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A comprehensive framework of E2-RING E3 interactions of the human ubiquitin-proteasome system.

van Wijk SJ, de Vries SJ, Kemmeren P, Huang A, Boelens R, Bonvin AM, Timmers HT - Mol. Syst. Biol. (2009)

Bottom Line: Both within the E2 and the E3 cohorts, several members were identified that are more versatile in their interaction behaviour than others.For validation we confirmed the interaction of several versatile E2s with E3s in in vitro protein interaction assays and we used mutagenesis to alter the E3 interactions of the E2 specific for K63 linkages, UBE2N(Ubc13), towards the K48-specific UBE2D2(UbcH5B).Our data provide a detailed, genome-wide overview of binary E2-E3 interactions of the human ubiquitination system.

View Article: PubMed Central - PubMed

Affiliation: Division of Biomedical Genetics, Department of Physiological Chemistry, University Medical Center Utrecht, Utrecht, The Netherlands.

ABSTRACT
Covalent attachment of ubiquitin to substrates is crucial to protein degradation, transcription regulation and cell signalling. Highly specific interactions between ubiquitin-conjugating enzymes (E2) and ubiquitin protein E3 ligases fulfil essential roles in this process. We performed a global yeast-two hybrid screen to study the specificity of interactions between catalytic domains of the 35 human E2s with 250 RING-type E3s. Our analysis showed over 300 high-quality interactions, uncovering a large fraction of new E2-E3 pairs. Both within the E2 and the E3 cohorts, several members were identified that are more versatile in their interaction behaviour than others. We also found that the physical interactions of our screen compare well with reported functional E2-E3 pairs in in vitro ubiquitination experiments. For validation we confirmed the interaction of several versatile E2s with E3s in in vitro protein interaction assays and we used mutagenesis to alter the E3 interactions of the E2 specific for K63 linkages, UBE2N(Ubc13), towards the K48-specific UBE2D2(UbcH5B). Our data provide a detailed, genome-wide overview of binary E2-E3 interactions of the human ubiquitination system.

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UBE2U physically interacts with MDM2. (A) HEK293T cells were transiently transfected with myc-tagged MDM2, lysed and incubated with GST–E2s immobilized on gluthathion-agarose beads. Bound material was resolved on 7.5% SDS–PAGE and proteins were visualized after immunoblotting with antibodies against myc. (B) Untransfected U2OS cells were lysed and combined with GST–E2s as in panel A. Immunoblotting was carried out using MDM2 antibodies (upper panel) and using p53 antibodies after reprobing (lower panel). Arrow indicates MDM2 signal, asterisk indicates background band.
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f6: UBE2U physically interacts with MDM2. (A) HEK293T cells were transiently transfected with myc-tagged MDM2, lysed and incubated with GST–E2s immobilized on gluthathion-agarose beads. Bound material was resolved on 7.5% SDS–PAGE and proteins were visualized after immunoblotting with antibodies against myc. (B) Untransfected U2OS cells were lysed and combined with GST–E2s as in panel A. Immunoblotting was carried out using MDM2 antibodies (upper panel) and using p53 antibodies after reprobing (lower panel). Arrow indicates MDM2 signal, asterisk indicates background band.

Mentions: Interestingly, we observed a high number of E3 interactions with an uncharacterized E2 annotated as UBE2U. The UBE2U protein (321 amino acids) is composed of a typical UBC-domain, with a C-terminal tail attached. No biological function has been described for this E2 yet, but microarray experiments showed that UBE2U mRNA transcripts have been identified mainly in tissues belonging to the urogenital tract. The UBE2U gene is only present in mammalian genomes. We looked in detail, which E3 enzymes were able to interact with this E2. One of the interacting E3s was MDM2. MDM2 has been identified as a major regulator of the cellular level and activity of the transcriptional regulator p53 (Momand et al, 1992). The significance of MDM2 in controlling normal cellular behaviour has become clear by the observation that in more than 50% of human tumours alterations in the p53 has been observed and in at least 7% of these cases, the MDM2 protein is affected (Momand et al, 1998). MDM2 has been identified as an E3 ligase, capable of ubiquitinating both itself and p53, targeting them for proteasomal degradation (Nakamura et al, 2000). For this action, MDM2 has a C-terminal Cys3–His2–Cys3 RING-finger, which was shown earlier to be able to interact with UBE2D2(UbcH5B) (Saville et al, 2004). To investigate whether other E2s identified in the two-hybrid screen can bind to MDM2, we focussed on the MDM2–UBE2U interaction that seemed quite strong from the Y2H. To investigate the MDM2–UBE2U interaction in more detail, we performed GST-pull-down assays with immobilized WT and catalytic mutant (C85A or C89A) GST–E2 UBC domains. As a negative control, GST–UBE2E1(UbcH6) was included. As shown in Figure 6A, MDM2 is captured by UBE2U and as expected also by UBE2D2(UbcH5B). Interestingly, UBE2U binds more efficiently to MDM2 than UBE2D2(UbcH5B). No binding could be observed with GST alone or GST–UBE2E1(UbcH6), reflecting the specific nature of MDM2–E2 interactions. As expected, mutation of the catalytic cysteine did not affect the interactions. Next, the GST–E2 enzymes were applied to lysates of human osteosarcoma cells (U2OS), which contain higher levels of MDM2 because of amplification or over-expression (Florenes et al, 1994). Figure 6B shows an efficient pull-down of endogenous MDM2 with both GST–UBE2U and GST–UBE2U C89A enzymes. Binding could also be observed when GST–UBE2D2(UbcH5B) WT and C85A where used, but no binding could be seen with GST alone or GST–UBE2E1(UbcH6). The GST–E2 bound MDM2 fractions were also tested by antibodies against p53. Figure 6B shows that p53 could be detected when MDM2 was captured on GST–E2 enzymes. These results indicate that UBE2U can bind to MDM2, confirming the Y2H results, and that the p53 substrate is part of the captured complex.


A comprehensive framework of E2-RING E3 interactions of the human ubiquitin-proteasome system.

van Wijk SJ, de Vries SJ, Kemmeren P, Huang A, Boelens R, Bonvin AM, Timmers HT - Mol. Syst. Biol. (2009)

UBE2U physically interacts with MDM2. (A) HEK293T cells were transiently transfected with myc-tagged MDM2, lysed and incubated with GST–E2s immobilized on gluthathion-agarose beads. Bound material was resolved on 7.5% SDS–PAGE and proteins were visualized after immunoblotting with antibodies against myc. (B) Untransfected U2OS cells were lysed and combined with GST–E2s as in panel A. Immunoblotting was carried out using MDM2 antibodies (upper panel) and using p53 antibodies after reprobing (lower panel). Arrow indicates MDM2 signal, asterisk indicates background band.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2736652&req=5

f6: UBE2U physically interacts with MDM2. (A) HEK293T cells were transiently transfected with myc-tagged MDM2, lysed and incubated with GST–E2s immobilized on gluthathion-agarose beads. Bound material was resolved on 7.5% SDS–PAGE and proteins were visualized after immunoblotting with antibodies against myc. (B) Untransfected U2OS cells were lysed and combined with GST–E2s as in panel A. Immunoblotting was carried out using MDM2 antibodies (upper panel) and using p53 antibodies after reprobing (lower panel). Arrow indicates MDM2 signal, asterisk indicates background band.
Mentions: Interestingly, we observed a high number of E3 interactions with an uncharacterized E2 annotated as UBE2U. The UBE2U protein (321 amino acids) is composed of a typical UBC-domain, with a C-terminal tail attached. No biological function has been described for this E2 yet, but microarray experiments showed that UBE2U mRNA transcripts have been identified mainly in tissues belonging to the urogenital tract. The UBE2U gene is only present in mammalian genomes. We looked in detail, which E3 enzymes were able to interact with this E2. One of the interacting E3s was MDM2. MDM2 has been identified as a major regulator of the cellular level and activity of the transcriptional regulator p53 (Momand et al, 1992). The significance of MDM2 in controlling normal cellular behaviour has become clear by the observation that in more than 50% of human tumours alterations in the p53 has been observed and in at least 7% of these cases, the MDM2 protein is affected (Momand et al, 1998). MDM2 has been identified as an E3 ligase, capable of ubiquitinating both itself and p53, targeting them for proteasomal degradation (Nakamura et al, 2000). For this action, MDM2 has a C-terminal Cys3–His2–Cys3 RING-finger, which was shown earlier to be able to interact with UBE2D2(UbcH5B) (Saville et al, 2004). To investigate whether other E2s identified in the two-hybrid screen can bind to MDM2, we focussed on the MDM2–UBE2U interaction that seemed quite strong from the Y2H. To investigate the MDM2–UBE2U interaction in more detail, we performed GST-pull-down assays with immobilized WT and catalytic mutant (C85A or C89A) GST–E2 UBC domains. As a negative control, GST–UBE2E1(UbcH6) was included. As shown in Figure 6A, MDM2 is captured by UBE2U and as expected also by UBE2D2(UbcH5B). Interestingly, UBE2U binds more efficiently to MDM2 than UBE2D2(UbcH5B). No binding could be observed with GST alone or GST–UBE2E1(UbcH6), reflecting the specific nature of MDM2–E2 interactions. As expected, mutation of the catalytic cysteine did not affect the interactions. Next, the GST–E2 enzymes were applied to lysates of human osteosarcoma cells (U2OS), which contain higher levels of MDM2 because of amplification or over-expression (Florenes et al, 1994). Figure 6B shows an efficient pull-down of endogenous MDM2 with both GST–UBE2U and GST–UBE2U C89A enzymes. Binding could also be observed when GST–UBE2D2(UbcH5B) WT and C85A where used, but no binding could be seen with GST alone or GST–UBE2E1(UbcH6). The GST–E2 bound MDM2 fractions were also tested by antibodies against p53. Figure 6B shows that p53 could be detected when MDM2 was captured on GST–E2 enzymes. These results indicate that UBE2U can bind to MDM2, confirming the Y2H results, and that the p53 substrate is part of the captured complex.

Bottom Line: Both within the E2 and the E3 cohorts, several members were identified that are more versatile in their interaction behaviour than others.For validation we confirmed the interaction of several versatile E2s with E3s in in vitro protein interaction assays and we used mutagenesis to alter the E3 interactions of the E2 specific for K63 linkages, UBE2N(Ubc13), towards the K48-specific UBE2D2(UbcH5B).Our data provide a detailed, genome-wide overview of binary E2-E3 interactions of the human ubiquitination system.

View Article: PubMed Central - PubMed

Affiliation: Division of Biomedical Genetics, Department of Physiological Chemistry, University Medical Center Utrecht, Utrecht, The Netherlands.

ABSTRACT
Covalent attachment of ubiquitin to substrates is crucial to protein degradation, transcription regulation and cell signalling. Highly specific interactions between ubiquitin-conjugating enzymes (E2) and ubiquitin protein E3 ligases fulfil essential roles in this process. We performed a global yeast-two hybrid screen to study the specificity of interactions between catalytic domains of the 35 human E2s with 250 RING-type E3s. Our analysis showed over 300 high-quality interactions, uncovering a large fraction of new E2-E3 pairs. Both within the E2 and the E3 cohorts, several members were identified that are more versatile in their interaction behaviour than others. We also found that the physical interactions of our screen compare well with reported functional E2-E3 pairs in in vitro ubiquitination experiments. For validation we confirmed the interaction of several versatile E2s with E3s in in vitro protein interaction assays and we used mutagenesis to alter the E3 interactions of the E2 specific for K63 linkages, UBE2N(Ubc13), towards the K48-specific UBE2D2(UbcH5B). Our data provide a detailed, genome-wide overview of binary E2-E3 interactions of the human ubiquitination system.

Show MeSH