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Identifying determinants of cullin binding specificity among the three functionally different Drosophila melanogaster Roc proteins via domain swapping.

Reynolds PJ, Simms JR, Duronio RJ - PLoS ONE (2008)

Bottom Line: Through domain swapping experiments, we demonstrate that Cullin binding specificity is strongly influenced by the Roc NH(2)-terminal domain, which forms an inter-molecular beta sheet with the Cullin.Substitution of the Roc1a RING domain with that of Roc1b results in a protein with similar Cullin binding properties to Roc1a that is active as an E3 ligase but cannot complement Roc1a mutant lethality, indicating that the identity of the RING domain can be an important determinant of CDL function.The Drosophila Roc proteins are highly similar, but have diverged during evolution to bind a distinct set of Cullins and to utilize RING domains that have overlapping, but not identical, function in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of North Carolina, Chapel Hill, North Carolina, United States of America.

ABSTRACT

Background: Cullin-dependent E3 ubiquitin ligases (CDL) are key regulators of protein destruction that participate in a wide range of cell biological processes. The Roc subunit of CDL contains an evolutionarily conserved RING domain that binds ubiquitin charged E2 and is essential for ubiquitylation. Drosophila melanogaster contains three highly related Roc proteins: Roc1a and Roc2, which are conserved in vertebrates, and Roc1b, which is specific to Drosophila. Our previous genetic data analyzing Roc1a and Roc1b mutants suggested that Roc proteins are functionally distinct, but the molecular basis for this distinction is not known.

Methodology/principal findings: Using co-immunoprecipitation studies we show that Drosophila Roc proteins bind specific Cullins: Roc1a binds Cul1-4, Roc1b binds Cul3, and Roc2 binds Cul5. Through domain swapping experiments, we demonstrate that Cullin binding specificity is strongly influenced by the Roc NH(2)-terminal domain, which forms an inter-molecular beta sheet with the Cullin. Substitution of the Roc1a RING domain with that of Roc1b results in a protein with similar Cullin binding properties to Roc1a that is active as an E3 ligase but cannot complement Roc1a mutant lethality, indicating that the identity of the RING domain can be an important determinant of CDL function. In contrast, the converse chimeric protein with a substitution of the Roc1b RING domain with that of Roc1a can rescue the male sterility of Roc1b mutants, but only when expressed from the endogenous Roc1b promoter. We also identified mutations of Roc2 and Cul5 and show that they cause no overt developmental phenotype, consistent with our finding that Roc2 and Cul5 proteins are exclusive binding partners, which others have observed in human cells as well.

Conclusions: The Drosophila Roc proteins are highly similar, but have diverged during evolution to bind a distinct set of Cullins and to utilize RING domains that have overlapping, but not identical, function in vivo.

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Ligase activity of RING swap proteins.A, Coomassie stained gel of purified GST-Roc proteins used in the ligase assays. Bracket indicates GST-Roc proteins. Asterisk indicates GST. Lower bands are degradation products. B, All Drosophila Roc proteins and chimeras were assessed for ligase activity in a substrate free assay. Briefly, 250 ng of Roc protein (or control GST) were added to a ubiquitin ligase mixture containing UbcH5, ubiquitin, and ligase buffer and incubated for 45 minutes. “−” and “+” indicate the absence or presence of UbcH5 in the reaction, respectively. Ubiquitin conjugates were detected by Western using an anti-Ub antibody (bracket indicates poly-ubiquitin chains), and GST-Roc proteins were detected using an anti-GST antibody.
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pone-0002918-g006: Ligase activity of RING swap proteins.A, Coomassie stained gel of purified GST-Roc proteins used in the ligase assays. Bracket indicates GST-Roc proteins. Asterisk indicates GST. Lower bands are degradation products. B, All Drosophila Roc proteins and chimeras were assessed for ligase activity in a substrate free assay. Briefly, 250 ng of Roc protein (or control GST) were added to a ubiquitin ligase mixture containing UbcH5, ubiquitin, and ligase buffer and incubated for 45 minutes. “−” and “+” indicate the absence or presence of UbcH5 in the reaction, respectively. Ubiquitin conjugates were detected by Western using an anti-Ub antibody (bracket indicates poly-ubiquitin chains), and GST-Roc proteins were detected using an anti-GST antibody.

Mentions: Chimeras between Roc1a and Roc2 behaved slightly differently than the ANBR and BNAR proteins. For instance, AN2R failed to bind any Cullin, even though it could be stably expressed (shown for Cul1 and Cul5 in Fig. 2C). There could be several reasons why AN2R fails to bind Cullin. There may be amino acids in the Roc1a RING domain necessary for Cul1 binding that are not present in the Roc2 RING domain. We swapped several potential specificity residues in the RING domain between Roc1a and Roc2, but were unable to alter the Cullin binding (data not shown). Alternatively, since ANBR binds to Cul1, the Roc2 RING domain may contain amino acids that prevent binding to Cul1. However, we show below that the AN2R protein is not active as an E3 ligase (for example, see Fig. 6 below), and thus this protein chimera is functionally inactive perhaps because it does not fold properly. In the reciprocal experiment, 2NAR bound strongly to Cul5, and more weakly to Cul1 (Fig. 2C). Interestingly, we could also detect some binding of 2NAR to Cul3 (Figure 2D). These data indicate that the Roc2 NH2-terminus confers strong binding preference to Cul5, and that the Roc1a RING domain contributes somewhat to the selection of Cul1 and Cul3.


Identifying determinants of cullin binding specificity among the three functionally different Drosophila melanogaster Roc proteins via domain swapping.

Reynolds PJ, Simms JR, Duronio RJ - PLoS ONE (2008)

Ligase activity of RING swap proteins.A, Coomassie stained gel of purified GST-Roc proteins used in the ligase assays. Bracket indicates GST-Roc proteins. Asterisk indicates GST. Lower bands are degradation products. B, All Drosophila Roc proteins and chimeras were assessed for ligase activity in a substrate free assay. Briefly, 250 ng of Roc protein (or control GST) were added to a ubiquitin ligase mixture containing UbcH5, ubiquitin, and ligase buffer and incubated for 45 minutes. “−” and “+” indicate the absence or presence of UbcH5 in the reaction, respectively. Ubiquitin conjugates were detected by Western using an anti-Ub antibody (bracket indicates poly-ubiquitin chains), and GST-Roc proteins were detected using an anti-GST antibody.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2500221&req=5

pone-0002918-g006: Ligase activity of RING swap proteins.A, Coomassie stained gel of purified GST-Roc proteins used in the ligase assays. Bracket indicates GST-Roc proteins. Asterisk indicates GST. Lower bands are degradation products. B, All Drosophila Roc proteins and chimeras were assessed for ligase activity in a substrate free assay. Briefly, 250 ng of Roc protein (or control GST) were added to a ubiquitin ligase mixture containing UbcH5, ubiquitin, and ligase buffer and incubated for 45 minutes. “−” and “+” indicate the absence or presence of UbcH5 in the reaction, respectively. Ubiquitin conjugates were detected by Western using an anti-Ub antibody (bracket indicates poly-ubiquitin chains), and GST-Roc proteins were detected using an anti-GST antibody.
Mentions: Chimeras between Roc1a and Roc2 behaved slightly differently than the ANBR and BNAR proteins. For instance, AN2R failed to bind any Cullin, even though it could be stably expressed (shown for Cul1 and Cul5 in Fig. 2C). There could be several reasons why AN2R fails to bind Cullin. There may be amino acids in the Roc1a RING domain necessary for Cul1 binding that are not present in the Roc2 RING domain. We swapped several potential specificity residues in the RING domain between Roc1a and Roc2, but were unable to alter the Cullin binding (data not shown). Alternatively, since ANBR binds to Cul1, the Roc2 RING domain may contain amino acids that prevent binding to Cul1. However, we show below that the AN2R protein is not active as an E3 ligase (for example, see Fig. 6 below), and thus this protein chimera is functionally inactive perhaps because it does not fold properly. In the reciprocal experiment, 2NAR bound strongly to Cul5, and more weakly to Cul1 (Fig. 2C). Interestingly, we could also detect some binding of 2NAR to Cul3 (Figure 2D). These data indicate that the Roc2 NH2-terminus confers strong binding preference to Cul5, and that the Roc1a RING domain contributes somewhat to the selection of Cul1 and Cul3.

Bottom Line: Through domain swapping experiments, we demonstrate that Cullin binding specificity is strongly influenced by the Roc NH(2)-terminal domain, which forms an inter-molecular beta sheet with the Cullin.Substitution of the Roc1a RING domain with that of Roc1b results in a protein with similar Cullin binding properties to Roc1a that is active as an E3 ligase but cannot complement Roc1a mutant lethality, indicating that the identity of the RING domain can be an important determinant of CDL function.The Drosophila Roc proteins are highly similar, but have diverged during evolution to bind a distinct set of Cullins and to utilize RING domains that have overlapping, but not identical, function in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of North Carolina, Chapel Hill, North Carolina, United States of America.

ABSTRACT

Background: Cullin-dependent E3 ubiquitin ligases (CDL) are key regulators of protein destruction that participate in a wide range of cell biological processes. The Roc subunit of CDL contains an evolutionarily conserved RING domain that binds ubiquitin charged E2 and is essential for ubiquitylation. Drosophila melanogaster contains three highly related Roc proteins: Roc1a and Roc2, which are conserved in vertebrates, and Roc1b, which is specific to Drosophila. Our previous genetic data analyzing Roc1a and Roc1b mutants suggested that Roc proteins are functionally distinct, but the molecular basis for this distinction is not known.

Methodology/principal findings: Using co-immunoprecipitation studies we show that Drosophila Roc proteins bind specific Cullins: Roc1a binds Cul1-4, Roc1b binds Cul3, and Roc2 binds Cul5. Through domain swapping experiments, we demonstrate that Cullin binding specificity is strongly influenced by the Roc NH(2)-terminal domain, which forms an inter-molecular beta sheet with the Cullin. Substitution of the Roc1a RING domain with that of Roc1b results in a protein with similar Cullin binding properties to Roc1a that is active as an E3 ligase but cannot complement Roc1a mutant lethality, indicating that the identity of the RING domain can be an important determinant of CDL function. In contrast, the converse chimeric protein with a substitution of the Roc1b RING domain with that of Roc1a can rescue the male sterility of Roc1b mutants, but only when expressed from the endogenous Roc1b promoter. We also identified mutations of Roc2 and Cul5 and show that they cause no overt developmental phenotype, consistent with our finding that Roc2 and Cul5 proteins are exclusive binding partners, which others have observed in human cells as well.

Conclusions: The Drosophila Roc proteins are highly similar, but have diverged during evolution to bind a distinct set of Cullins and to utilize RING domains that have overlapping, but not identical, function in vivo.

Show MeSH
Related in: MedlinePlus