The 20S proteasome processes NF-κB1 p105 into p50 in a translation-independent manner
2006; Springer Nature; Volume: 25; Issue: 9 Linguagem: Inglês
10.1038/sj.emboj.7601081
ISSN1460-2075
AutoresAnu K. Moorthy, О. В. Савинова, Jessica Q. Ho, Vivien Ya‐Fan Wang, Don Vu, Gourisankar Ghosh,
Tópico(s)interferon and immune responses
ResumoArticle13 April 2006free access The 20S proteasome processes NF-κB1 p105 into p50 in a translation-independent manner Anu K Moorthy Anu K Moorthy Search for more papers by this author Olga V Savinova Olga V Savinova Search for more papers by this author Jessica Q Ho Jessica Q Ho Search for more papers by this author Vivien Ya-Fan Wang Vivien Ya-Fan Wang Search for more papers by this author Don Vu Don Vu Search for more papers by this author Gourisankar Ghosh Corresponding Author Gourisankar Ghosh Department of Chemistry and Biochemistry, University of California-San Diego, La Jolla, CA, USA Search for more papers by this author Anu K Moorthy Anu K Moorthy Search for more papers by this author Olga V Savinova Olga V Savinova Search for more papers by this author Jessica Q Ho Jessica Q Ho Search for more papers by this author Vivien Ya-Fan Wang Vivien Ya-Fan Wang Search for more papers by this author Don Vu Don Vu Search for more papers by this author Gourisankar Ghosh Corresponding Author Gourisankar Ghosh Department of Chemistry and Biochemistry, University of California-San Diego, La Jolla, CA, USA Search for more papers by this author Author Information Anu K Moorthy, Olga V Savinova, Jessica Q Ho, Vivien Ya-Fan Wang, Don Vu and Gourisankar Ghosh 1 1Department of Chemistry and Biochemistry, University of California-San Diego, La Jolla, CA, USA *Corresponding author. Department of Chemistry and Biochemistry, University of California-San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0359, USA. Tel.: +1 858 822 0375; Fax: +1 858 822 1408; E-mail: [email protected] The EMBO Journal (2006)25:1945-1956https://doi.org/10.1038/sj.emboj.7601081 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The NF-κB p50 is the N-terminal processed product of the precursor, p105. It has been suggested that p50 is generated not from full-length p105 but cotranslationally from incompletely synthesized molecules by the proteasome. We show that the 20S proteasome endoproteolytically cleaves the fully synthesized p105 and selectively degrades the C-terminus of p105, leading to p50 generation in a ubiquitin-independent manner. As small as 25 residues C-terminus to the site of processing are sufficient to promote processing in vivo. However, any p105 mutant that lacks complete ankyrin repeat domain (ARD) is processed aberrantly, suggesting that native processing must occur from a precursor, which extends beyond the ARD. Remarkably, the mutant p105 that lacks the internal region including the glycine-rich region (GRR) is completely degraded by 20S proteasome in vitro. This suggests that the GRR impedes the complete degradation of the p105 precursor, thus contributing to p50 generation. Introduction NF-κB dimers play a central role in orchestrating diverse biological processes through their transcriptional activity (Ghosh et al, 1998). The NF-κB family has five members, p50, p52, p65, c-Rel, and RelB. p50 homo- and heterodimers carry out broad and diverse cellular functions. All cellular p50 is generated from its precursor p105, and thus the ultimate composition of potent NF-κB dimers is controlled at the level of processing of p105 into p50. This processing event has generated significant attention since the discovery of p105 because of its unique nature. It has been well established that the proteasome is responsible for p105 processing (Coux and Goldberg, 1998; Ciechanover et al, 2001). The generation of p50 from p105 is one of the few examples in which the substrate undergoes processing instead of complete degradation by the proteasome. The other examples of proteasome-mediated processing that generate products from the precursors are the inducible processing of yeast transcription factors SPT23 and MGA2 in response to metabolic state of yeast cells and the regulated processing of Drosophila Ci transcription factor (Chen et al, 1999; Hoppe et al, 2000). p105 is a multidomain protein consisting of four structural domains connected and flanked by flexible segments. These domains are the N-terminal domain (NTD), dimerization domain (DimD), ankyrin repeat domain (ARD), and death domain (DD) (Figure 1A). The DimD and ARD are connected by a 180-residue-long largely unstructured region, which contains the site of processing and critical elements regulating processing. The 22-amino-acid segment (351–372) immediately C-terminal to the DimD contains the nuclear localization sequence (NLS). Together the NTD, DimD, and NLS polypeptide constitute the p50 Rel homology region (RHR). A glycine-rich region (GRR) follows the RHR and ends at the site of processing (Lin and Ghosh, 1996; Orian et al, 1999). From this study, it appears that the site of processing is at or near residue 430 of mouse p105 sequence. The segment between the GRR and the ARD (roughly from residue 431 to 530) is not homologous to any known structural domain and its role in p105 processing has not been well defined. The C-terminus of p105 (residues 365–971) is also referred to as IκBγ (Inoue et al, 1992). The full-length p105 functions as an inhibitor of NF-κB transcriptional activity (Liou et al, 1992). Specific stimulus activates IκB kinase β (IKKβ), which phosphorylates p105 C-terminal serine residues 927 and 932 allowing for degradation of entire p105 and release of the bound NF-κB subunits (Lang et al, 2003). A number of studies have attempted to elucidate the mechanism of p105 processing. The accepted mechanism of p105 processing is the cotranslational processing model (Lin et al, 1998, 2000). This model proposes that the random pausing of the ribosome after synthesizing approximately 530 residues (around the beginning of ARD) leads to the generation of p50. This has been suggested to occur by proteasomal processing while the synthesized protein is still bound to the ribosome. However, when the ribosome does not pause, a complete p105 molecule is synthesized, which cannot undergo further processing. Several other studies have shown that ubiquitination was also essential for constitutive p105 processing (Coux and Goldberg, 1998; Sears et al, 1998; Ciechanover et al, 2001). Figure 1.Processing of p105(1–971) and GST-p105(365–971) to p50 or p50-like product in vitro by the 20S proteasome. (A) Domain organization of mouse p105 showing NTD, DimD, ARD, and death domain (DD). Positions of NLS, GRR, and phosphorylation sites (P) are indicated. (B) Processing of His-tagged full-length p105(1–971) by 20S proteasome. Reaction products were separated by SDS–PAGE and visualized by Western blotting with p50(NLS) antibody. (C) Processing of GST-tagged p105(365–971) by the 20S proteasome. Reaction products were separated by SDS–PAGE and visualized by Western blotting with GST antibody (left panel) or CTp105 antibody (right panel). (D) Proteasome activity assays to test activity of 20S proteasome toward a fluorogenic peptide substrate. The activity with and without 0.03% SDS is indicated by solid and dotted lines, respectively. Download figure Download PowerPoint It is generally believed that the physiologically functional proteasome is primarily the 26S proteasome and hence both constitutive processing and signal-dependent degradation of p105 are attributed to this protein complex. The 26S proteasome is composed of three large moieties: the catalytic 20S core particle and two 19S regulatory particles (Voges et al, 1999). The two gates of the barrel-like structure of the 20S proteasome are closed by interdigitating N-terminal residues of the α-type subunits restricting access for cellular proteins to the catalytic sites that reside in the inner cavity (Groll et al, 2000; Orlowski and Wilk, 2000). Two 19S regulatory particles bind to each end of the 20S core forming the 26S proteasome, which is involved in ubiquitin (Ub)-dependent and Ub-independent proteolysis. The 19S regulatory particle recognizes the target proteins, unfolds them in an energy-dependent manner, opens the entry gate of the core particle, and transfers the unfolded proteins to the sites of catalysis. A new study demonstrates a functional role for REGγ, a member of the 11S family of proteasome activators, in enhancing the Ub-independent degradation of transcriptional coactivator protein SRC-3 (Li et al, 2006). Biological activity of the 20S proteasome, independent of the regulatory subunits, did not receive significant attention until recently. An increasing number of cellular proteins including p21Cip1 as well as translation initiation factors eIF3α and eIF4G were reported to be degraded by the 20S proteasome both in vivo and in vitro (Verma and Deshaies, 2000; Touitou et al, 2001; Liu et al, 2003; Orlowski and Wilk, 2003; Baugh and Pilipenko, 2004). In this study, we wanted to test if the 20S proteasome can process p105 into p50. In vitro results using pure proteins show that indeed the 20S proteasome can generate p50 from p105. We also show that the proteasome can function as an endoprotease, and that it preferentially degrades the entire C-terminal end of the molecule. The GRR serves as a stop signal for the proteasome and increases the stability of the processed product. We tested that p50 generation is independent of translation and does not require ubiquitination. Results 20S proteasome processes p105 into p50 To test whether the 20S proteasome could generate p50 from p105 in vitro, we used recombinantly expressed and purified p105 and highly pure 20S proteasome for the processing reaction (Supplementary Figures 1 and 2). The reaction products were separated by SDS–PAGE and visualized by Western blotting with p50 (NLS) antibody. We observe that the amount of p105 decreased over time with concomitant increase of p50 (Figure 1B). These experiments were repeated reproducibly several times with different preparations of p105 and with proteasome from different sources: human cultured cells, bovine liver, and rabbit reticulocytes. We do not detect any products that would correspond to free C-terminus of p105, which is consistent with earlier in vivo studies, suggesting that the C-terminal region is completely degraded (data not shown). This would also imply that the isolated C-terminal region of p105 should be readily degraded by the 20S proteasome. To test this, we purified N-terminal glutathione-S-transferase (GST) fused to the C-terminus of p105, GST-p105(365–971), from Escherichia coli and tested its degradation by the proteasome. Reaction products were detected by Western blotting with GST antibody. We observe that the 20S proteasome generates a free GST-related product (Figure 1C, left panel). The same reaction products were probed with antibody against the extreme C-terminal peptide of p105. We do not detect any products corresponding to free C-terminus (Figure 1C, right panel). To eliminate the possibility that the 20S proteasome, used in our assays, had been artificially activated during purification or freezing, we did a fluorogenic peptide activity assay. We compared proteasomal activity in the absence and presence of 0.03% SDS. If the proteasome was artificially activated, one would expect peptide degradation even in the absence of SDS with no significant enhancement in activity with SDS. Our results, however, show that the 20S proteasome used in our p105 processing assay is indeed latent (Figure 1D). Furthermore, to confirm that the 20S proteasome did not contain other contaminating proteases, a mass spectrometric analysis was carried out. Trypsin digestion combined with LC/MS showed that the proteasome sample contained only 14 proteins, corresponding to the seven α- and seven β-subunits of mammalian 20S proteasome (Supplementary Figure 2). Thus, the activity observed in our assay must come from the 20S proteasome only. Together, these results suggest that p105 is a natural substrate of latent 20S proteasome and that p50 can be generated from the full-length precursor. p50 generation is independent of translation Generation of p50 from pure full-length p105 by the 20S proteasome prompted us to revisit the current cotranslational processing model, which excludes a precursor–product relationship between p105 and p50. Earlier studies by Fan and Maniatis (1991) had shown that p50 is generated from p105. Hence it appeared that the results of pulse–chase experiments had been interpreted differently. We therefore performed a similar pulse–chase radiolabeling experiment. HEK293T cells were transfected with a vector expressing full-length p105 as an N-terminal Flag fusion. Consistent with Fan and Maniatis's observation, we do see generation of p50 from the full-length p105 precursor (Figure 2A). However, even after a prolonged period of chase, only a fraction of p105 undergoes processing, suggesting that a significant pool of p105 is resistant to processing. Because unprocessed p105 serves specific function in vivo, there must be a mechanism to partially protect p105 from processing. Figure 2.p50 generation in vivo is independent of translation and requires ankyrin repeat containing precursors for precise processing. (A) HEK293T cells transfected (right panel) or untransfected (left panel) with Flag-tagged full-length p105 were pulse-radiolabeled with 35S-Met for 30 min and chased for the indicated time. Cell lysates were immunoprecipitated with Flag antibody and separated by SDS–PAGE and visualized by fluorography. The schematic diagram for p105 is given as a residue numbering guideline. (B) HEK293T cells transfected with various YFP-tagged p105 truncations or with the empty vector. The precursors and processed products were separated on 10% (15% in insets) SDS–PAGE gel and visualized by Western blotting with GFP antibody. The schematic diagram for p105 is given as a residue numbering guideline. (C) HEK293T cells transfected with various YFP-tagged p105 constructs, and analyzed at indicated times after transfection. Levels of precursor and product proteins were assessed by Western blotting with GFP antibody. The schematic representation of constructs used is given. Download figure Download PowerPoint We also generated a series of C-terminal deletion p105 mutants and tested if p50 is generated from these precursors by transfecting HEK293T cells with vectors expressing the truncated proteins as N-terminal yellow fluorescence protein (YFP) fusions (Figure 2B). Comparing the migration distances of the processed YFP-p50 and mutant YFP-p105(1–430) protein on SDS–PAGE gel, we concluded that the processing site appears to be at or near residue 430 (Figure 2B). Hereafter, we shall refer to 430 as the site of processing. Although we failed to resolve the unprocessed and processed bands for YFP-p105(1–445) fusion protein, the presence of the processed product from the YFP-p105(1–455) construct is clear. All proteins longer than residue 455 also undergo processing. Hence our experiments reveal that p50 can be generated from a precursor as short as ∼25 residues extended beyond the site of processing. It has been known for over 40 years, and reconfirmed recently with the elucidation of the 50S ribosome subunit crystal structure, that the peptide channel in the ribosome covers 35–50 residues of the growing polypeptide chain. Thus, such a short extension of 25 residues beyond the processing site is too small for the proteasome to make direct contact and catalyze the processing reaction while the precursor is still bound to the ribosome, which refutes the very essence of the cotranslational model. Indeed, our results are consistent with earlier observations made by Blank et al (1991) where they showed that a truncated p105 encompassing only 435 residues underwent processing. To test if the NTD has any effect in processing, we made these C-terminal deletion constructs with or without the NTD (1–244). Removal of the NTD does not affect the processing of p105, and the p50-like product, p50(∼245–430), is generated from all N-terminally truncated p105 mutants amenable for processing in vivo (Figure 2B, right panel). Our results also show that the efficiency of processing varies between shorter and longer precursors. While processing of C-terminally truncated p105 proteins is clearly evident, the full-length p105 appeared to be the most efficient substrate for generation of the processed p50 product. We do not know the reason behind this differential processing. We and others have shown that although full-length p105 is cytoplasmic, the shorter C-terminally truncated (shorter than ∼800 residues) constructs are predominantly nuclear (Blank et al, 1991; Moorthy and Ghosh, 2003). It is possible that the proteasome that processes p105 is less abundant in the nucleus and it is only during their short stay in the cytoplasm that they get processed. Interestingly, we observe an additional cryptic, smaller p50-related product that is generated only from truncated p105 precursors, which lack complete ARD and sequences beyond ARD (Figures 2C and 4E). Based on the similar molecular weights between this smaller cleavage product and an expressed fragment ending at residue 363 and the recognition of the smaller product by the p50(NLS) antibody, the aberrant cleavage site appears to be located near but C-terminal to the NLS (data not shown). We do not observe such a cryptic product when the C-terminus of the precursor ends beyond the ARD. Thus, it appears that shorter precursors are processed aberrantly. Overall, our experiments show that even a short precursor, containing only 25 residues beyond the processing site, is sufficient for p50 generation. However, although p50 is the major product of processing from these shorter precursors, another minor aberrant product is also generated. Both these observations contradict the cotranslational model. Figure 3.p50 generation in vivo is Ub-independent. (A) Temperature-sensitive E1 mutant ts20 cells maintained at the permissive (P) or restrictive (R) temperatures. Levels of p50 and p105 in lysates from cells grown in P and R temperatures were assessed by Western blotting with p50(NLS) antibody (top panel). Ub-dependent cellular p53 accumulated at the R temperature in ts20 cells (bottom panel). (B) HEK293T cells were transfected with expression plasmids encoding wild-type p105 (WT) and lysine to arginine mutant (KtoR) of p105 fused to YFP. The minimal processing labile p105, 1–465 and 245–465, were used. Proteins were separated on 15% (left panel) and 10% (right panel) SDS–PAGE gel and visualized by Western blotting with GFP antibody. The schematic diagram for the p105 constructs with the positions of the mutated lysines is given. Download figure Download PowerPoint Figure 4.p105 processing is an endoproteolytic event. (A) 20S proteasome endoproteolytically cleaves GST-p105(365–971)-GFP in vitro as detected by generation of GST-related products by Western blotting with GST antibody. (B, C) 20S proteasome endoproteolytically cleaves GST-p105(365–971)-GFP in vitro as detected by generation of GFP-related products by Western blotting with GFP antibody. GST-GFP, GST, and GFP remain stable over the course of the reaction. The schematic diagram of proteins used is given. (D) GFP fluorescence (excitation 395 nm, emission 508 nm) of GST-p105(365–971)-GFP and GST-GFP was monitored during the 20S proteasomal reaction. (E) HEK293T cells transfected with various YFP-p105-GFP double fusion proteins as indicated schematically on top. Lane 9 represents the empty GFP vector control transfection. The precursors and processed products were visualized by Western blotting with p50(NLS) antibody. Download figure Download PowerPoint To test if the minor processed product is also generated from the shorter precursors by the 20S proteasome in vitro, we expressed and purified p105(1–495) and p105(245–495) from E. coli. Indeed, a smaller p50-related product is generated from the shorter precursors by the 20S proteasome in vitro (Supplementary Figure 3). Together these in vitro and in vivo experiments demonstrate that accurate processing requires full-length p105 precursor or at least a precursor that extends beyond ARD. Similar observations both in vivo and in vitro reiterate two facts: firstly, the smaller cryptic product may not be a mere artifact of random proteolysis and, secondly, the 20S proteasome may indeed be the cellular proteasome involved in the constitutive processing of p105. p105 processing is Ub-independent Our in vitro processing reaction did not require ubiquitination. To test whether cellular constitutive processing of p105 requires ubiquitination, we used mouse ts20 cell line. These cells have a temperature-sensitive E1 Ub-activating enzyme and, at the restrictive temperature, protein degradation by the classical Ub–26S proteasome pathway is drastically reduced (Ciechanover et al, 1984; Finley et al, 1984; Chowdary et al, 1994; Salvat et al, 2000). At the permissive temperature, we observe the processing of p105 to p50. Interestingly, processing of p105 in the ts20 cell line is unaffected at the restrictive temperature (Figure 3A, top panel). As a control, the level of the transcription factor p53 was monitored at the two temperatures in the ts20 cells. At the permissive temperature, the unstable p53 is completely absent, but at the restrictive temperature, p53 is present at an appreciable level (Chowdary et al, 1994; Salvat et al, 2000) (Figure 3A, bottom panel). These results imply that ubiquitination may not be required for constitutive p105 processing. To further monitor p50 generation from p105 in these E1-deficient cells, we attempted to determine the half-life of p105. The cells were grown for different lengths of time followed by treatment with the protein synthesis inhibitor cycloheximide (CHX). Toxicity seemed to be enhanced when ts20 cells at the restrictive temperature were treated with CHX for even the relatively short time period of 4 h (Supplementary Figure 4). Because the level of p50 was already very high at the point of CHX treatment, processing of p105, if any, could not be monitored above the background level. Our attempts to carry out pulse–chase experiment to monitor endogenous processing were not successful owing to the absence of a good p50 antibody for immunoprecipitation. As an additional proof of Ub-independent processing of p105, we took the two processing compatible precursors, p105(1–465) and p105(245–465), and mutated lysines to arginines. It is known that in Ub-mediated proteasomal degradation, lysines located close to the flexible portions of a protein substrate are the ones that undergo ubiquitination (Prakash et al, 2004). Therefore, we mutated all ubiquitination susceptible lysines around the processing region, K423/K436/K438/K442/K446/K458, to arginines. The wild-type and mutant constructs were transfected into HEK293T cells as YFP fusions, and processing of wild type and mutants was compared. We observe that replacing the lysines did not affect the processing of p105 (Figure 3B). Not only did both the wild type and the mutants show processing, but the level of processing was comparable. Hence, p105 processing appears to be a Ub-independent event. The 20S proteasome functions as an endoprotease Next we addressed if p50 is generated as a result of internal cleavage of p105 polypeptide by the 20S proteasome. We created a fusion protein in which the p105 C-terminal fragment is flanked by the stable GST and green fluorescent protein (GFP) domains at the N- and C-termini, respectively, GST-p105(365–971)-GFP. As a control, we also created a GST and GFP fusion protein containing only 20 random intervening residues derived form the cloning site, GST-GFP. These proteins were expressed in E. coli and purified to near homogeneity and subjected to 20S proteasome digestion. We observed that 20S proteasome endoproteolytically cleaves GST-p105(365–971)-GFP recombinant protein and generates GST- and GFP-related products as detected by Western blotting with GST and GFP antibodies (Figure 4A–C). Although the generation of GFP-related products was not very prominent with short exposures (Figure 4B, left panel, lanes 6 and 7), we repeated the experiment, and longer exposures showed clear generation of smaller GFP-related products (Figure 4C, right panel, lanes 6 and 7). Additionally, the GFP fluorescence did not change significantly during the course of this degradation reaction (Figure 4D). This implies that, perhaps, a spectrum of C-terminal p105 polypeptides of different lengths, each retaining intact GFP, is generated during the course of this degradation reaction. The control fusion protein, GST-GFP, exhibits no cleavage and no change in GFP fluorescence in the duration of the proteasomal reaction (Figures 4A–D). We further tested if the isolated GST and GFP proteins are substrates of the 20S proteasome. As expected, no substantial degradation of free GST and GFP substrates was observed (Figures 4A–C). To test if processing of p105 is initiated through an endoproteolytic cleavage in vivo, HEK293T cells were transfected with expression vectors encoding different C-terminally truncated p105 proteins with both termini fused to stably folded domains, YFP at the N-terminus and GFP at the C-terminus. We observe the generation of YFP-p50(∼245–430) in all cases where the C-terminus of the linker is at residue 465 or beyond (Figure 4E). However, as shown earlier, the substrate must retain at least 812 residues to avoid the generation of the cryptic processed product. The fact that p105 with ‘blocked’ termini, protected by the stable domains, can undergo processing suggests that processing initiates through an endoproteolytic cleavage event. The GRR is the proteasomal stop signal To further test the role of the GRR and other p105 C-terminal sequence elements in processing, we have generated two additional GST fusion proteins: one lacking the C-terminal 171 residues, GST-p105(365–800), and the other lacking GRR, GST-p105(435–971). Like GST-p105(365–971), 20S proteasome efficiently utilizes GST-p105(365–800) in vitro and generates an identical stable product that is larger in size than GST (Figure 5A, left panel). This product is recognized by the GST antibody and its size appears to be equivalent to the recombinant, purified GST-GRR, which suggests that the product contains the GRR. Considering this and our in vivo results (Figure 4E), we conclude that the last 171 residues are not essential for p105 processing. Figure 5.GRR protects upstream domains from degradation by the 20S proteasome. (A) The left panel shows that in vitro 20S proteasome generates stable products from GST-p105(365–971) and GST-p105(365–800), but not from GST-p105(435–971), which lacks the GRR. The size of the product generated is equivalent to the size of recombinant GST-GRR. The right panel shows that the GST-GRR generated from GST-p105(365–971) is stable over a prolonged period of incubation. Precursors and products were visualized by Western blotting with GST antibodies. The schematic diagram of proteins used is given. (B) Stable p50-like product, His-p50(∼1–430), is generated from the full-length p105, but not from mutant p105, which lacks GRR, p105Δ(356–498) (left and middle panels). RHR, p105(39–363) was stable over the same period of incubation with 20S proteasome (right panel). Proteins were visualized by both Coomassie staining and Western blotting with p50 antibody. The schematic diagram of proteins used in the in vitro 20S proteasome assay is given on top. (C) HEK293T cells were transfected with full-length p105 or p105 deletion mutant p105Δ(356–498) as HA (left panel) or YFP (right panel) fusion proteins. Cell extracts were separated by SDS–PAGE followed by Western blotting with HA and GFP antibodies. ns refers to nonspecific band. (D) HEK293T cells transfected (right and middle panels) or untransfected (left panel) with Flag-tagged full-length p105 and p105Δ(356–498) were pulse-radiolabeled with 35S-Met for 30 min and chased for the indicated time. Cell lysates were immunoprecipitated with Flag antibody and separated by SDS–PAGE and visualized by fluorography. Download figure Download PowerPoint When GST-p105(435–971) was subjected to 20S proteasome, we observed similar reduction of the full-length protein with increasing reaction time, but no significant amount of GST accumulated during the course of the reaction (Figure 5A, left panel). It is possible that in the absence of the GRR, the entire GST-p105(435–971) is degraded by the 20S proteasome. An alternative explanation would be that in the absence of the GRR sequence, the proteasome makes multiple cleavages within GST-p105(435–971), and the amount of each of these products is too low to be detected by Western blotting. Overall, these results indicate that the GRR is not a good substrate for the proteasome and serves as an effective stop signal, protecting the NTDs from degradation. GST-GRR generated by the 20S proteasome from GST-p105(365–971) was resistant to further degradation, confirming that the GRR is a poor substrate for proteasomal degradation (Figure 5A, right panel). To further test the role of the GRR in proteasomal degradation, we used an internal deletion mutant of p105 where the entire processing region containing GRR was removed, p105Δ(356–498) (Fu et al, 2004). We cloned p105Δ(356–498) into a bacterial expression vector and expressed it in E. coli. Unfortunately, we failed to purify this deletion mutant to homogeneity. A contaminant approximately 50 kDa remained associated with it. Based on the immunoreactivity of this 50 kDa protein, we concluded that this molecule is p105-related. Its approximate molecular
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