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Previous Article | Next Article 
Molecular and Cellular Biology, July 2006, p. 4958-4969, Vol. 26, No. 13
0270-7306/06/$08.00+0 doi:10.1128/MCB.00160-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Department of Molecular Pharmacology,1 Department of Structural Biology and Genetics/Tumor Cell Biology, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, Tennessee 38105,2 Howard Hughes Medical Institute3
Received 27 January 2006/ Accepted 29 March 2006
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ABSTRACT |
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INTRODUCTION |
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-amino group of the substrate lysine residue. Isopeptidases catalyze de-SUMOylation, and thus a balance of Ubc9 conjugation and isopeptidase activities modulate steady-state levels of SUMO conjugates.
In budding yeast and mammalian cells, Ubc9 is the sole SUMO-conjugating enzyme and is essential for cell viability (13, 25, 38). In contrast to Ub conjugation, where substrate specificity is dictated by E3 ligases, SUMO target recognition is determined, in part, by the E2 Ubc9. Structural and biochemical data indicate selective binding of a consensus 
KX(D/E) motif by Ubc9, where is a hydrophobic residue, X is any amino acid, and K is the lysine modified with SUMO (2, 17, 36). However, the relative simplicity of this pathway belies the complexity of protein targets modified by SUMO. The limited substrate specificity imparted by Ubc9 interactions with KXE residues is insufficient to account for the increasing number of proteins modified by SUMO and the modification of proteins at nonconsensus SUMO sites (5, 11, 24, 28, 37, 43, 44, 48). Ubc9-target interactions may also be facilitated by E3 ligases, such as the Siz/PIAS family, RanBP2, and Polycomb Pc2 protein (12, 16, 24, 31). Siz/PIAS E3 ligases appear to act as substrate adaptors. In contrast, recent structural studies suggest that the IR1-M E3 ligase domain of RanBP2 binds SUMO and Ubc9 to optimize the orientation of the SUMO-Ubc9 thioester, thereby enhancing conjugation (35, 40). Thus, E3 ligases may function as chaperones to facilitate SUMO conjugation in the absence of direct substrate interactions. However, whether Ubc9 contains additional determinants of substrate specificity and what role E3 ligases play in the recognition of select targets remain unclear.
Covalent modification with SUMO can alter target protein subcellular localization, activity, stability, or protein-protein interactions, thereby affecting various processes, including gene transcription, apoptosis, cell cycle progression, chromatin organization, and DNA repair (6, 10, 12, 16). Proteomic approaches have defined a large number of SUMO substrates in yeast and human cells (5, 11, 24, 28, 37, 43, 44, 48). Yet, it appears that only a small percentage of a given protein is covalently modified by SUMO at any time. Thus, the functional analysis of SUMO-modified substrates is complicated, as persistent alterations in protein function may be induced by relatively transient cycles of SUMO modification.
Eukaryotic cell sensitivity to DNA-damaging agents is also affected by SUMO conjugation. Postreplicative DNA repair is regulated, in part, by the addition of mono-Ub or poly-Ub to K164 in proliferating cell nuclear antigen (PCNA) in response to DNA damage (30, 42). Surprisingly, Ubc9 conjugates SUMO to the same residue in PCNA, thereby implicating distinct modifications in response to different replication stresses. Although UBC9 is essential for cell viability, conditional yeast ubc9 mutants exhibit increased sensitivity to DNA-damaging agents (15, 22). We reported the isolation of the ubc9P123L mutant in a yeast genetic screen for conditional mutants exhibiting enhanced sensitivity to DNA lesions induced by drugs that target DNA topoisomerase I (Top1) (15). At the nonpermissive temperature, 36°C, global SUMO conjugates were severely suppressed in cells expressing Ubc9P123L, yet cell viability was retained. Relative to wild-type UBC9 strains, ubc9P123L cells exhibited enhanced sensitivity to a wide range of DNA-damaging agents (including drugs that target Top1, hydroxyurea [HU], the alkylating agent methyl methanesulfonate [MMS], and UV light) but not to other environmental stress. Although Top1 is modified by SUMO, the enhanced sensitivity of ubc9P123L cells to DNA damage was not dependent on Top1 SUMO conjugation or expression. Rather, a lower threshold of select SUMO-target conjugates was required to maintain cell viability in the absence of genotoxic stress. This premise was further supported by the observation that the viability, but not the HU resistance, of cells deleted for the Ulp2 SUMO-isopeptidase was restored by the reduced activity of Ubc9P123L at 36°C (15).
In order to define functional domains in Ubc9 that dictate cellular responses to DNA-damaging agents versus those necessary for cell viability, we took advantage of the conservation between human Ubc9 and yeast Ubc9 (hUbc9 and yUbc9, respectively) to assess the consequences of subtle structural alterations in SUMO E2 enzyme activity. We solved the structure of yUbc9 and, based on comparisons with crystal structures of hUbc9 (41), constructed a series of chimeric enzymes to define the functions of specific amino acid residues in regulating enzyme activity in vivo. Here we report that differences in the geometries of divergent side chain residues within protein domains implicated in (i) binding of substrate residues flanking the canonical SUMO site, (ii) interaction with the RanBP2 E3 ligase, and (iii) binding of the heterodimeric E1 and Smt3 have distinct effects on cell growth and resistance to DNA-damaging agents.
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MATERIALS AND METHODS |
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Isogenic S. cerevisiae strains FY250 (MAT
ura3-52 his3
200 leu21 trp163), EKY3 (FY250 top1::TRP1), RRY82 (EKY3 ubc9-10), HJY13 (FY250 ubc9-10), and PTY30 (EKY3 ubc9:: his+, YCpUBC9) were described previously (15). Yeast cells were transformed and cultured using standard methods.
Yeast plasmids are listed in Table 1. The Pro123-to-Leu mutation in hUbc9P123L was generated in YCpSchUBC9 by using the QuikChange site-directed mutagenesis kit (Stratagene). Yeast-human UBC9 chimeras were generated by homologous recombination (29) of PCR-generated chimeric junctions (Table 2).
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Antibodies and immunoblots.
Ubc9 antibodies (A645 from Boston Biochem) recognize yUbc9 and hUbc9. Rat -tubulin antibodies were from Accurate Chemical and Scientific Corp (Westbury, NY), anti-Myc antibodies (clone 9E10) were from Roche Diagnostics Corp. (Indianapolis, IN), anti-Flag M2 antibodies were from Sigma, and anti-Smt3 antibodies were provided by E. S. Johnson (Thomas Jefferson University) or raised against purified Smt3 in rabbits. Anti-yUbc9-specific antibodies were described previously (15). Anti-hUbc9 specific antibodies were raised against residues 59 to 71 in rabbits and affinity purified on peptide-coupled Sepharose 4B using standard techniques as described previously (15).
To assess the levels of yUbc9, hUbc9, and Smt3 conjugates, galactose-induced cultures were grown for 6 h at 26°C or 36°C and NaOH-trichloroacetic acid extracts were prepared as described previously (15). Proteins were resolved in 12% or 4 to 12% Bis-Tris polyacrylamide gels with MOPS (morpholinepropanesulfonic acid) running buffer (NuPAGE; Invitrogen) and blotted onto activated polyvinylidene difluoride membranes (Perkin-Elmer, LifeScience). Ubc9 proteins or Smt3 conjugates were visualized by immunostaining and chemiluminescence. Immunostaining with -tubulin served as a loading control.
In vitro SUMO conjugation assays. The heterodimeric yE1 (yAos1/Uba2), yUbc9, and a truncated version of Smt3, which terminates at G98 to represent the mature form, were expressed in BL21(DE3) cells (Novagen) and purified as described previously (1). A cDNA clone expressing full-length hUbc9 was obtained by PCR amplification of human cDNA (Invitrogen) and cloned into pGEX4T3. Following expression in BL21(DE3) cells, hUbc9 was purified by glutathione affinity chromatography as described previously (1). The N-terminal glutathione S-transferase tag was removed by overnight treatment with thrombin at 4°C, and Ubc9 proteins were purified by ion-exchange chromatography using a Resource Q column pressured by an AKTA fast protein liquid chromatography system (GE Healthcare) and by size exclusion chromatography using an SD200 column (GE Healthcare), concentrated by ultrafiltration, frozen in liquid nitrogen, and stored at –80°C.
Formation of yUbc9-Smt3, hUbc9-Smt3, and Smt3-Smt3 conjugates was assessed following incubation of 10 µg of hUbc9 or yUbc9, 40 µg of Smt3, and either 0.1 µg (1x), 1 µg (10x), or 10 µg (100x) of Aos1/Uba2 in 30 to 50 mM Tris (pH 7.5), 100 mM NaCl, 10 mM MgCl2, and 4 mM ATP in a volume of 35 to 50 µl. Reaction mixtures were incubated at room temperature for 0, 15, or 60 min and terminated with sodium dodecyl sulfate sample buffer lacking dithiothreitol (DTT). The samples were equally divided, with half treated with 100 mM DTT prior to boiling. Reaction products were resolved in 4 to 12% Bis-Tris polyacrylamide gels (Novagen) in MES (morpholineethanesulfonic acid) buffer and visualized by Coomassie or SYPRO-Ruby staining. The identities of specific bands, previously defined by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (1), were confirmed by immunostaining.
Crystallization, data collection, and structure determination. Initial crystals of yUbc9 were obtained in 21% polyethylene glycol 4000 (PEG 4000), 0.1 M MES (pH 6.0), 0.1 M NaBr, and 5 mM DTT by using the hanging-drop vapor diffusion method at 18°C. High-quality crystals for X-ray diffraction were grown by microseeding into 18% PEG 6000, 0.1 M HEPES (pH 7.5), 0.1 M NaBr, and 5 mM DTT. The crystals belong to the P21 space group with unit cell dimensions a = 59.3 Å, b = 60.1 Å, c = 95.3 Å, and ß = 105.0o and four monomers per asymmetric unit. Crystals were flash frozen in 21% PEG 6000, 50 mM HEPES (pH 7.5), 0.1 M NaBr, 5 mM DTT, and 30% glycerol prior to data collection at the National Synchrotron Light Source beamline X25. Data were collected from a single crystal to 1.75-Å resolution with 1,502,049 total reflections and 65,526 unique reflections. The data were indexed with HKL2000 and scaled with SCALEPACK (27) to a final Rmerge of 4.8% and overall completeness of 99.5%. Phases were obtained by molecular replacement using polyalanine hUbc9 (PDB accession no. 1U9A [41]) as the search model in AMORE (26). The model was refined using CNS version 1.1 (3), with real-space model refinement and visualization performed in O version 9.0 (19). The refinement proceeded until convergence with an R factor of 22.1% and an Rfree of 24.5%. The average B factor for 5,022 protein atoms was 27.9 Å2, and that for 471 solvent atoms located in the asymmetric unit was 35.3Å2. The fraction of residues in the core and additionally allowed regions of the Ramachandran plot was 99.2%, with no residues in the disallowed region, as determined by PROCHECK (20). A summary of diffraction data and refinement statistics are provided in Tables 3 and 4. All figures were made with PyMol (DeLano Scientific, San Carlos CA).
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RESULTS |
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Binding of substrates or E3 ligases to Ubc9 contributes to efficient SUMOylation; nevertheless, the role that the SUMO E2 itself plays in discriminating between substrates remains unclear. To address the role of Ubc9 residues in dictating cell survival and response to genotoxic stress, we exploited the similarities between yUbc9 and hUbc9 to ask how subtle alterations in structure would affect cell growth and survival. hUbc9 and yUbc9 are highly conserved proteins, exhibiting 56% identity (75% similarity) in amino acid sequence (Fig. 1A) (46). Using a plasmid shuffle in a ubc9 strain, hUbc9 complemented the essential function of yUbc9 when overexpressed from the GAL1 promoter, albeit at a reduced rate of cell growth (Fig. 1B). However, the introduction of a C-terminal Myc epitope suppressed this slow-growth phenotype. Papillae in vector control and hUBC9 patches result from recombination between URA3 and LEU2-based vectors. Similar results were obtained when hUBC9 was overexpressed from a strong constitutive promoter (GPD), obviating concerns about medium effects. In contrast, low constitutive expression of hUBC9 failed to maintain ubc9 viability (data not shown).
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We then asked if hUbc9 could complement the increased sensitivity of ubc9-10 mutant cells to DNA-damaging agents. The slow-growth phenotype and enhanced HU or CPT sensitivity of ubc9-10 cells at 36°C were suppressed by constitutive expression of wild-type yUbc9 and partially suppressed by leaky expression of the GAL1-yUBC9 construct on dextrose (Fig. 2). In contrast, GAL1-promoted hUbc9 expression had little effect on ubc9-10 cell sensitivity to HU or CPT, with only a slight increase in colony formation relative to the vector control. Thus, hUbc9 provides sufficient function to maintain yeast ubc9 cell viability yet apparently lacks determinants necessary to restore cellular resistance to genotoxic stress. Nevertheless, the phenotypic consequences of the P123L mutation were conserved: while overexpression of hUbc9P123L maintained ubc9 cell viability (data not shown), these cells were hypersensitive to HU and CPT (compare hUBC9 with hubc9P123L in Fig. 2).
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To determine if the phenotypic consequences of the P123L mutation or addition of a C-terminal epitope resulted from alterations in Ubc9 thioester transfer, the formation of Smt3 isopeptide linkages with Smt3 and Ubc9 was assessed in vitro. The extents of substrate SUMOylation catalyzed by yUbc9P123L, hUbc9P123L, and hUbc9-Myc were comparable to those obtained with relevant wild-type controls (Fig. 3E). Under these conditions (36°C, the nonpermissive temperature for ubc9-10 cells), both Smt3 and Ubc9 were efficiently conjugated with Smt3. Moreover, as in Fig. 1C, reactions containing hUbc9 proteins were assayed with a 100-fold excess of yE1. Thus, mutation of Pro123 or a C-terminal tag had no detectable effect on Ubc9 catalysis in vitro. Rather, these findings prompted us to consider that alterations in substrate specificity, dictated by either differences in Ubc9 determinants or E3 ligase interactions, induced distinct cellular responses to genotoxic stress.
Structure determination of yeast Ubc9 and comparison with human Ubc9.
The structure of yUbc9 (Fig. 4A) was determined from data collected at the National Synchrotron Light Source beamline X25 from a single crystal, with four monomers per asymmetric unit (Tables 3 and 4). Phases were obtained by molecular replacement using polyalanine hUbc9 (PDB accession no. 1U9A [41]) as a search model. The model was refined to 1.75 Å, with an R factor of 22.1 and Rfree of 24.5. A least-squares fit of all four copies of yUbc9 in the asymmetric unit revealed a root mean square (RMS) deviation over all C atoms of 0.35 Å. As seen in Fig. 4B, further comparison of yUbc9 with hUbc9 (PDB accession no. 1A3S [41]) showed a considerable conservation of backbone structures, with an RMS deviation of 0.7 Å over all C atoms between the yeast and human forms.
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KXE motif of RanGAP1 (2, 35), including Ubc9 residues K74, Y87, S89, and T91. Moreover, the flat surface immediately preceding residue 131, which interacts with the hydrophobic of the consensus motif, is superimposable in both structures (Fig. 4B [expanded in Fig. 4C]).
In hUbc9, residues 131 to 143 comprise helix C (Fig. 4B), which interacts with additional residues in RanGAP1 that lie outside the consensus KXE site and have been implicated in select substrate binding (2, 35). Residues spanning the loop that precedes helix C include the P123 mutated in ubc9-10 cells; however, as shown in Fig. 4C, 11 of the 13 residues comprising helix C are not conserved from yeast to human. Indeed, differences in residue side chains at positions 131 (Ala to Glu in yeast), 135 (Thr to Arg in yeast), and 139 (Gln to Arg in yeast) result in considerable alterations in surface charge and structure (Fig. 4C), which would clash with RanGAP1 residues in the SUMO-RanGAP1-Ubc9-RanBP2 structure (35) and disrupt critical hydrophobic interactions between A131 of hUbc9 and F564 of RanGAP1. Based on these considerations, we asked if such alterations in side chain geometry and charge would dictate differences in SUMO E2 activity in vivo.
C-terminal human/yeast Ubc9 chimeras.
To address the functions of Ubc9 domains, homologous recombination (as described previously [29]) was used to generate reciprocal swaps of the C-terminal domains of yUbc9 and hUbc9, beginning at residue P123 or E143 (Fig. 5). In the designations, the first letter refers to the N-terminal sequence (h or y) and the number indicates the first residue of the C-terminal h or y Ubc9 component. The chimeras were expressed in ubc9 cells, using the plasmid shuffle shown in Fig. 1, to assess chimera E2 function in vivo. N-terminal proximal binding sites for yE1 and Smt3 were previously defined within yUbc9 (1). Thus, we reasoned that efficient E1-E2 interactions dictated by the yeast N terminus would obviate more subtle effects of substrate binding domains imparted by the human E2. Indeed, as seen with y123hUbc9 or y143hUbc9, expression of either chimera sufficed to maintain cell viability as well as wild-type yUbc9 (Fig. 5). Overexpression of hUbc9 also maintained cell viability, albeit with a slow-growth phenotype. The cell growth defect was suppressed by the introduction of the C-terminal yUbc9 domain, beginning at residue P123 in h123yUbc9. However, this effect was not due to alterations of C-terminal sequences, as induced by the Myc tag (Fig. 1B), since defective cell growth was still seen with h143yUbc9. Rather, the introduction of yeast residues 123 to 143, which span the P123N124 loop and helix C depicted in Fig. 4, enhanced hUbc9 function in maintaining yeast cell viability. Thus, in addition to N-terminal residues, two other determinants affect SUMO E2 function: alterations in the C terminus and residues spanning 123 to 143.
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cells, also affected cell resistance to genotoxic stress. As seen in Fig. 6, the introduction of yeast residues 123 to 143 in h123y143hUbc9 dramatically increased ubc9-10 cell resistance to HU at 36°C, relative to that observed with cells expressing wild-type hUbc9. However, none of these constructs restored the levels of global Smt3 conjugates detected in cells expressing yUbc9 (Fig. 6B). In contrast, the introduction of human C-terminal residues into yUbc9 abrogated the HU resistance induced by leaky expression on dextrose plates (compare yUBC9 with the C-terminal chimeras, y123hUBC9 and y143hUBC9). This effect was not due to differences in protein expression, as antibodies recognizing yeast residues 134 to 145 (anti-yUbc9) revealed comparable levels of wild-type yUbc9 and y143hUbc9 (Fig. 6B). Moreover, similar levels of global Smt3 conjugates were detected in ubc9-10 cells expressing wild-type yUbc9 or either of the y/h C-terminal chimeras. Thus, modified target selection, rather than the extent of global SUMOylation, appears to determine cellular resistance to DNA damage.
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SIZ1 encodes a PIAS family E3 ligase that directly interacts with Ubc9 and its targets to facilitate substrate SUMOylation (18). Other PIAS E3 ligases in yeast include Siz2 and Mms21 (18, 47). SIZ1 and SIZ2 are nonessential genes implicated in SUMO conjugation of septins and DNA topoisomerase II, while MMS21 is an essential gene that regulates cell sensitivity to a wide range of DNA-damaging agents (18, 39, 47). In our hands, increased expression of MMS21 or SIZ2 genes failed to restore ubc9-10 cell resistance to HU or Top1 poisons at 36°C (data not shown). In contrast, increased gene dosage of SIZ1 on a multicopy vector restored ubc9-10 cell resistance to the DNA damage induced by Top1T722A (Fig. 7A). Yet, increased levels of SIZ1 failed to stabilize the Ubc9P123L protein or to restore global SUMO conjugates at 36°C (Fig. 7B). Moreover, the activity of Siz1 appeared to be specific for Top1-induced toxicity, as YEpSIZ1 failed to restore ubc9-10 cell resistance to HU and decreased the low level of HU resistance conferred by overexpression of hUbc9 (Fig. 7C). In these experiments, hUbc9 was constitutively overexpressed, rather than expressed from the galactose-inducible promoter as in Fig. 2 and 3.
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A third prediction is that increasing substrate interactions with hUbc9 chimeras containing yeast N-terminal sequences, via elevated levels of an E3 ligase, would enhance the growth defects induced by E1/Smt3 sequestration by y22hUbc9 or y38hUbc9. As seen in Fig. 9, increased dosage of the Siz1 E3 ligase, sufficient to increase ubc9-10 cell resistance to Top1 poisons (Fig. 7A), did enhance the cytotoxicity of y22hUbc9 and y38hUbc9. In contrast, YEpSIZ1 failed to alter the pattern of ubc9-10 cell sensitivity to HU induced by expression of any of the N-terminal chimeras. Similar results were obtained with increased SIZ2 gene dosage (data not shown). As these SUMO E3 ligases directly interact with select Ubc9 substrates, these findings support the premise that E1/Smt3 interactions with Ubc9, in concert with alterations in substrate binding, have distinct effects on cell viability and cellular responses to different genotoxic stresses.
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DISCUSSION |
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KX(D/E) motif in RanGAP1 (2). Moreover, the E3 ligase domain of RanBP2 interacts with hUbc9 and SUMO to facilitate thioester bond formation, rather then directly interacting with RanGAP1 (35, 40). Although substrate or E3 ligase binding by Ubc9 contributes to efficient SUMOylation, the role that the SUMO E2 plays in discriminating between substrates has remained unclear. We previously reported that substitution of Leu for Pro123 in yeast Ubc9P123L induced a synthetic lethal phenotype, whereby a global reduction in SUMO conjugates at 36°C sufficed to sustain cell viability yet enhanced cell sensitivity to a wide range of DNA-damaging agents. Thus, alterations in select target SUMOylation, induced by a single amino acid substitution in Ubc9, had an adverse impact on cell survival, but only in the context of genotoxic stress. Here we defined the functional domains in Ubc9 that dictate cellular responses to DNA-damaging agents versus those necessary to maintain cell viability in the absence of genotoxic stress, by assessing the biological consequences of subtle structural alterations in SUMO E2 activity.
A comparison of yeast and human Ubc9 activity in isogenic yeast strains and in in vitro SUMOylation assays illustrated distinct functional interactions of these highly conserved SUMO E2 enzymes. For instance, a 100-fold excess of yE1 was required to achieve Ubc9-Smt3 and Smt3-Smt3 conjugates with hUbc9 that were comparable to those with yUbc9, and hUbc9 was not as efficient a substrate for SUMOylation as yUbc9 (Fig. 1C). Recent biochemical studies demonstrated that Smt3 was efficiently activated and conjugated to human E1 in vitro (21). However, as the transfer of Smt3 to hUbc9 or yUbc9 was defective, those authors considered that surfaces within the E1-SUMO thioester adduct provide some determinants for E2-thioester adduct formation. Our findings suggest that Ubc9 also provides determinants of E2 thioester transfer and that the differences between yeast and human Ubc9 proteins could be exploited to define differences in biological function.
Consistent with this premise, the slow-growth phenotype of ubc9 cells expressing hUbc9 was suppressed by the addition of C-terminal Myc or Flag tags, possibly as a consequence of increased protein stability or more efficient interactions with other components of the yeast SUMO pathway. Moreover, we found that N- or C-terminal modifications of hUbc9, sufficient to allow protein detection in immunoblots, also affected hUbc9 activity in yeast. In the structure of the SUMO-RanGAP1-Ubc9-RanBP2 complex, RanBP2 E3 ligase interactions span the C and N termini of hUbc9 (35, 40). Thus, it is tempting to speculate that similar interactions with an, as-yet-unidentified, yeast SUMO E3 could bridge N and C termini of Ubc9 to affect E2 function.
Structure/function of Ubc9.
We also solved the structure of yUbc9, which in comparison with known structures of hUbc9 showed a considerable conservation of backbone architecture. The P123 residue, mutated in yUbc9P123L and hUbc9P123L, occupies the same position in both enzymes. Hydrogen-bonding interactions between the E2 side chain of N85 with backbone atoms of E2 residue N124 and the C-terminal SUMO-1 G97 residue within the E2 active site were revealed in the cocrystal structure of hUbc9 in complex with SUMO-1 conjugated to RanGAP1 (35). As these residues are conserved in yUbc9 and Smt3, such interactions are consistent with biochemical data indicating a role for N85 in stabilizing the transition state during E2 catalysis (45). We previously showed that substitution of Ala for Pro123 had no effect on yUbc9 function in vivo, while the introduction of the bulky hydrophobic side chain of Leu at this position reduced global SUMO conjugation and enhanced cell sensitivity to DNA damage at 36°C (15). Such a structural distortion in the P123N124 loop might compromise E2 catalysis by disrupting the coordination of N85 within the active site. We think this unlikely, however, as both yUbc9P123L and hUbc9P123L proteins were able to maintain ubc9 cell viability and were active in vitro (Fig. 2 and 3 and data not shown). Alternatively, binding of the extended Smt3 C terminus might suppress structural distortions induced by a temperature shift to 36°C.
Another possibility is that alterations in the geometry of the P123N124 loop might affect substrate binding by more distal C-terminal Ubc9 residues, such as residues 131 to 143. These largely nonconserved residues comprise helix C (Fig. 4B), which interacts with residues in RanGAP1 that flank the consensus KXE site in cocrystal structures with hUbc9 (2, 35). Bernier-Villamor et al., (2) reported that mutation of several of these hUbc9 residues (A131 and Y134) abolished efficient SUMOylation of RanGAP1 in vitro yet had limited effects on SUMO modification of two other substrates, p53 and I
B. Thus, interactions of residues in RanGAP1 with helix C residues in hUbc9, required for efficient substrate modification, appeared to be substrate specific and, as such, were posited to provide binding surfaces for Ubc9 that could substitute for an E3 ligase. These considerations prompted the construction of yeast/human chimeras to determine whether such differences in side chain geometry would distinguish substrate SUMOylation necessary for cellular resistance to distinct genotoxic stresses and/or cell viability.
Indeed, differences in the geometries and charges of divergent side chains defined three determinants of Ubc9 activity in suppressing cell sensitivity to DNA-damaging agents, as follows.
(i) Ubc9 residues 123 to 143, implicated in binding of nonconserved residues within substrates that flank the SUMO consensus site. As mutation of human residues I125 and Q126 to match the P125 and N126 residues found in yUbc9 did not affect enzyme activity in vivo (data not shown), these findings support a model whereby residues 131 to 143 in helix C dictate select substrate interactions, while residues 123 to 130 contribute to the recognition of consensus SUMO site residues in substrate proteins.
(ii) Ubc9 residues 22 to 38, which interact with human RanBP2 E3 ligase. These data support a model whereby Ubc9 interactions with an E3 ligase, other than Siz1 or Siz2, facilitate substrate SUMOylation necessary to confer resistance to MMS and HU. This contrasts with the ability of Siz1 to act as a dosage suppressor of Top1-induced DNA damage. This model does not exclude additional interactions between Ubc9 residues 22 to 38 and SUMO E1, as suggested by the structure of Ubc12 and the E2 binding domain of NEDD8 E1 (14), although E1 and E3 binding to this Ubc9 domain may be mutually exclusive, as reported for other Ubl E2-E3 protein pairs (7).
(iii) N-terminal Ubc9 residues implicated in the binding of E1 and SUMO. Moreover, these studies demonstrate a functional interaction between N-terminal and substrate-binding domains of Ubc9 and support a model whereby chimeras that contain the N-terminal domain of yUbc9 and the substrate-binding determinants of hUbc9 act as dominant negative mutants by sequestering yE1/Smt3 to reduce global SUMOylation. This effect was exacerbated by increased dosage of Siz1 or Siz2 E3 ligase. However, this defect in Ubc9 activity was completely suppressed by introducing yUbc9 substrate-binding residues (123 to 143) into the same N-terminal chimeras. Thus, the introduction of yUbc9 residues implicated in E1/Smt3 binding (residues 1 to 22) and substrate binding (residues 123 to 143) redirects the catalytic activity of hUbc9 to maintain cell viability and restore cellular resistance to genotoxic stress.
In contrast, increased dosage of Siz1 E3 ligase (but not Siz2) partially suppressed ubc9-10 cell sensitivity to Top1 poisons, with no effect on cell sensitivity to HU or MMS. Moreover, the extent of global SUMOylation supported by distinct E2 enzymes did not predict cellular resistance to genotoxic stress. Rather, our findings indicate that select substrate SUMOylation, dictated by specific E2/E3 interactions, distinguishes cellular resistance to distinct genotoxic stresses and provide a compelling rationale for developing novel chemotherapeutics that target specific components of the SUMO conjugation pathway.
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ACKNOWLEDGMENTS |
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This work was supported in part by NIH grants CA23099 and CA111542 (to M.-A.B.); the Pew Scholars Program, Beckman Young Investigator Award, Howard Hughes Medical Institute, and NIH grant GM069530 (to B.A.S.); an American Cancer Society fellowship (to D.M.D.); NCI Cancer Center core grant CA21765; and the American Lebanese Syrian Associate Charities.
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FOOTNOTES |
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