), respectively (Fig. 2B). In cells incubated with proteasome inhibitor lactacystin or MG-132, the degradation of PC2 was suppressed. Tunicamycin (Tm) is a known ER stress inducer that enhances the degradation of ERAD substrates in both yeast and mammals (24–26). We found that the endogenous PC2 level is indeed reduced in IMCD and HeLa cells incubated with Tm, but not in cells incubated with DMSO (as a vehicle control) (Fig. 2C and D). Because both the mRNA level and protein synthesis of PC2 were not significantly affected by Tm, as assessed by RT–PCR and 35S-methionine labeling, respectively (data not shown), this result indicates that Tm promotes PC2 degradation. In this experiment, the expression of immunoglobulin heavy-chain binding protein (BiP, also called GRP78), an ER chaperone protein activated during the ER stress, was used as a marker of the Tm-induced ER stress. We also examined the effect of Tm on GFP-tagged human PC2 stably expressed in MDCK cells and found similar results (Fig. 2E). The effect of Tm on PC2 degradation was confirmed by another ER stress inducer dithiothreitol (DTT) (data not shown). As expected, the Tm-promoted degradation was inhibited by proteasome inhibitors MG-132 and lactacystin (Fig. 2C–E). Together with the accelerated PC2 degradation promoted by ubiquitin expression (Fig. 1C), our data indicate that PC2 turnover is regulated by the ubiquitin–proteasome system.


Figure 2
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  		Figure 2. Polycystin-2 (PC2) degradation is regulated by proteasome system. (A) and (B) Effects of proteasome inhibitors on PC2 degradation. HeLa cells (A) and stable Madin-Darby canine kidney (MDCK) cells expressing GFP-PC2 (B) were treated by 50 µg/ml cycloheximide (CHX) (Sigma-Aldrich Canada) along with 10 µM proteasome inhibitor lactacystin or MG-132 (Sigma-Aldrich Canada) for 8 h, followed by immunoblotting of the resulting cell extracts with the PC2 antibody. (C), (D) and (E) Effect of proteasome inhibitors on the Tm-induced degradation of PC2. Inner medullary collecting duct (IMCD) (C), HeLa (D) and stable MDCK cells expressing GFP-PC2 (E) were treated by 2 µg/ml Tm with or without 10 µM MG-132 or lactacystin for 8 h, followed by immunoblotting of cell extracts with antibodies, as indicated. Arrow in (E) suggests the unglycosylated form of GFP-PC2. The expressions of BiP were used as an indication of unfolded protein response (UPR).

 
PC2 interacts with components of the ERAD pathway
Given the ER membrane localization of PC2 we reasoned that ERAD components might be involved in the PC2 degradation through the 26S ubiquitin–proteasome system. Numerous studies have demonstrated that the p97 complex (p97-Ufd1-Npl4) plays a key role in the transport of ERAD substrates from the ER to the cytosol (7–9,27,28). To test a potential association of PC2 with p97, we immunoprecipitated the extracts of IMCD cells using the anti-PC2 antibody. We found that p97 was indeed co-precipitated with PC2 (Fig. 3A, left panel, lanes 2 versus 1) and that MG-132 substantially increased the amount of the precipitated p97 (Fig. 3A, left panel, lanes 3 versus 2), which should be due at least in part to an increased amount of ubiquitinated PC2 (Fig. 1). This result is paralleled by a previous report that proteasome inhibitors enhance the level of the association of substrate MHC with the ERAD component Derlin-1 that mediates substrate retro-translocation (10). In negative control experiments we found that ER membrane proteins ATF6{alpha} (29) and Sec61{alpha}, a subunit of the Sec61 translocon complex (30), are not co-precipitated with PC2 (Fig. 3A). Further, p97 was not immunoprecipitated by the PC2 antibody using PC2-knockout mouse embryos (31) or HeLa cells in which PC2 expression was substantially reduced (to 20%) by small interference RNA (siRNA) (Fig. 3B). The data on the PC2–p97 specific interaction in vivo thus support that PC2 is an ERAD substrate.


Figure 3
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  		Figure 3. Polycystin-2 (PC2) interacts with p97 and the 26S proteasome. (A) Immunoprecipitates by PC2 were prepared similarly as in Figure 1A and used for immunoblotting with a p97, S12 or ATF6{alpha} antibody. Cell extracts (Input, 10%) were utilized for detecting p97, S12, Sec61{alpha} and ATF6{alpha} by immunoblotting. (B) Immunoprecipitates using a PC2 antibody were obtained from extracts of E13.5 embryos of PC2 knockout (–/–) or wild type (+/+) mice, and of HeLa cells with PC2 or control (Ctrl) small interference RNA (siRNA). These precipitates were then used for immunoblotting with a p97 or S12 antibody. Mouse tissue and HeLa cell extracts (Input, 10%) were utilized to detect p97 and S12 by immunoblotting. (C) Co-localization between PC2 and p97 determined by immunofluorescence. Madin-Darby canine kidney (MDCK) cells stably expressing GFP-PC2 or GFP were stained with the p97 antibody (panels 3 and 7). PC2 was monitored by GFP (panels 2 and 6). Cells were also stained with 4,6-diamidino-2-phenylindole (DAPI) (panels 1 and 5). (D) Effect of over-expressed PC2 on the expression of the endogenous p97 in MDCK cells. Proteins extracted from MDCK cells stably expressing GFP-PC2 or GFP were subjected to immunoblotting with the p97 antibody. Lysis buffer was used as a negative control.

 
Immunofluorescence microscopy was performed using MDCK cells stably expressing GFP-PC2 to examine the co-localization of PC2 and p97. GFP-PC2 was mainly distributed to perinuclear regions typical of ER localization, whereas GFP alone had a different pattern of distribution (Fig. 3C, panels 2 versus 6). Interestingly, in GFP-PC2-expressing cells, but not in GFP-expressing cells, the p97 distribution pattern was similar to that of PC2 (Fig. 3C). These data together with the complexing between p97 and PC2 (Fig. 3A and B) indicate that the PC2–p97 interaction may have immobilized p97 in the perinuclear regions, while leaving its steady state level unaffected (Fig. 3D).

Since ERAD substrates are eventually destroyed by the 26S proteasome after their retro-translocation from the ER to the cytosol, we also examined potential association of PC2 with the proteasome. We found that S12, a subunit of the 19S proteasome believed to recognize ubiquitinated substrates for degradation by the 20S proteasome (32), is in the same complex as PC2, and like p97, its complexing with PC2 substantially augmented in the presence of MG-132 (Fig. 3A, left panel, lanes 3 versus 2). Similar to p97, S12 was not co-immunoprecipitated with PC2 in the PC2-knockout embryos or PC2-knockdown cells (Fig. 3B). The data indicate that poly-ubiquitinated PC2 is indeed retro-translocated from the ER to the cytosol where it is recognized by the 19S proteasome for degradation.

Herp associates with PC2 and regulates its degradation
Herp (or Mif1) is a single-transmembrane ER membrane protein with its N- (amino acids 1–285) and C-termini (amino acids 308–391) putatively localized to the cytosol, and possesses an ubiquitin-like (UBL) domain (amino acids 14–85) on the N-terminus (33,34). Since Herp is highly up-regulated during UPR and known to be implicated in ERAD (12,33,35,36), we examined whether Herp is involved in the PC2 degradation. For this HeLa cells were transfected with vector pcDNA3.1 or pcDNA3.1-mHerpf in which the Herp N- and C-termini are tagged with Myc and FLAG, respectively (33,37). It was reported that the full-length mHerpf is ~61 kDa and resides on the ER (33). In cells over-expressing Herp PC2 degradation accelerated (Figs 4A and 5A, lanes 3 versus 1). Multiple cleaved N-terminal fragments, at 50, 37, 30 and 15 kDa, were present in the cells (as detected by a Myc antibody), of which the 50-kDa fragment has comparable high abundance as the full-length Herp (Fig. 4A, blot Myc, lanes 3 and 4). Fractionation analyses showed that the 50-kDa proteolytic fragment of Herp associates with the ER membrane (Fig. 4B). As a method control, IP3R-1, a subunit of the ER IP3R Ca channel complex (38), was purified from the membrane fraction. In contrast, only the full-length Herp was detected with an antibody against the C-terminal FLAG (Fig. 4A, blot FLAG, lanes 3 and 4), indicating that removal of N-terminal fragments makes the rest of the protein (tagged with FLAG) unstable. Our data suggest that a fragment of ~15 kDa at the Herp N-terminal end, that contains the UBL domain (amino acids 14–85), is important for the protein stability. Next, we tested possible association of PC2 with Herp in these HeLa cells. Indeed, both the full-length and the 50-kDa fragment, but not smaller fragments (perhaps due to their lower abundance), of Herp were found to interact with PC2 (Fig. 4C, blots FLAG and Myc, lanes 3 and 4). Reciprocally, PC2 was co-immunoprecipitated with Herp and the level of the Herp-associated PC2 seemed to increase by MG-132 (Fig. 4D, blot PC2, lanes 4 versus 3), presumably by delaying the retro-translocation of PC2 from the ER. Of note, a band corresponding to a size slightly smaller than that of the full-length PC2 was present in the precipitates of cells over-expressing Herp (Fig. 4C, blot PC2, lane 3, arrow). Because this band was undetectable when cells were treated with MG-132 (Fig. 4C, blot PC2, lanes 4 versus 3) it may represent an intermediate form of PC2 during its degradation by the proteasome. Thus our data show that the UBL protein Herp interacts with PC2 and promotes its degradation through the ERAD pathway.


Figure 4
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  		Figure 4. Herp interacts with polycystin-2 (PC2) on the endoplasmic reticulum (ER) membrane. (A) Cleavage of Herp. Extracts of HeLa cells transfected with pcDNA3.1-mHerpf or vector pcDNA3.1 were subjected to immunoblotting with an FLAG or Myc (rabbit) antibody to detect the full-length Herp (blots FLAG and Myc) and cleaved Herp (blot Myc). The bands with the largest molecular weight indicated by an arrow may correspond to a non-specific signal as they are present in control cells as well (also see panel B). (B) Solubility of cleaved fragments of Herp. Membrane and cytosolic fractions were separated from the lysates of HeLa cells transiently transfected with pcDNA3.1-mHerpf or the pcDNA3.1 vector (5 µg in 100-mm dishes) for 40 h. Full-length and cleaved fragments of Herp were detected by immunoblotting with the Myc (rabbit), IP3R-1 or β-actin antibody. (C) Complexing between Herp and PC2 determined by co-immunoprecipitation (IP). IP with the PC2 antibody using cell extracts in (A) was performed as in Figure 1A. Immunoprecipitates were blotted with the FLAG, Myc (rabbit), or PC2 antibody. (D) Confirmation of the specific interaction of PC2 with Herp. Immunoprecipitates from the extracts used in (A) with the Myc (rabbit) antibody were immunoblotted with the PC2 or Myc (mouse) antibody.

 


Figure 5
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  		Figure 5. Herp promotes polycystin-2 (PC2) degradation. (A) Effect of Herp over-expression on PC2 degradation. HeLa cells were transfected with pcDNA3.1-mHerpf or pcDNA3.1 vector (1 µg in 35-mm dishes) for 40 h before immunoblotting. Treatment with Tm (8 h) was used as a positive control. (B) Effect of Herp knockdown on PC2 degradation. HeLa cells were transfected with Herp small interference RNA (siRNA) or control siRNA (Ctrl) (20 µl of 20 µM in 35-mm dishes) for 40 h before immunoblotting. Treatment with Tm (8 h) was used to induce PC2 degradation. (C) Effects of Herp on the degradation of PC2 mutants. Extracts of HeLa cells co-transfected with plasmid GFP-PC2 or a PC2 mutant, and with pcDNA3.1-mHerpf (+) [or vector pcDNA3.1 (–), as a control] were subjected to immunoblotting with a GFP, FLAG or β-actin antibody. (D) Interaction of PC2 mutants with Herp. Extracts of HeLa cells co-transfected with pcDNA3.1-mHerpf and with pEGFP, pEGPF-PC2 or a PC2 mutant plasmid for 48 h were subjected to immunoprecipitation (IP) with the GFP antibody and immunoblotting with the FLAG or GFP antibody. Cell extracts (Input, 10%) were utilized for detecting transfection efficiency.

 
Note that, although an increase in the Herp expression, like the treatment with an ER stress inducer, accelerates PC2 degradation, it does not induce ER stress to the cells, as assessed by the BiP expression (Fig. 5A). We next tested the effect of Herp knockdown, by siRNA, on PC2 degradation. A robust reduction in the Herp expression was obtained in HeLa cells with Herp siRNA (Fig. 5B, lanes 3 versus 1). The amount of PC2 augmented when Herp expression was inhibited by siRNA in both normal cells and those under ER stress triggered by Tm (lanes 3 versus 1 and 4 versus 2). Thus, the rate of PC2 degradation correlates with the Herp level. To further document this correlation, instead of directly altering the Herp level by over-expression or siRNA, we made use of ER stress inducers, which are known to increase Herp expression by inducing ER stress (33). Indeed, the Herp level increased and the PC2 level decreased by Tm (Fig. 5A and B, lanes 2 versus 1) and DTT (data not shown) in HeLa cells. Similar results were also obtained in cells with Herp knockdown (Fig. 5B, lanes 4 versus 3). Taken together, our data obtained under various conditions demonstrate that Herp regulates PC2 degradation, presumably via their physical interaction and its promotion of retro-translocation.

Next, we tested whether the degradation of pathogenic mutants of PC2 is mediated by Herp, using HeLa cells co-transfected with a plasmid encoding pEGFP, pEGFP-PC2 or a mutant PC2, and with pcDNA3.1-mHerpf (or pcDNA3.1 vector, as a control). Similar to PC2, the degradation of PC2 pathogenic mutants R872X and E837X, and PC2 mutants lacking the intracellular C-terminus (PC2{Delta}C, amino acids 1–688 or S689X) or N-terminus (PC2{Delta}N, amino acids 209–968) was promoted by Herp (Fig. 5C). However, Herp exhibited no effect on the turnover of PC2 mutant lacking both N- and C-termini (PC2{Delta}NC, amino acids 209-688) (Fig. 5C). Because PC2{Delta}C and PC2{Delta}N, but not PC2{Delta}NC, interact with Herp (Fig. 5D), our data together suggest that the Herp–PC2 physical interaction is critical for PC2 degradation by the ERAD pathway.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND MATHODS
 FUNDING
 REFERENCES
 
The present study shows that PC2, an ER Ca release channel, is a novel ERAD substrate, using several pieces of data, including ubiquitination of PC2, degradation inhibition by proteasome inhibitors, interaction with the ERAD components (p97 and Herp) and the 26S proteasome. PC2 is ubiquitinated under the physiological condition, and PC2 ubiquitination, as well as the amounts of the PC2–p97 and PC2-S12 complexes, significantly augments when PC2 retro-translocation and degradation are blocked by proteasome inhibitors, indicating that these forms of PC2 are located at the upstream of the proteasome. ATPase p97, which provides a driving force for retro-translocation of ERAD substrates from the ER, aggregates to perinuclear regions in the presence of over-expressed PC2, presumably due to and in favor of its physical binding with PC2. ER stress inducers increase the expression of Herp, which directly promotes PC2 degradation by increasing the retro-translocation of PC2 from the ER to the proteasome complex in the cytosol. Whether other changes induced by ER stress inducers also contribute to the increased PC2 degradation remains to be determined.

Our data demonstrate that Herp regulates PC2 degradation. Herp, a known ER stress response protein (12,33,35,36), is implicated in ERAD. However, it has so far been unclear as to how this UBL protein regulates the ERAD pathway. Our data show that Herp is in the same complex as the ERAD substrate PC2. Direct changes in the Herp level, by over-expression or siRNA knockdown, positively correlate with the rate of PC2 degradation. This correlation is further supported by the effect of indirect changes in the Herp level induced by ER stress inducers with or without simultaneous Herp siRNA. Thus, the amount of Herp is critical in determining the cellular level of PC2, which may account, at least in part, for the Herp-induced reduction in the ER Ca release in ER-stressed cells (37).

Interestingly, an increase in PC2 degradation is accompanied by Herp C-terminal cleavage in cells either transiently over-expressing this ER membrane protein or under ER stress. The 50-kDa fragment of Herp corresponds likely to a mutant missing the C-terminus, based on its size and attachment to the membrane (Fig. 4), indicating that the PC2–Herp complexing is via the N-terminus or the transmembrane domain of Herp. Of note, the difference in the detected fragments between the over-expressed, tagged Herp (Fig. 4A) and the increased Herp by Tm (Fig. 5B) should be due to different antibodies used. It will be interesting to determine whether Herp cleavage plays a role in its regulation of PC2 degradation; for example, whether its C-terminus may have an inhibitory effect on its activity with respect to PC2 degradation. The observation of Herp cleavage under these conditions is consistent with our previous study (35). Also, it was reported that ATF6 and Luman, two ER membrane-localized transcription factors mediating UPR, are activated through cleavage upon UPR or when they are transiently expressed in cells (35,39,40).

We propose a six-state model to illustrate the proteasome-dependent PC2 degradation (Fig. 6) in which the involvements or roles of ubiquitin, Herp, p97, the 26S proteasome complex, ER stress inducers and proteasome inhibitors, are indicated. On the ER membrane, PC2 interacts with Herp (state 1) and is ubiquitinated (state 2), for example, by TAZ-regulated SCFβ-Trcp E3 ligase complex (23). Next, ATPase p97 is recruited onto ubiquitinated PC2 (state 3). An increase in the Herp level, induced either directly (by transfection) or indirectly (by ER stress), promotes Herp cleavage and transition from state 2 to 3. The retro-translocation of PC2 from the ER membrane to the cytosol (state 4) may be driven by ATP hydrolysis of p97 and Herp cleavage, and leads to its recognition by the proteasome complex (state 5) for ultimate degradation (state 6). Proteasome inhibitors delay PC2 retro-translocation and increase the population of PC2 in upstream states, for example, the PC2-proteasome and PC2–p97 complexes.


Figure 6
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  		Figure 6. A model for polycystin-2 (PC2) degradation through the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway. State 1, PC2 in complex with Herp. State 2, TAZ-dependent PC2 ubiquitination. State 3, binding of p97 to ubiquitinated PC2 and Herp cleavage. State 4, retro-translocated PC2 (from the ER membrane) in complex with p97 in the cytosol. State 5, PC2 in complex with the proteasome. State 6, destructed PC2.

 
It is commonly accepted, and as supported by numerous studies including those using animal or cellular models, that loss of function of PC2 or polycystin-1 (PC1), a PC2 interacting partner mutated in ~80% of ADPKD patients, results in cystogenesis. Interestingly, increased PC2 expression in TAZ-knockout mice and increased PC1 expression in pkd1 transgenic mice are both associated with renal cystogenesis (23,41). Thus, together it seems that an altered (either increased or decreased) PC1 or PC2 expression/function from their normal ranges may lead to disease. Our present study found that the degradation of pathogenic mutants R872X and E837X and truncation mutants PC2{Delta}C and PC2{Delta}N is all enhanced by Herp though the enhancement may be of different degrees (Fig. 5C), possibly due to varied strength of interaction with Herp. On the other hand, the level of PC2{Delta}NC is not affected by Herp and this mutant has no interaction with Herp. These data together indicate that the PC2–Herp physical interaction is essential to the regulation of PC2 expression (and thus, function). Thus, the ERAD-regulated PC2 turnover may be important for the pathogenesis of ADPKD. Future studies should examine (i) whether/how pathogenic point mutations in PC2 affect its interaction with and degradation by Herp, and (ii) whether/how mutations in PC2 impact the ERAD machinery, which then affects cell’s normal properties.


   MATERIALS AND MATHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND MATHODS
FUNDING
 REFERENCES
 
Cell culture, DNA constructs and gene transfection
IMCD, MDCK and HeLa cells were cultured in Dulbecco’s Modified Eagle’s Medium (high glucose; Invitrogen) containing 10% (v/v) fetal bovine serum, 1% penicillin and streptomycin at 37°C and 5% CO2. Plasmids pEGFP-PC2, pEGFP-R872X, pEGFP-E837X, pEGFP-PC2{Delta}C, pEGFP-PC2{Delta}N, and pEGFP-PC2{Delta}NC were constructed based on a method described previously (21). HeLa cells were grown to ~70% confluency prior to transfection using Lipofectamine 2000 (Invitrogen). MDCK cells stably expressing GFP-PC2 or GFP were selected as previously described (42) and maintained using G418 (300 µg/ml).

Immunoprecipitation, immunoblotting, immunofluorescence microscopy
Protein extraction, immunoblotting, immunoprecipitation and microscopy were performed as described (22). Typically, 20 and 200 µg of total cellular protein were used for immunoblotting and immunoprecipitation, respectively. HeLa cells were transiently transfected with pEGFP, pEGFP-Ub (43), pEGFP-PC2, pcDNA3.1 (Invitrogen) or pcDNA3.1-mHerpf (33) for immunoprecipitation. At 20 h post-transfection, cells were split into two equal fractions for treatment with a proteasome inhibitor. To examine the degradation of PC2 mutants we transfected HeLa cells with pEGFP-PC2, pEGFP-R872X, pEGFP-E837X, pEGFP-PC2{Delta}C, pEGFP-PC2{Delta}N, pEGFP-PC2{Delta}NC, or vector pEGFP. At 6 h post-transfection, cells were split into two equal fractions for subsequent co-transfection with pcDNA3.1-mHerpf or pcDNA3.1 at 20 h post-transfection. For the interaction of Herp with PC2 mutants, HeLa cells were transfected with pcDNA3.1-mHerpf. At 6 h post-transfection, cells were split into five equal fractions for subsequent co-transfection with PC2 or its mutant plasmids 20 h post-transfection. Fluorescent images were captured on a motorized Olympus IX81 microscopy installed with a CCD cooling RT SE6 monochrome camera (Diagnostic Instruments). Final composite images were made using Image-Pro Plus 5.0 (Media Cybernetics).

Cycloheximide chase assays
In CHX chase experiments, IMCD cells were incubated with 50 µg/ml of CHX in the presence or absence of a proteasome inhibitor for 0 or 8 h. Cells were harvested for protein preparation and immunoblotting, as described (22).

Antibodies
Rabbit antibodies against p97, Myc or FLAG, and mouse anti-ubiquitin antibody were purchased from Cell Signaling Technology. Mouse anti-Myc antibody was from Chemicon. Anti-S12, -IP3R and -Sec61{alpha} antibodies were from Affinity BioReagents. β-actin antibody was from Sigma-Aldrich Canada. Goat anti-BiP and rabbit anti-ATF6{alpha} were purchased from Santa Cruz. GFP antibodies were a gift from Dr Luc Berthiaume (University of Alberta, Canada; also available at www.eusera.com). Mouse anti-PC2 and rabbit anti-Herp antibodies were described as before (21,22,33,35). Secondary antibodies were from Amersham or Promega.

Subcellular fractionation
HeLa cells over-expressing Herp were homogenized in a cold fractionation buffer (33). The homogenate was subjected to a serial of centrifugations, at 1000 g for 10 min, 10 000 g for 10 min and 100 000 g for 60 min. The supernatants and pellets from the 100 000 g centrifugation that contained the cytosolic and membrane fractions, respectively, were collected. The pellets were dissolved in the CellLyticTMM Cell Lysis Reagent (Sigma-Aldrich Canada).

Herp and PC2 knockdown by small interference RNA
Herp Stealth siRNA (sense, 5'-UCAGAAUGCUGCUCCUCAAUU and antisense, 5'-UUGAGGAGCAGCAUUCUGAUU) and its specific control siRNA (sense, 5'-ACAUAGCCAUGCGUUACUCUU and antisense, 5'-GAGUAACGCAUGGCUAUGUUU) were used to transfect HeLa cells using Lipofectamine 2000 reagent following the manufacturer’s instructions. PC2 knockdown was described previously (22). The efficiency of the siRNA knockdown was assessed by immunoblotting.


   FUNDING
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND MATHODS
 FUNDING
REFERENCES
 
This work was supported by the Canadian Institutes of Health Research and the Kidney Foundation of Canada (to X.-Z.C.). Q.L. is a recipient of the Polycystic Kidney Disease Foundation Fellowship. X.-Z.C. is a Senior Scholar of the Alberta Heritage Foundation for Medical Research.

Conflict of Interest statement. None declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND MATHODS
 FUNDING
 REFERENCES
 

  1. Ellgaard L., Helenius A. Quality control in the endoplasmic reticulum. Nat. Rev. Mol. Cell Biol (2003) 4:181–191.[CrossRef][ISI][Medline]

  2. Rutkowski D.T., Kaufman R.J. A trip to the ER: coping with stress. Trends Cell Biol (2004) 14:20–28.[CrossRef][ISI][Medline]

  3. McCracken A.A., Brodsky J.L. Assembly of ER-associated protein degradation in vitro: Dependence on cytosol, calnexin, and ATP. J. Cell Biol (1996) 132:291–298.[Abstract/Free Full Text]

  4. Meusser B., Hirsch C., Jarosch E., Sommer T. ERAD: the long road to destruction. Nat. Cell Biol (2005) 7:766–772.[CrossRef][ISI][Medline]

  5. Fang S.Y., Ferrone M., Yang C.H., Jensen J.P., Tiwari S., Weissman A.M. The tumor autocrine motility factor receptor, gp78, is a ubiquitin protein ligase implicated in degradation from the endoplasmic reticulum. Proceedings of the National Academy of Sciences of the United States of America (2001) 98:14422–14427.[Abstract/Free Full Text]

  6. Kikkert M., Doolman R., Dai M., Avner R., Hassink G., van Voorden S., Thanedar S., Roitelman J., Chau V., Wiertz E. Human HRD1 is an E3 ubiquitin ligase involved in degradation of proteins from the endoplasmic reticulum. J. Biol. Chem (2004) 279:3525–3534.[Abstract/Free Full Text]

  7. Jarosch E., Taxis C., Volkwein C., Bordallo J., Finley D., Wolf D.H., Sommer T. Protein dislocation from the ER requires polyubiquitination and the AAA-ATPase Cdc48. Nat. Cell Biol (2002) 4:134–139.[CrossRef][ISI][Medline]

  8. Ye Y.H., Meyer H.H., Rapoport T.A. The AAA ATPase Cdc48/p97 and its partners transport proteins from the ER into the cytosol. Nature (2001) 414:652–656.[CrossRef][Medline]

  9. Ye Y.H., Meyer H.H., Rapoport T.A. Function of the p97-Ufd1-Npl4 complex in retrotranslocation from the ER to the cytosol: dual recognition of nonubiquitinated polypeptide segments and polyubiquitin chains. J. Cell Biol (2003) 162:71–84.[Abstract/Free Full Text]

  10. Lilley B.N., Ploegh H.L. A membrane protein required for dislocation of misfolded proteins from the ER. Nature (2004) 429:834–840.[CrossRef][Medline]

  11. Loureiro J., Lilley B.N., Spooner E., Noriega V., Tortorella D., Ploegh H.L. Signal peptide peptidase is required for dislocation from the endoplasmic reticulum. Nature (2006) 441:894–897.[CrossRef][Medline]

  12. Schulze A., Standera S., Buerger E., Kikkert M., van Voorden S., Wiertz E., Koning F., Kloetzel P.M., Seeger M. The ubiquitin-domain protein HERP forms a complex with components of the endoplasmic reticulum associated degradation pathway. J. Mol. Biol (2005) 354:1021–1027.[CrossRef][ISI][Medline]

  13. Sun F., Zhang R.L., Gong X.Y., Geng X.H., Drain P.F., Frizzell R.A. Derlin-1 promotes the efficient degradation of the cystic fibrosis transmembrane conductance regulator (CFTR) and CFTR folding mutants. J. Biol. Chem (2006) 281:36856–36863.[Abstract/Free Full Text]

  14. Ye Y.H., Shibata Y., Yun C., Ron D., Rapoport T.A. A membrane protein complex mediates retro-translocation from the ER lumen into the cytosol. Nature (2004) 429:841–847.[CrossRef][Medline]

  15. Clapham D.E. TRP channels as cellular sensors. Nature (2003) 426:517–524.[CrossRef][Medline]

  16. Koulen P., Cai Y., Geng L., Maeda Y., Nishimura S., Witzgall R., Ehrlich B.E., Somlo S. Polycystin-2 is an intracellular calcium release channel. Nat. Cell Biol (2002) 4:191–197.[CrossRef][ISI][Medline]

  17. McGrath J., Somlo S., Makova S., Tian X., Brueckner M. Two populations of node monocilia initiate left-right asymmetry in the mouse. Cell (2003) 114:61–73.[CrossRef][ISI][Medline]

  18. Nauli S.M., Alenghat F.J., Luo Y., Williams E., Vassilev P., Lil X.G., Elia A.E.H., Lu W.N., Brown E.M., Quinn S.J., et al. Polycystins 1 and 2 mediate mechanosensation in the primary cilium of kidney cells. Nature Genetics (2003) 33:129–137.[CrossRef][ISI][Medline]

  19. Luo Y., Vassilev P.M., Li X.G., Kawanabe Y., Zhou J. Native polycystin 2 functions as a plasma membrane Ca2+-permeable cation channel in renal epithelia. Mol. Cell. Biol (2003) 23:2600–2607.[Abstract/Free Full Text]

  20. Torres V.E., Harris P.C. Mechanisms of disease: autosomal dominant and recessive polycystic kidney diseases. Nat. Clin. Pract. Nephrol (2006) 2:40–55.[CrossRef][ISI][Medline]

  21. Li Q., Montalbetti N., Shen P.Y., Dai X.Q., Cheeseman C.I., Karpinski E., Wu G.Q., Cantiello H.F., Chen X.Z. Alpha-actinin associates with polycystin-2 and regulates its channel activity. Hum. Mol. Genet (2005) 14:1587–1603.[Abstract/Free Full Text]

  22. Wu Y.L., Dai X.Q., Li Q., Chen C.X., Mai W.Y., Hussain Z., Long W.T., Montalbetti N., Li G.C., Glynne R., et al. Kinesin-2 mediates physical and functional interactions between polycystin-2 and fibrocystin. Hum. Mol. Genet (2006) 15:3280–3292.[Abstract/Free Full Text]

  23. Tian Y., Kolb R., Hong J.H., Carroll J., Li D., You J., Bronson R., Yaffe M.B., Zhou J., Benjamin T. TAZ promotes PC2 degradation through a SCF beta-Trcp E3 ligase complex. Mol. Cell. Biol (2007) 27:6383–6395.[Abstract/Free Full Text]

  24. Friedlander R., Jarosch E., Urban J., Volkwein C., Sommer T. A regulatory link between ER-associated protein degradation and the unfolded-protein response. Nat. Cell Biol (2000) 2:379–384.[CrossRef][ISI][Medline]

  25. Lass A., McConnell E., Nowis D., Mechref Y., Kang P., Novotny M.V., Wojcik C. A novel function of VCP (valosin-containing protein; p97) in the control of N-glycosylation of proteins in the endoplasmic reticulum. Arch. Biochem. Biophys (2007) 462:62–73.[CrossRef][ISI][Medline]

  26. Travers K.J., Patil C.K., Wodicka L., Lockhart D.J., Weissman J.S., Walter P. Functional and genomic analyses reveal an essential coordination between the unfolded protein response and ER-associated degradation. Cell (2000) 101:249–258.[CrossRef][ISI][Medline]

  27. Ye Y.H., Shibata Y., Kikkert M., van Voorden S., Wiertz E., Rapoport T.A. Recruitment of the p97 ATPase and ubiquitin ligases to the site of retrotranslocation at the endoplasmic reticulum membrane. Proceedings of the National Academy of Sciences of the United States of America (2005) 102:14132–14138.[Abstract/Free Full Text]

  28. Zhong X.Y., Pittman R.N. Ataxin-3 binds VCP/p97 and regulates retrotranslocation of ERAD substrates. Hum. Mol. Genet (2006) 15:2409–2420.[Abstract/Free Full Text]

  29. Yoshida H., Haze K., Yanagi H., Yura T., Mori K. Identification of the cis-acting endoplasmic reticulum stress response element responsible for transcriptional induction of mammalian glucose-regulated proteins - Involvement of basic leucine zipper transcription factors. J. Biol. Chem (1998) 273:33741–33749.[Abstract/Free Full Text]

  30. Wiertz E.J.H.J., Tortorella D., Bogyo M., Yu J., Mothes W., Jones T.R., Rapoport T.A., Ploegh H.L. Sec61-mediated transfer of a membrane protein from the endoplasmic reticulum to the proteasome for destruction. Nature (1996) 384:432–438.[CrossRef][Medline]

  31. Wu G.Q., Somlo S. Molecular genetics and mechanism of autosomal dominant polycystic kidney disease. Molecular Genetics and Metabolism (2000) 69:1–15.[CrossRef][ISI][Medline]

  32. Pickart C.M., Cohen R.E. Proteasomes and their kin: Proteases in the machine age. Nature Reviews Molecular Cell Biology (2004) 5:177–187.[CrossRef][ISI][Medline]

  33. Kokame K., Agarwala K.L., Kato H., Miyata T. Herp, a new ubiquitin-like membrane protein induced by endoplasmic reticulum stress. J. Biol. Chem (2000) 275:32846–32853.[Abstract/Free Full Text]

  34. van Laar T., Schouten T., Hoogervorst E., van Eck M., van der Eb A.J., Terleth C. The novel MMS-inducible gene Mif1/KIAA0025 is a target of the unfolded protein response pathway. FEBS Lett (2000) 469:123–131.[CrossRef][ISI][Medline]

  35. Liang G.Q., Audas T.E., Li Y., Cockram G.P., Dean J.D., Martyn A.C., Kokame K., Lu R. Lumam/CREB3 induces transcription of the endoplasmic reticulum (ER) stress response protein Herp through an ER stress response element. Mol. Cell. Biol (2006) 26:7999–8010.[Abstract/Free Full Text]

  36. Nogalska A., Engel W.K., McFerrin J., Kokame K., Komano H., Askanas V. Homocysteine-induced endoplasmic reticulum protein (Herp) is up-regulated in sporadic inclusion-body myositis and in endoplasmic reticulum stress-induced cultured human muscle fibers. J. Neurochem (2006) 96:1491–1499.[CrossRef][ISI][Medline]

  37. Chan S.L., Fu W.M., Zhang P.S., Cheng A.W., Lee J.W., Kokame K., Mattson M.P. Herp stabilizes neuronal Ca2+ homeostasis and mitochondrial function during endoplasmic reticulum stress. J. Biol. Chem (2004) 279:28733–28743.[Abstract/Free Full Text]

  38. Foskett J.K., White C., Cheung K.H., Mak D.O.D. Inositol trisphosphate receptor Ca2+ release channels. Physiol. Rev (2007) 87:593–658.[Abstract/Free Full Text]

  39. Haze K., Yoshida H., Yanagi H., Yura T., Mori K. Mammalian transcription factor ATF6 is synthesized as a transmembrane protein and activated by proteolysis in response to endoplasmic reticulum stress. Mol. Biol. Cell (1999) 10:3787–3799.[Abstract/Free Full Text]

  40. Ye J., Rawson R.B., Komuro R., Chen X., Dave U.P., Prywes R., Brown M.S., Goldstein J.L. ER stress induces cleavage of membrane-bound ATF6 by the same proteases that process SREBPs. Mol. Cell (2000) 6:1355–1364.[CrossRef][ISI][Medline]

  41. Thivierge C., Kurbegovic A., Couillard M., Guillaume R., Cote O., Trudel M. Overexpression of PKD1 causes polycystic kidney disease. Mol. Cell. Biol (2006) 26:1538–1548.[Abstract/Free Full Text]

  42. Li Q., Dai X.D., Shen P.Y., Cantiello H.F., Karpinski E., Chen X.Z. A modified mammalian tandem affinity purification procedure to prepare functional polycystin-2 channel. FEBS Lett (2004) 576:231–236.[CrossRef][ISI][Medline]

  43. Bergink S., Salomons F.A., Hoogstraten D., Groothuis T.A.M., de Waard H., Wu J., Yuan L., Citterio E., Houtsmuller A.B., Neefjes J., et al. DNA damage triggers nucleotide excision repair-dependent monoubiquitylation of histone H2A. Genes Dev (2006) 20:1343–1352.[Abstract/Free Full Text]


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