The knowledge of transporter protein expression and function at the human blood-brain barrier (BBB) is critical to prediction of drug BBB penetration and design of strategies for improving drug delivery to the brain or brain tumor. This study determined absolute transporter protein abundances in isolated microvessels of human normal brain (N = 30), glioblastoma (N = 47), rat (N = 10) and mouse brain (N = 10), and cell membranes of MDCKII cell lines, using targeted proteomics. In glioblastoma microvessels, efflux transporters (ABCB1 and ABCG2), monocarboxylate transporter 1 (MCT1), glucose transporter 1 (GLUT1), sodium-potassium pump (Na/K ATPase), and Claudin-5 protein levels were significantly reduced, while large neutral amino acid transporter 1 (LAT1) was increased and GLU3 remained the same, as compared with human normal brain microvessels. ABCC4, OATP1A2, OATP2B1, and OAT3 were undetectable in microvessels of both human brain and glioblastoma. Species difference in BBB transporter abundances was noted. Cellular permeability experiments and modeling simulations suggested that not a single apical uptake transporter but a vectorial transport system consisting of an apical uptake transporter and basolateral efflux mechanism was required for efficient delivery of poor transmembrane permeability drugs from the blood to brain.
Abstract BACKGROUND Mechanistic understanding and quantitative prediction of drug penetration across the human blood-brain barrier (BBB) is critical to rational drug development and treatment for brain cancer especially glioblastoma. However, prediction of drug brain/tumor penetration has been significantly hindered mainly due to the lack of quantitation data on transporter protein expression levels at the human BBB. This study was to determine protein expression levels of major transporters and markers at the BBB of human brain and glioblastoma. METHOD The absolute protein expression levels of major transporters and markers were determined in isolated microvessels of human brain (N=30), glioblastoma (N=47), rat (N=10) and mouse brain (N=10), using liquid chromatography with tandem mass spectrometry (LC-MS/MS) based targeted proteomics. RESULTS In isolated microvessels of 30 human brain specimens, the median protein abundances for ABCB1, ABCG2, GLUT1, GLUT3, LAT1, MCT1, Na/K ATPase, and Claudin-5 were 3.38, 6.21, 54.51, 7.17, 3.42, 5.71, 32.14, and 1.15 fmol/µg protein, respectively. In glioblastoma microvessels, ABCB1, ABCG2, MCT1, GLUT1, Na/K ATPase, and Claudin-5 protein levels were significantly reduced, while LAT1 was increased and GLU1 remained the same. ABCC4, OATP1A2, OATP2B1, and OAT3 were undetectable in isolated microvessels of both human brain and glioblastoma. There was species difference in transporter protein expression levels in isolated microvessels of human, rat and mouse brain. Specifically, rodent BBB expressed significantly higher ABCB1 but similar ABCG2, as compared to human BBB. CONCLUSION The physical and biochemical barriers of the BBB in glioblastomas are largely disrupted, as indicated by the loss or significant reduction in protein expression of the tight junction marker (claudin-5), brain endothelial cell marker (GLUT1), and major efflux transporters (ABCB1 and ABCG2) as compared to normal human BBB. Differential BBB transporter protein expression levels provides mechanistic and quantitative basis for the prediction of heterogeneous drug penetration into human normal brain and glioblastoma.
Epithelial-mesenchymal transition (EMT) is a key process in tumor progression and metastasis.Previous studies have shown that MCF10A human mammary epithelial cells undergo EMT when cultured at low cell density, but the underlying mechanism remains poorly understood.Here we show that the expression of proteasome genes and the proteasome activity increase in response to low cell density.Moreover, the proteasome inhibitor MG132 blocks EMT in sparse MCF10A cells.The transcription of E-cadherin gene is repressed in sparse MCF10A cells, but is recovered upon the treatment with MG132.While the mRNA levels of Snail and Slug are markedly elevated at low cell density, the steady state protein levels are similar between sparse and confluent cells.These results suggest that the suppression of E-cadherin gene expression in sparse MCF10A cells is likely caused by downregulation of its transcription activator(s) rather than upregulation of its repressors such as Snail and Slug.This study reveals that cell density-dependent EMT in MCF10A cells is regulated by proteasome activity.
Substrates of the ubiquitin system are degraded by the 26 S proteasome, a complex protease consisting of at least 32 different subunits. Recent studies showed that RPN4 (also named SON1 and UFD5) is a transcriptional activator required for normal expression of the Saccharomyces cerevisiae proteasome genes. Interestingly, RPN4 is extremely short-lived and degraded by the 26 S proteasome, establishing a feedback circuit that controls the homeostatic abundance of the 26 S proteasome. The mechanism underlying the degradation of RPN4, however, remains unclear. Here we demonstrate that the proteasomal degradation of RPN4 is mediated by two independent degradation signals (degron). One degron leads to ubiquitylation on internal lysine(s), whereas the other is independent of ubiquitylation. Stabilization of RPN4 requires inhibition of internal ubiquitylation and inactivation of the ubiquitin-independent degron. RPN4 represents the first proteasomal substrate in S. cerevisiae that can be degraded through ubiquitylation or without prior ubiquitylation. This finding makes it possible to use both yeast genetics and biochemical analysis to investigate the mechanism of ubiquitin-independent proteolysis. Substrates of the ubiquitin system are degraded by the 26 S proteasome, a complex protease consisting of at least 32 different subunits. Recent studies showed that RPN4 (also named SON1 and UFD5) is a transcriptional activator required for normal expression of the Saccharomyces cerevisiae proteasome genes. Interestingly, RPN4 is extremely short-lived and degraded by the 26 S proteasome, establishing a feedback circuit that controls the homeostatic abundance of the 26 S proteasome. The mechanism underlying the degradation of RPN4, however, remains unclear. Here we demonstrate that the proteasomal degradation of RPN4 is mediated by two independent degradation signals (degron). One degron leads to ubiquitylation on internal lysine(s), whereas the other is independent of ubiquitylation. Stabilization of RPN4 requires inhibition of internal ubiquitylation and inactivation of the ubiquitin-independent degron. RPN4 represents the first proteasomal substrate in S. cerevisiae that can be degraded through ubiquitylation or without prior ubiquitylation. This finding makes it possible to use both yeast genetics and biochemical analysis to investigate the mechanism of ubiquitin-independent proteolysis.
The 26S proteasome is the major protease responsible for degradation of regulatory and abnormal proteins in the cell. Proteasomal degradation controls many cellular processes including, but not limited to, cell cycle control, transcription, DNA repair, apoptosis, quality control and antigen presentation. To elucidate how the proteasome is regulated is crucial to our understanding of the molecular details of proteasomal degradation and its functions in diverse biological pathways. In this article, I will highlight recent advances in understanding the proteasome structure and assembly and the regulation of proteasome gene expression. The implications of these new developments in cancer therapy will also be discussed.
<p>SUPPLEMENTARY METHODS Supplementary Figure 1 Overall metabolic kinetics of AZD1775 in human liver and intestinal microsomes (HLM and HIM). Supplementary Figure 2 PBPK model simulations showing the impact of BBB integrity (i.e., PSB), transporter activity (i.e., CLefflux,vivo and CLuptake,vivo), and drug binding to brain tissues (i.e., fu,br) on the extent of AZD1775 BBB penetration (assessed by Kp,uu) and the rate of penetration (assessed by Tmax,br).</p>
Eukaryotes contain a highly conserved multienzyme system which covalently links a small protein, ubiquitin, to a variety of intracellular proteins that bear degradation signals recognized by this system. The resulting ubiquitin-protein conjugates are degraded by the 26S proteasome, an ATP-dependent protease. Pathways that involve ubiquitin play major roles in a huge variety of processes, including cell differentiation, cell cycle, and responses to stress. In this article we briefly review the design of the ubiquitin system, and describe two recent advances, the finding that ubiquitin ligases interact with specific components of the 26S proteasome, and the demonstration that peptides accelerate their uptake into cells by activating the N-end rule pathway, one of several proteolytic pathways of the ubiquitin system.
Lysine selection is a long-standing problem in protein ubiquitination catalyzed by the RING ubiquitin ligases. It is well known that many substrates carry multiple lysines that can be ubiquitinated. However, it has seldom been addressed whether one lysine is preferred for ubiquitin conjugation when all other lysines exist. Here we studied the mechanism underlying ubiquitin-dependent degradation of Rpn4, a transcription activator of the Saccharomyces cerevisiae proteasome genes. We found that the ubiquitin-dependent degradation of Rpn4 can be mediated by six different lysines. Interestingly, we showed through in vivo and in vitro assays that lysine 187 is selected for ubiquitination when all other lysines are available. To the best of our knowledge, this is the first demonstration of a preferential ubiquitination site chosen from a group of lysines susceptible for ubiquitination. We further demonstrated that lysine 187 and a proximal acidic domain constitute a portable degradation signal. The implications of our data are discussed. Lysine selection is a long-standing problem in protein ubiquitination catalyzed by the RING ubiquitin ligases. It is well known that many substrates carry multiple lysines that can be ubiquitinated. However, it has seldom been addressed whether one lysine is preferred for ubiquitin conjugation when all other lysines exist. Here we studied the mechanism underlying ubiquitin-dependent degradation of Rpn4, a transcription activator of the Saccharomyces cerevisiae proteasome genes. We found that the ubiquitin-dependent degradation of Rpn4 can be mediated by six different lysines. Interestingly, we showed through in vivo and in vitro assays that lysine 187 is selected for ubiquitination when all other lysines are available. To the best of our knowledge, this is the first demonstration of a preferential ubiquitination site chosen from a group of lysines susceptible for ubiquitination. We further demonstrated that lysine 187 and a proximal acidic domain constitute a portable degradation signal. The implications of our data are discussed. The ubiquitin (Ub) 2The abbreviations used are: Ub, ubiquitin; E1, Ub-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin-protein isopeptide ligase; E4, Ub chain elongation factor; HA, hemagglutinin; GST, glutathione S-transferase; NAD, N-terminal acidic domain; CAD, C-terminal acidic domain. system is responsible for selectively marking abnormal and regulatory proteins for degradation by the proteasome (1Hershko A. Ciechanover A. Varshavsky A. Nat. Med. 2000; 10: 1073-1081Crossref Scopus (570) Google Scholar, 2Pickart C.M. Cell. 2004; 116: 181-190Abstract Full Text Full Text PDF PubMed Scopus (588) Google Scholar, 3Conaway R.C. Brower C.S. Conaway J.W. Science. 2002; 296: 1254-1258Crossref PubMed Scopus (346) Google Scholar). Ubiquitination of a protein substrate is a consecutive process involving multiple enzymes (4Pickart C.M. Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (2920) Google Scholar). Ub is first activated by the Ub-activating enzyme (E1), forming a thioester between the C-terminal carboxyl group of Ub and a specific cysteine of the E1. The Ub moiety of the E1∼Ub thioester is thereafter transferred to one of the Ub-conjugating enzymes (E2). The Ub moiety of the E2∼Ub thioester is conjugated, via an isopeptide bond, to the ϵ-amino group of a lysine residue (Lys) of a substrate or a preceding Ub molecule conjugated to the substrate, the latter reaction resulting in a substrate-linked multi-Ub chain. Most E2s function in complex with one of the E3 enzymes or Ub ligases. A Ub ligase also denotes an E2·E3 complex. Besides this common scenario, multiubiquitination of a subset of substrates also requires a Ub chain elongation factor (E4) (5Koegl M. Hoppe T. Schlenker S. Ulrich H.D. Mayer T.U. Jentsch S. Cell. 1999; 96: 635-644Abstract Full Text Full Text PDF PubMed Scopus (845) Google Scholar). Ubiquitination of a specific substrate is mainly regulated through modulation of its degradation signal (degron) and through control of the activity of its cognate E3 (4Pickart C.M. Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (2920) Google Scholar, 6Petroski M.D. Deshaies R.J. Nat. Rev. Mol. Cell Biol. 2005; 6: 9-20Crossref PubMed Scopus (1682) Google Scholar, 7Weissman A.M. Nat. Rev. Mol. Cell Biol. 2001; 2: 169-178Crossref PubMed Scopus (1257) Google Scholar, 8Tyers M. Jorgensen P. Curr. Opin. Genet. Dev. 2000; 10: 54-64Crossref PubMed Scopus (270) Google Scholar). The isopeptide bond between Ub and a substrate can be hydrolyzed by deubiquitinating enzymes, which provides yet another layer of regulation for substrate ubiquitination (9Amerik A.Y. Hochstrasser M. Biochim. Biophys. Acta. 2004; 1695: 189-207Crossref PubMed Scopus (744) Google Scholar). Most E3s are grouped into two families (HECT E3 and RING E3) based on their catalytic modules and features of sequence and structure (4Pickart C.M. Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (2920) Google Scholar, 7Weissman A.M. Nat. Rev. Mol. Cell Biol. 2001; 2: 169-178Crossref PubMed Scopus (1257) Google Scholar). A HECT E3 can accept Ub moiety from an associated E2∼Ub thioester, forming an E3∼Ub thioester intermediate and acting as a proximal Ub donor to the substrate that it selects. By contrast, formation of thioester between a RING E3 and Ub has not been detected. The precise mechanism by which a RING E3 catalyzes the transfer of Ub from the E2∼Ub thioester to the substrate is still poorly understood. Many known substrates of the RING E3s carry multiple lysines that can be ubiquitinated (10Petroski M.D. Deshaies R.J. Mol. Cell. 2003; 11: 1435-1444Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 11Nakamura S. Roth J.A. Mukhopadhyay T. Mol. Cell. Biol. 2000; 20: 9391-9398Crossref PubMed Scopus (161) Google Scholar, 12Kornitzer D. Raboy B. Kulka R.G. Fink G.R. EMBO J. 1994; 13: 6021-6030Crossref PubMed Scopus (219) Google Scholar). It is unclear whether one lysine is preferred for Ub conjugation when all other lysines susceptible for ubiquitination are also available in the substrate. This question remains one of the central issues in regard to the mechanism of substrate ubiquitination by the RING E3s. It is also uncertain whether or not formation of multiple Ub chains is required for substrate degradation by the proteasome. Failure to address this problem partly results from biased experimental designs that emphasized the essential but not the sufficient lysine for substrate ubiquitination and degradation. Rpn4 (also named Son1 and Ufd5) is a transcription activator required for normal expression of the Saccharomyces cerevisiae proteasome genes (13Mannhaupt G. Schnall R. Karpov V. Vetter I. Feldmann H. FEBS Lett. 1999; 450: 27-34Crossref PubMed Scopus (264) Google Scholar, 14Xie Y. Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3056-3061Crossref PubMed Scopus (345) Google Scholar). Interestingly, Rpn4 is extremely short-lived and degraded by the proteasome (14Xie Y. Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3056-3061Crossref PubMed Scopus (345) Google Scholar). These observations and subsequent reports demonstrated that the proteasome homeostasis is regulated by a negative feedback circuit in which Rpn4 up-regulates the proteasome genes and is destroyed by the proteasome (15Ju D. Wang L. Mao X. Xie Y. Biochem. Biophys. Res. Commun. 2004; 321: 51-57Crossref PubMed Scopus (65) Google Scholar, 16London M. Keck B.I. Ramos P.C. Dohmen R.J. FEBS Lett. 2004; 567: 259-264Crossref PubMed Scopus (75) Google Scholar). Intriguingly, the proteasomal degradation of Rpn4 can be mediated by two distinct pathways (17Ju D. Xie Y. J. Biol. Chem. 2004; 279: 23851-23854Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). One pathway is Ub-independent, whereas the other involves lysine ubiquitination. Recently, we demonstrated that the Ub-dependent degradation of Rpn4 is mediated by the Rad6/Ubr2 Ub ligase (18Wang L. Mao X. Ju D. Xie Y. J. Biol. Chem. 2004; 279: 55218-55223Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Ubr2, a RING E3, is a sequence homolog of Ubr1, the E3 component of the N-end rule pathway (18Wang L. Mao X. Ju D. Xie Y. J. Biol. Chem. 2004; 279: 55218-55223Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 19Hochstrasser M. Annu. Rev. Genet. 1996; 30: 405-439Crossref PubMed Scopus (1459) Google Scholar, 20Xie Y. Varshavsky A. EMBO J. 1999; 18: 6832-6844Crossref PubMed Scopus (143) Google Scholar). The Ubr2-mediated ubiquitin-dependent degradation of Rpn4 has been shown to play an important role in controlling the steady-state levels of Rpn4 and the proteasome (18Wang L. Mao X. Ju D. Xie Y. J. Biol. Chem. 2004; 279: 55218-55223Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). In the current work, we investigated the molecular mechanism underlying the Ub-dependent degradation of Rpn4. We found that the Ub-dependent degradation of Rpn4 can be mediated by six different lysines. Remarkably, among these six lysines, Lys-187 is the preferential ubiquitination site. We further demonstrated that Lys-187 and a proximal acidic domain constitute a portable Ubr2-dependent degradation signal. Yeast Strains and Plasmids—The yeast strains used were JD52 (MATa his3-Δ200 leu2–3, 112 lys2–801 trp1-Δ63 ura3–52) and YXY274 (a ubr2Δ::HIS3 derivative of JD52; see Ref. 18Wang L. Mao X. Ju D. Xie Y. J. Biol. Chem. 2004; 279: 55218-55223Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Details of plasmid constructs are available upon request. All mutants carrying point mutations and/or deletions were generated by PCR-mediated mutagenesis and confirmed by DNA sequencing. Pulse-Chase and Immunoprecipitation Analysis—S. cerevisiae cells from 10-ml cultures (A600 of 0.8–1.0) in SD medium containing 0.1 mm CuSO4 and essential amino acids were harvested. The cells were resuspended in 0.3 ml of the same medium supplemented with 0.15 mCi of [35S]methionine/cysteine (Expre35S35S labeling mix, PerkinElmer Life Sciences) and incubated at 30 °C for 5 min. The cells were then pelleted and resuspended in the same SD medium with cycloheximide (0.2 mg/ml) and excessive cold l-methionine/l-cysteine (2 mg/ml l-methionine and 0.4 mg/ml l-cysteine) and chased at 30 °C. An equal volume of sample was withdrawn at each time point. Labeled cells were harvested, lysed in an equal volume of 2× SDS buffer (2% SDS, 30 mm dithiothreitol, 90 mm Na-HEPES, pH 7.5), and incubated at 100 °C for 3 min. The supernatants were diluted 20-fold with buffer A (1% Triton X-100, 150 mm NaCl, 1 mm EDTA, 50 mm Na-HEPES, pH 7.5) before being applied to immunoprecipitation with anti-HA antibody (Sigma) or anti-β-galactosidase antibody (Promega) combined with protein A-agarose (Calbiochem) or anti-FLAG M2 affinity-agarose (Sigma). The volumes of supernatants used in immunoprecipitation were adjusted to equalize the amounts of 10% trichloroacetic acid-insoluble 35S. The immunoprecipitates were washed three times with buffer A and resolved by SDS-PAGE followed by autoradiography and quantitation with a PhosphorImager (Amersham Biosciences). Pulldown-Ubiquitination Assay—The pulldown-ubiquitination assay was carried out as described previously (18Wang L. Mao X. Ju D. Xie Y. J. Biol. Chem. 2004; 279: 55218-55223Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Specifically, ubr2Δ cells harboring plasmid 425GAL1FLAGUBR2 overexpressing N-terminally FLAG-tagged Ubr2 from the GAL1 promoter in a high copy vector were grown in synthetic selective medium containing 2% galactose to an A600 of 1.8. Cells were spun down and manually ground to fine powder with a pestle. The pellet being ground was kept frozen by liquid nitrogen. Crude extracts were prepared by incubation of the powder in buffer B (0.2% Triton X-100, 150 mm NaCl, 50 mm HEPES, pH 7.5) plus protease inhibitor mix (Roche Diagnostics). Approximately, 0.2 mg of crude extract was used for each pulldown. After a standard pulldown, the beads were washed three times with buffer C (25 mm HEPES, pH 7.5, 25 mm KCl, 5 mm MgCl2, 2 mm ATP, and 0.1 mm dithiothreitol) and then subjected to ubiquitination reactions containing 100 nm Uba1 and 1 μm Rad6 with or without 7 μm ubiquitin in buffer C at 30 °C for 45 min. The GST fusion proteins were resolved by SDS-PAGE followed by immunoblotting with anti-GST antibody (Sigma). Rad6 was overexpressed and purified from vector pET-11d in bacteria, whereas N-terminally hexahistidine-tagged Uba1 was overexpressed from YEplac181 in S. cerevisiae and purified by nickel-nitrilotriacetic acid chromatography according to the manufacturer's instructions (Qiagen). Ub-dependent Degradation of Rpn4 Mediated by Different Lysines with Lys-187 Being the Most Efficient—Our early work has shown that proteasomal degradation of Rpn4 can be mediated by two distinct mechanisms, Ub-dependent and -independent (17Ju D. Xie Y. J. Biol. Chem. 2004; 279: 23851-23854Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Taking advantage of the observation that deletion of the N-terminal 10 amino acids substantially inhibits the Ub-independent degron but displays no effect on the Ub-dependent degradation of Rpn4 (17Ju D. Xie Y. J. Biol. Chem. 2004; 279: 23851-23854Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 18Wang L. Mao X. Ju D. Xie Y. J. Biol. Chem. 2004; 279: 55218-55223Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), we adopted Rpn4Δ1–10 to study the molecular mechanism underlying the Ub-dependent degradation of Rpn4. We first tried to define the lysine(s) required for Rpn4Δ1–10 degradation. Rpn4 possesses 35 lysines throughout its 531-amino acid sequence. Lys-9 was already deleted in Rpn4Δ1–10. Previous study and current pulse-chase analysis showed that the turnover of Rpn4Δ1–10/10R, a mutant of Rpn4Δ1–10 with 10 N-terminal lysines (Lys-47 to Lys-187, referred to the positions in wild type Rpn4, see Fig. 1A) mutated to arginines, was severely inhibited (Fig. 1, B and E), indicating that the major ubiquitination site or sites are among the 10 N-terminal lysines. Note that the band just above the position of Rpn4Δ1–10 and Rpn4Δ1–10/10R represents a phosphorylated form of these two proteins, which is sensitive to treatment with calf intestinal alkaline phosphatase (data not shown). To determine whether all 10 N-terminal lysines are required for Rpn4Δ1–10 degradation, we constructed two Rpn4Δ1–10 mutants that bear Lys-to-Arg mutations for the first four lysines (N-4R) and for the fifth to tenth lysines (C-6R), respectively. Pulse-chase experiments were carried out to measure the stability of these two proteins. Although C-6R remained unstable, its turnover was much slower than that of N-4R, which was degraded as rapidly as Rpn4Δ1–10 (Fig. 1C). These results indicate that whereas the four N-terminal lysines are dispensable, one or more of the next six lysines (Lys-123 to Lys-187) are essential for wild type level degradation of Rpn4Δ1–10. To determine whether any one of the six lysines (Lys-123, Lys-132, Lys-137, Lys-141, Lys-158, and Lys-187) is sufficient to sustain the degradation of Rpn4Δ1–10 at a normal rate, an individual lysine was added back into Rpn4Δ1–10/10R, and the turnover rates of the resulting mutants were measured by pulse-chase analysis. As shown in Fig. 1D, all Rpn4Δ1–10/10R derivatives carrying one of the six lysines were short-lived (lanes 1–18). Remarkably, the mutant with Lys-187 (Rpn4Δ1–10/K187) was clearly degraded faster than others (Fig. 1D, compare lanes 4–6 with lanes 1–3 and 7–18). The half-life of Rpn4Δ1–10/K187 is ∼3 min, which is similar to that of Rpn4Δ1–10, whereas the half-lives of other derivatives are ∼8 min (Fig. 1, B, D, and E). Thus, the degradation of Rpn4Δ1–10 can be efficiently mediated by any one of the six lysines in the N-terminal region (Lys-123, Lys-132, Lys-137, Lys-141, Lys-158, and Lys-187). Lys-187, however, is the only one that can sustain wild type level degradation of Rpn4Δ1–10. Lys-187 Is the Preferential Ubiquitination Site of Rpn4—The more efficient degradation of Rpn4Δ1–10 mediated by Lys-187 than by other lysines promoted us to examine if Lys-187 is a preferential ubiquitination site. We first sought to compare the ubiquitination efficiency of various Rpn4Δ1–10/10R derivatives that carry one of the lysines from Lys-132 to Lys-187. As the measurement of multiubiquitinated substrates formed in vivo can be complicated because of their degradation by the proteasome and rapid deubiquitination by the deubiquitinating enzymes, we decided to assess the efficiency of conjugation of the first Ub moiety to different lysines added back to Rpn4Δ1–10/10R. To this end, we took advantage of UbK48R/G76A, a ubiquitin mutant that inhibits the formation of a Lys-48-linked multi-Ub chain (21Finley D. Sadis S. Monia B.P. Boucher P. Ecker D.J. Crook S.T. Chau V. Mol. Cell. Biol. 1994; 14: 5501-5509Crossref PubMed Scopus (303) Google Scholar). Our early work has shown that the Ub-dependent degradation of Rpn4 is through Lys-48-linked multiubiquitination (17Ju D. Xie Y. J. Biol. Chem. 2004; 279: 23851-23854Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Moreover, UbK48R/G76A is a poor substrate for deubiquitinating enzymes (22Hodgins R.R.W. Ellison K.S. Ellison M.J. J. Biol. Chem. 1992; 267: 8807-8812Abstract Full Text PDF PubMed Google Scholar) such that substrates conjugated with a moiety of UbK48R/G76A can be well preserved in a short pulse-labeling period. Therefore, the ratio of monoubiquitinated to nonubiquitinated species of a specific substrate largely reflects the ubiquitination efficiency at a defined lysine. Cells co-overexpressing UbK48R/G76A with N-terminally HA-tagged Rpn4Δ1–10, Rpn4Δ1–10/10R, or Rpn4Δ1–10/10R derivatives carrying one of the lysines from Lys-132 to Lys-187 were metabolically labeled with [35S]methionine for 5 min. To facilitate the determination of monoubiquitinated substrates, a transformant co-overexpressing N-terminally HA-tagged Rpn4Δ1–10 and N-terminally His6-Myc-tagged UbK48R/G76A (His -Myc-UbK48R/G76A6) was used as control. Substrates conjugated with epitope-tagged Ub have been shown to migrate slower than those with untagged Ub (17Ju D. Xie Y. J. Biol. Chem. 2004; 279: 23851-23854Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 23Ellison M.J. Hochstrasser M. J. Biol. Chem. 1991; 266: 21150-21157Abstract Full Text PDF PubMed Google Scholar). 35S-Labeled cell extracts were incubated with anti-HA antibody, and the precipitated substrates were separated by SDS-PAGE. As expected, a characteristic difference in mobility was observed between the Rpn4Δ1–10 conjugates attached with His6-Myc-UbK48R/G76A and UbK48R/G76A (Fig. 2A, compare lanes 1 and 2). Although no ubiquitinated species of Rpn4Δ1–10/10R was observed, monoubiquitinated species of the Rpn4Δ1–10/10R derivatives were readily detected (Fig. 2A, compare lanes 3 and 4–8), consistent with the observation that any one of these lysines can mediate Rpn4Δ1–10 degradation (Fig. 1D). Remarkably, the ratio of monoubiquitinated to nonubiquitinated Rpn4Δ1–10/K187 was comparable with that of Rpn4Δ1–10 and much higher (∼3–5-fold) than those of other Rpn4Δ1–10/10R derivatives bearing a different lysine (Fig. 2A, lanes 2–8). This observation is in line with the expectation of Lys-187 as a preferential Ub acceptor. Interestingly, monoubiquitinated Rpn4Δ1–10/K187 migrated noticeably faster than the monoubiquitinated species of other Rpn4Δ1–10/10R derivatives, whereas all nonubiquitinated substrates had a similar mobility (Fig. 2A, compare lanes 8 and 4–7), suggesting that the Lys-187-Ub conjugate exhibits a different configuration compared with the Ub conjugates at other lysines. Strikingly, only one type of monoubiquitinated species of Rpn4Δ1–10 was detected based on mobility, and this mobility was the same as that of the monoubiquitinated Rpn4Δ1–10/K187 (Fig. 2A, compare lanes 2 and 8). These observations reveal that Lys-187 is selected for ubiquitination from multiple lysines that could serve as Ub acceptors in Rpn4Δ1–10. It is noteworthy that small but noticeable amounts of Rpn4Δ1–10 and Rpn4Δ1–10/K187 appeared to bear two Ub moieties (Fig. 2A, lanes 2 and 8). Given that Rpn4Δ1–10/10R was not ubiquitinated, Lys-187 was therefore the only Ub-acceptor in Rpn4Δ1–10/K187. It is likely that the diubiquitinated species of Rpn4Δ1–10/K187 was generated by attachment of an endogenous Ub molecule to Lys-187 followed by conjugation of a UbK48R/G76A moiety to Lys-48 of the preceding endogenous Ub. The detection of diubiquitinated species of Rpn4Δ1–10/K187 but not other Rpn4Δ1–10/10R derivatives carrying Lys-132, Lys-137, Lys-141, or Lys-158 reflects the higher ubiquitination efficiency at Lys-187, further supporting that Lys-187 is the preferential ubiquitination site of Rpn4. To verify the results of the in vivo ubiquitination experiments, we performed a pulldown-ubiquitination assay, which has successfully shown that Ubr2, pulled down by GST fusion of Rpn4Δ1–10 (GST-Rpn4Δ1–10) from yeast extract, multiubiquitinated GST-Rpn4Δ1–10 in the presence of Rad6 and Uba1 (18Wang L. Mao X. Ju D. Xie Y. J. Biol. Chem. 2004; 279: 55218-55223Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Agarose beads preloaded with GST-Rpn4Δ1–10, GST-Rpn4Δ1–10/10R, GST-Rpn4Δ1–10/K187, and GST-Rpn4Δ1–10/K158 were incubated with extract from ubr2Δ cells overexpressing Ubr2 from the GAL1 promoter in a high copy plasmid. After a standard pulldown, the beads were further incubated with purified Rad6 and Uba1 in the presence (lanes 1, 3, 5, and 7) or absence (lane 2, 4, 6, and 8) of ubiquitin (Fig. 2B). The GST fusions were then separated by SDS-PAGE and subjected to immunoblotting analysis with anti-GST antibody. As expected, the ubiquitination was specific to the N-terminal lysines of Rpn4 as GST-Rpn4Δ1–10/10R was not ubiquitinated (Fig. 2B, lanes 3 and 4). Consistent with the in vivo ubiquitination assay, GST-Rpn4Δ1–10/K187 was ubiquitinated more efficiently than GST-Rpn4Δ1–10/K158 (Fig. 2B, compare lanes 5 and 7). Moreover, the ubiquitination pattern of GST-Rpn4Δ1–10/K187 was virtually identical to the major ubiquitinated species of GST-Rpn4Δ1–10, which is somewhat different from that of GST-Rpn4Δ1–10/K158 (Fig. 2B, compare lanes 1, 5, and 7). The difference in mobility of the monoubiquitinated species of GST-Rpn4Δ1–10/K187 and GST-Rpn4Δ1–10/K158 was also observed in the pull-down-ubiquitination assay (Fig. 2B, compare lanes 5 and 7; upper and lower panels). Taken together, the results from in vivo and in vitro assays demonstrate that Lys-187 is the preferential ubiquitination site of Rpn4, whereas the other five lysines serve as alternative ubiquitination sites. The N-terminal Acidic Domain (NAD) Is Essential for the Ub-dependent Degradation of Rpn4—Our recent study has located the major, if not exclusive, degron of Rpn4 to its N-terminal 229-residue domain (17Ju D. Xie Y. J. Biol. Chem. 2004; 279: 23851-23854Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). To further define the minimal sequences required for the Ub-dependent degradation of Rpn4, we created a series of N-terminally truncated mutants (Fig. 3A). Pulse-chase analysis showed that deletion up to the N-terminal 148 amino acids had no effect on the turnover of Rpn4 (Fig. 3B, lanes 1–12), implying that the Ub-dependent degron was located within residues 149–229. Not surprisingly, further deletion to residue 190 or 205 inhibited the degradation (Fig. 3B, lanes 13–18) as the preferential and all alternative ubiquitination sites were removed. Interestingly, the sequence of residues 211–229 is rich in acidic amino acids, a characteristic of transcription activation domain (Fig. 3A). We suspect that this domain might act as a ubiquitination signal because the ubiquitination signals of transcription activators are often overlapped with their activation domains (24Muratani M. Tansey W.P. Nat. Rev. Mol. Cell Biol. 2003; 4: 1-10Crossref Scopus (677) Google Scholar). To test this hypothesis, we first compared the stability of two truncated Rpn4 mutants containing residues 11–229 (Rpn411–229) and 11–210 (Rpn411–210), respectively, using pulse-chase analysis (Fig. 3C). Whereas Rpn411–229 was extremely short-lived, Rpn411–210 was barely degraded, suggesting that residues 211–229 serve as a ubiquitination signal in Rpn411–229. We then examined the role of residues 211–229 in the context of Rpn4Δ1–10. Sequence analysis showed that Rpn4 carries two domains rich in acidic amino acids (Fig. 3A). NAD consists of residues 211–229, whereas the C-terminal acidic domain (CAD) lies between residues 300–312. We, therefore, generated two mutants from Rpn4Δ1–10 via internal deletion of residues 211–229 and 291–313, respectively (Fig. 3A). Remarkably, pulse-chase analysis showed that deletion of NAD but not CAD inhibited the degradation of Rpn4Δ1–10 (Fig. 3D, compare lanes 1–3 with 4–6), indicating that NAD is required for the Ub-dependent degradation of Rpn4. The proximal position of NAD to Lys-187 may functionally distinguish itself from CAD. To examine whether NAD also plays a role in the Ub-independent degradation of Rpn4, we deleted NAD in the context of full-length Rpn4 and measured the turnover of Rpn4Δ211–229 by pulse-chase analysis. As shown in Fig. 3D (lanes 7–9), deletion of NAD displayed no effect on the Ub-independent degradation of Rpn4, indicating that NAD is specific for the Ub-dependent degradation of Rpn4. Lys-187 and NAD Constitute a Portable Ubr2-dependent Degron—A Ub-dependent degron is composed of a ubiquitination site and a ubiquitination signal, usually a short primary sequence or a structural feature recognized by a specific Ub ligase. The identification of Lys-187 as the preferential ubiquitination site and NAD as an essential sequence for the Ub-dependent degradation of Rpn4 prompted us to determine whether Lys-187 and NAD (including minimal flanking sequences) constitute a sufficient degron. To this end, we fused residues 172–229 of Rpn4 to the otherwise stable β-galactosidase reporter protein and measured the stability of the resulting fusion protein Rpn4172–9-β-galactosidase. As shown in Fig. 4 (lanes 1–4), Rpn4172–229-β-galactosidase Ubr2 was rapidly degraded, indicating that Lys-187 and NAD do form a sufficient and portable degron. Interestingly, substitution of Lys-187 with arginine inhibited the degradation of Rpn4172–229-β-galactosidase (Fig. 4, lanes 9–12), indicating that the implanted Lys-187 functions as the ubiquitination site in the fusion protein. To rule out the possibility that a cryptic degron was created in the Rpn4172–229-β-galactosidase fusion, we evaluated the stability of Rpn4172–229-β-galactosidase in ubr2Δ, where Ubr2, the cognate Ub ligase for Rpn4, was absent. Pulse-chase analysis revealed that Rpn4172–229-β-galactosidase was stabilized in the ubr2Δ mutant (Fig. 4, lanes 5–8). Thus, Lys-187 and NAD constitute a portable degron specifically targeted by Ubr2. We postulate that NAD acts as the ubiquitination signal. Lysine selection by the RING E3s is a central question in the field of protein ubiquitination. Although several models exemplified by the "effective concentration" and the "hit-and-run" models have been proposed to explain the process of lysine selection (25Deffenbaugh A.E. Scaglione K.M. Zhang L. Moore J.M. Buranda T. Sklar L.A. Skowyra D. Cell. 2003; 114: 611-622Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 26Orlicky S. Tang X. Willems A. Tyers M. Sicheri F. Cell. 2003; 112: 243-256Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar, 27Wu G. Xu G. Schulman B.A. Jeffrey P.D. Harper J.W. Pavletich N.P. Mol. Cell. 2003; 11: 1445-1456Abstract Full Text Full Text PDF PubMed Scopus (516) Google Scholar, 28Schulman B.A. Carrano A.C. Jeffrey P.D. Bowen Z. Kinnucan E.R.E. Finnin M.S. Elledge S.J. Harper J.W. Pagano M. Pavletich N. Nature. 2000; 408: 381-386Crossref PubMed Scopus (490) Google Scholar), these models can only interpret the data of ubiquitination of one substrate but not the other. It is well known that many substrates of the Ub-proteasome system can be ubiquitinated at multiple lysines. However, in every case that we are aware of, it has never been determined if one lysine is preferred when all other lysines are also available. Extensive analysis of several substrates including Gcn4, p53, and c-Jun show that no single Lys-to-Arg substitution affects protein degradation, suggesting that it is rather nonselective with respect to which lysine of these substrates is ubiquitinated (11Nakamura S. Roth J.A. Mukhopadhyay T. Mol. Cell. Biol. 2000; 20: 9391-9398Crossref PubMed Scopus (161) Google Scholar, 12Kornitzer D. Raboy B. Kulka R.G. Fink G.R. EMBO J. 1994; 13: 6021-6030Crossref PubMed Scopus (219) Google Scholar, 29Treier M. Staszewski L.M. Bohmann D. Cell. 1994; 78: 787-798Abstract Full Text PDF PubMed Scopus (846) Google Scholar). However, strictly speaking, this type of analysis only demonstrates whether one of the lysines is essential for substrate ubiquitination but is unable to define a potential preferential ubiquitination site. Using an approach of adding back individual lysines to a lysine-free Sic1 allele, Petroski and Deshaies (10Petroski M.D. Deshaies R.J. Mol. Cell. 2003; 11: 1435-1444Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar) recently demonstrated that each one of the six N-terminal lysines is ubiquitinated and is sufficient to mediate the degradation of Sic1. Although these authors showed that the Ub chains attached at different N-terminal lysines influence the rate of in vitro proteasomal proteolysis, the in vivo half-lives of the Sic1 mutants carrying different lysines are quite similar (10Petroski M.D. Deshaies R.J. Mol. Cell. 2003; 11: 1435-1444Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). It is unknown whether one of these N-terminal lysines of Sic1 behaves as a preferred ubiquitination site. Using a similar "adding-back" approach, we identified six lysines in the N-terminal domain of Rpn4, each of which was able to sustain the degradation of Rpn4. Remarkably, among these six lysines, Lys-187 was ubiquitinated more efficiently than the others, and the turnover rate of Rpn4 via Lys-187 was also markedly higher than that by other individual lysines. Consistently, Lys-187 and NAD constitute an efficient and portable Ubr2-dependent degron. More importantly, both in vivo and in vitro ubiquitination assays demonstrated that Lys-187 is selected for ubiquitination when all other lysines are also available. To the best of our knowledge, this is the first demonstration of a preferential ubiquitination site that is selected from a group of lysines susceptible for ubiquitination in a substrate. It is currently unclear how the Ub ligase Ubr2/Rad6 selects Lys-187 over other lysines. One possibility is that Lys-187 is proximally positioned to the active center of the Ubr2/Rad6 Ub ligase, as the primary sequence of Rpn4 suggests that Lys-187 is closer to the NAD ubiquitination signal than other lysines. Further investigation of the molecular details of Rpn4 ubiquitination, e.g. deciphering the atomic structure of the Ubr2·Rad6·Rpn4 complex, will shed new light on the general mechanism of lysine selection. It remains an open question whether the presence of multiple Ub chains on a protein substrate is essential for its degradation by the proteasome. Our current study demonstrates that the Ub-dependent degradation of Rpn4 can be mediated efficiently by six individual lysines. Consistent with our data, a recent report has shown that each one of the six N-terminal lysines is sufficient for the degradation of Sic1 (10Petroski M.D. Deshaies R.J. Mol. Cell. 2003; 11: 1435-1444Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Therefore, it appears that a single multi-Ub chain is sufficient to sustain efficient degradation of at least a subset of proteasomal substrates. What would be the physiological meaning of having multiple Ub chains on a substrate? Given the presence of multiple Ub chain receptors including the intrinsic proteasome subunits (Rpn10 and Rpt5) and proteasome-associated UBA domain proteins in the cell (30Madura K. Trends Biochem. Sci. 2004; 29: 637-640Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), it is likely that multiple Ub chains may slow the dissociation of a substrate from the proteasome, providing the proteasome with sufficient time to unfold and translocate the substrate into the proteolytic chamber. This high affinity conferred by multiple Ub chains, although not an absolute requirement for the degradation of some substrates such as Rpn4 and Sic1, may be essential for others, especially those that are difficult to unfold. We reason that the structural features of protein substrates determine whether a single Ub chain is enough or multiple Ub chains are required for their degradation. For instance, a single multi-Ub chain may be sufficient for the degradation of substrates with loosely structured segments such as unfolding initiation sites. We thank Michael Ellison for plasmids.
To clarify the role of the Shc-Grb2-Sos trimer in the oncogenic signaling of the point mutation-activated HER-2/neu receptor tyrosine kinase (named p185), we interfered with the protein-protein interactions in the Shc.Grb2.Sos complex by introducing Grb2 mutants with deletions in either amino- (delta N-Grb2) or carboxyl-(delta C-Grb2) terminal SH3 domains into B104-1-1 cells derived from NIH3T3 cells expressing the point mutation-activated HER-2/neu. We found that the transformed phenotypes of the B104-1-1 cells were largely reversed by the delta N-Grb2. The effect of the delta C-Grb2 was much weaker. Biochemical analysis showed that the delta N-Grb2 was able to associate Shc but not p185 or Sos, while the delta C-Grb2 bound to Shc, p185, and Sos. The p185-mediated Ras activation was severely inhibited by the delta N-Grb2 but not the delta C-Grb2. Taken together, these data demonstrate that interruption of the interaction between Shc and the endogenous Grb2 by the delta N-Grb2 impairs the oncogenic signaling of the activated p185, indicating that (i) the delta N-Grb2 functions as a strong dominant-negative mutant, and (ii) Shc/Grb2/Sos pathway plays a major role in mediating the oncogenic signal of the activated p185. Unlike the delta N-Grb2, delta C-Grb2 appears to be a relatively weak dominant-negative mutant, probably due to its ability to largely fulfill the biological functions of the wild-type Grb2.
'/%3e%3cuse xlink:href='%23a' transform='rotate(23.036 .093 25.536)'/%3e%3cuse xlink:href='%23a' transform='rotate(45.87 1.273 16.18)'/%3e%3cuse xlink:href='%23a' transform='rotate(69.945 .996 12.078)'/%3e%3cuse xlink:href='%23a' transform='rotate(20.66 -19.689 31.932)'/%3e%3c/svg%3e)