The human class III E2s (UBE2E1, UBE2E2, and UBE2E3) engage in complexes with damaged DNA binding protein 1 and de-etiolated 1, substrate adaptor components for the CUL4a E3 ligases (36
). However, the functions of the enzymes in these complexes remain to be determined. It is also not known whether the class III E2s function with other cullins. To gain further insight into these issues, we carried out a yeast two-hybrid screen with UbcM2, the mouse counterpart of human UBE2E3. The two enzymes are 100% identical. Four interacting proteins were identified, and each interacted with both wild type and catalytically inactive (C145S) UbcM2 (). The enzyme interacted with the RING-finger domain-containing fragments of FLJ10597, RNF5, and Ariadne-2 (). The other prey, RCBTB1, is a BTB domain-containing protein genetically implicated in hematological malignancies (32
). Three independent, overlapping clones of RCBTB1 were retrieved with the smallest common domain consisting of the carboxy-terminal 87 amino acids, residues 445−531 (). This region partially overlaps with the BTB domain (residues 359−465 ()).
We used binding studies to characterize the interaction between RCBTB1and UbcM2. The human form of RCBTB1 is 96% identical to its mouse counterpart and was used for all subsequent experiments. 35
S-RCBTB1 was expressed in a rabbit reticulocyte lysate by TNT and combined with different forms of GST-UbcM2. The C145A mutant is catalytically inactive (33
), and the F122A and P155R/A156R mutants are deficient in E3 binding ( and refs 37
). These experiments confirmed that wild type and catalytically inactive UbcM2 bound full-length RCBTB1 (, lanes 2 and 3). Unexpectedly, the F122A and P155R/A156R UbcM2 mutants interacted with RCBTB1 more robustly than wild type enzyme (, lanes 4 and 5). In contrast, neither mutant appreciably precipitated the RING-finger protein AO7 (, lanes 9 and 10), nor interacted with the RING-finger protein RNF5 (). Expression and proper folding of the mutants was demonstrated by their interaction with importin-11 (). Together, these data show that UbcM2 interacts with full-length RCBTB1 independent of the active site cysteine and that UbcM2 mutants which do not productively associate with RING-finger containing proteins have an increased affinity for RCBTB1. This prompted us to test the idea that RING-finger proteins and RCBTB1 bind mutually exclusively to UbcM2. We tested this by attempting to dissociate a bead-bound UbcM2 /35
S-RCBTB1 complex with a large molar excess of recombinant AO7. Excess AO7 did not compete the binding of RCBTB1 to UbcM2 (data not shown), and we conclude that RING-finger containing proteins do not compete with RCBTB1 for a common binding site on UbcM2.
To further map the domains mediating the RCBTB1–UbcM2 interaction, we tested and quantitated the binding of a series of 35S-labeled RCBTB1 truncation mutants () to recombinant myc-UbcM2-His6. Nonspecific binding of the truncations was minimal (, lanes 7−12). These experiments revealed that, on average, (i) removal of the C-terminal 87 residues of RCBTB1 decreased UbcM2 binding by 63%, (ii) removal of the BTB domain and the C-terminal 87 residues decreased UbcM2 binding by 78%, and (iii) deletion of the BTB domain alone decreased binding by 55%. In addition, a fragment encompassing the isolated BTB domain and C-terminal 87 residues bound UbcM2 twice as well as full-length RCBTB1, and a fragment representing the C-terminal 87 residues of RCBTB1 bound comparably to wild type. This 87-residue fragment only has a single methionine and is difficult to visualize (, lane 6, asterisk). These data confirm that the C-terminal 87 residues of RCBTB1 are sufficient to mediate the interaction with UbcM2, but that the adjacent BTB domain contributes to and/or stabilizes the interaction. The finding that a RCBTB1 fragment lacking residues 1−359 has increased binding to UbcM2 indicates that one or more domains within the N-terminal 359 residues of RCBTB1 may negatively regulate the interaction.
Because RCBTB1 harbors a BTB domain, we next tested if the protein had the properties of a CUL3 substrate adaptor (19
). RCBTB1 and CUL3 were coexpressed in human embryonic kidney 293T (HEK293T) cells to determine if the two proteins could be coprecipitated. The results from these experiments () showed that an RCBTB1–CUL3 complex can form and be isolated from cell lysates. The isolation of a complex between the endogenous proteins was precluded by a failure of our α-RCBTB1 antisera to detect endogenous RCBTB1 in several cell lines (data not shown).
Figure 2 RCBTB1 has the biochemical properties of a CUL3 substrate adaptor. (A) A RCBTB1–CUL3 complex can be recovered from cell lysates. HEK293T cells were transfected with plasmids encoding triple HA-tagged CUL3 (HA3-CUL3) (lane 1) alone or with Flag-tagged (more ...)
Biochemical and structural studies of cullin-based E3 ligases have demonstrated that substrate adaptors such as skp1/2, elongin B/C, and BTB domain proteins bind the N-terminal domains of their cognate cullins (10
). This binding is mediated by a highly conserved hydrophobic helix (e.g., referred to as H2 helix) present in all cullins (e.g., refs 10
). In CUL3, helix 2 spans residues 51 to 67 (31
). To determine if RCBTB1 interacts with CUL3 through helix 2, we generated a series of N-terminal truncations of CUL3 and assessed their capacity to coprecipitate 35
S-labeled RCBTB1. RCBTB1 was coprecipitated by wild type CUL3 and CUL3 (Δ24), a truncation lacking the N-terminal 24 residues (, lanes 2 and 3), but not by CUL3 (Δ57) and CUL3 (Δ106), mutants which lack an intact helix 2 (, lanes 4 and 5). As shown in , all of the CUL3 truncations folded properly as evidenced by their binding to Roc1.
Figure 3 UbcM2 interacts with the domain of CUL3 that recruits substrate adaptors. (A) 35S-labeled CUL3 was incubated with GSH-sepharose and either GST or the indicated GST-UbcM2 fusion proteins. Bead-bound proteins were resolved by SDS–PAGE. 35S-labeled (more ...)
We extended these findings by testing the binding of RCBTB1 to CUL3 (S53A, F54A), a double point mutant lacking the capacity to interact stably with substrate adaptors (31
). Wild type CUL3 coprecipitated RCBTB1 whereas the mutant did not (, top panel). We next tested if the BTB domain of RCBTB1 was required for the RCBTB1–CUL3 interaction using recombinant, His6
-S-tagged CUL3 (1−417) and a panel of 35
S-labeled RCBTB1 proteins. CUL3 (1−417) is sufficient for substrate adaptor binding (19
) and, as expected, bound wild type RCBTB1 but not to various carboxy-terminal truncations (, lanes 2−5). Deletion of the BTB domain from RCBTB1 also largely abrogated CUL3 binding (, lane 6). CUL3, however, bound robustly to a fragment of RCBTB1 encompassing the BTB domain and the carboxy-terminal tail (, lane 7). This same RCBTB1 fragment interacted with UbcM2 (, lane 5). In contrast, disruption or removal of the BTB domain from this fragment dramatically reduced binding to CUL3 (, lanes 8 and 9). Nonspecific binding of the various 35
S-labeled RCBTB1 proteins is shown in Supplemental Figure 1
in the Supporting Information. These data indicate that RCBTB1 interacts with CUL3 in a BTB domain-dependent fashion and that the 87-residue, carboxy-terminal tail of RCBTB1 contributes to the interaction. These results further support the conclusion that RCBTB1 has the properties of a CUL3 substrate adaptor.
The finding that a putative CUL3 substrate adaptor (RCBTB1) was a binding partner of UbcM2 indicated that the enzyme might engage CUL3 ligase complexes. This link was further strengthened by the finding that both UbcM2 and CUL3 bound the same domain of RCBTB1 (, lane 5, and , lane 7). To investigate the relationship between UbcM2 and CUL3, we did pulldown assays using GST-UbcM2 and 35
S-labeled CUL3 as we did not detect a direct interaction between bacterially expressed UbcM2 and CUL3 (data not shown). We found that wild type and catalytically inactive UbcM2 precipitated CUL3 (). Unexpectedly, we also found that a mutant enzyme, UbcM2 (F122A), which lacks the capacity to stably interact with RING-finger proteins (see ), bound CUL3 (, lane 4). This result was surprising because the architecture of a prototypical CUL3-based ligase () shows that E2s are recruited into the complex through a bridging RING-finger protein, namely, Roc1/Rbx/Hrt1 (11
To further investigate the specificity of the UbcM2–CUL3 interaction, we tested the capacity of different GST-E2 fusion proteins to precipitate CUL3. E2s are broadly grouped into 4 classes, and representatives from each were tested. All class III E2s (UbcM2, UBE2E1, and UBE2E2) bound CUL3 (, lanes 5−7), as did hCDC34, a class II E2 (, lane 4). UbcH5b, a class I E2, bound weakly to CUL3 (, lane 2). UbcH10 and UbcH7 did not bind the cullin (, lanes 3 and 8) above background (, lane 1). These data indicate that a subset of E2s selectively interacts with CUL3 in this assay.
Based on our data showing that UbcM2 does not require a bridging RING-finger protein to interact with CUL3 (, lane 4), we next tested if the class III E2s bind the N-terminal half of CUL3 (residues 1−417). This CUL3 fragment lacks the binding site for Roc1 (i.e., residues 597−615 (19
)) but encompasses the domain that mediates substrate adaptor binding (19
). Pulldowns were done with GST-E2 proteins and 35
S-labeled-CUL3 (1−417). UbcM2, UBE2E2, and to a lesser extent UBE2E1 bound the N-terminal half of CUL3 (, lanes 5−7) whereas the other E2s did not (, lanes 2 and 4).
We fine-mapped the interaction of UbcM2 within the N-terminal half of CUL3 using CUL3 truncations expressed in the TNT system. GST-UbcM2 bound wild type CUL3 and a truncation lacking the N-terminal 24 residues (, lanes 5 and 6), but the mutants bearing larger deletions were not appreciably precipitated (, lanes 7 and 8). All the truncations were functional as indicated by their interaction with GST-Roc1 (, lanes 9−12). These data corroborate our findings that UbcM2 associates with the N-terminal half of CUL3 (, lane 7). Interestingly, the domain of CUL3 that interacts with UbcM2 overlaps with the region bound by RCBTB1 () and other BTB substrate adaptors (19
The binding of UbcM2 and RCBTB1 to a common domain of CUL3 lead us to test if the interaction of UbcM2 with CUL3 was coupled to substrate adaptor recruitment. This was done by determining if UbcM2 could bind the double point mutant of CUL3 that fails to associate with substrate adaptors (e.g., ). Wild type or (S53A, F54A) CUL3 were expressed in separate TNT reactions and combined with GST, GST-UbcM2, or GST-Roc1. GST-UbcM2 bound wild type CUL3 but not the mutant (). The mutant was not globally misfolded as demonstrated by its capacity to bind GST-Roc1. These data demonstrate that UbcM2 and substrate adaptors associate with a common domain of CUL3 and imply that the interaction of UbcM2 with CUL3 either requires the presence of a substrate adaptor or alternatively, is mutually exclusive with substrate adaptor binding.
We next tested if UbcM2 interacted with other BTB-containing substrate adaptors using recombinant His6
-S-UbcM2 and 35
S-labeled BTB proteins expressed in individual TNT reactions. The proteins tested were (i) RCBTB1, (ii) the closely related RCBTB2, (iii) Ctb9, (iv) Ctb101, and (v) SPOP (41
). To demonstrate that each BTB protein could bind CUL3, aliquots of the TNT reactions were also incubated with His6
-S-tagged CUL3 (1−417). These experiments demonstrated that both CUL3 (1−417) (, lanes 6−10) and UbcM2 (, lanes 11−15) specifically bound all of the BTB proteins. These findings imply that UbcM2 can engage multiple CUL3 ligase complexes and has the properties of a general cofactor.
Figure 4 UbcM2 interacts with a panel of CUL3 substrate adaptors. (A) 35S-labeled, BTB-containing substrate adaptors were combined with either control S-protein beads (lanes 1−5), or recombinant, immobilized His6-S-CUL3 (1−417) (lanes 6−10), (more ...)
Because UbcM2 and substrate adaptors associate with a common domain of CUL3, we next tested if the binding of UbcM2 to CUL3 influenced substrate adaptor docking. Using the same experimental approach shown in , binding reactions were supplemented with 10 μM GST (, lanes 1−12) or 10 μM GST-UbcM2 (, lanes 13−18) and the amount of bound BTB-domain proteins was analyzed. We found that a large excess of UbcM2 did not compete with the binding of substrate adaptors to CUL3 (, compare lanes 7−12 with 13−18). Thus, the interaction of UbcM2 with CUL3 is not mutually exclusive with substrate adaptor recruitment indicating that UbcM2 likely binds substrate adaptor-loaded CUL3 complexes.
Additional support for this interpretation came from an experiment testing if substrate adaptor coexpression influenced the amount of CUL3 precipitated by UbcM2. RCBTB1 and CUL3 were overexpressed alone or together in HEK293T cells and the transfected-cell lysates were used for pulldowns with GST, GST-UbcM2, or GST-UbcH5b. UbcM2 precipitated RCBTB1 (, bottom panel) and CUL3 (, top panel) when each was expressed individually. Further, when RCBTB1 and CUL3 were coexpressed, UbcM2 precipitated a greater amount of CUL3 (). Interestingly, the CUL3 precipitated from the coexpression lysate showed a nearly 1:1 ratio of neddylated cullin to unmodified cullin. Overexpression of SPOP gave the same results (data not shown). The identification of the slower migrating band as neddylated CUL3 was demonstrated using green fluorescent protein (GFP)-tagged Nedd8 (data not shown). These data reveal that UbcM2 associates with both unmodified and neddylated CUL3, a heterodimer of which represents the activated form of CUL3 ligases (20
). In contrast, UbcH5b showed no capacity to precipitate RCBTB1 and minimal capacity to stably associate with CUL3 (). These results are consistent with UbcM2 and substrate adaptors associating simultaneously with CUL3, suggesting that UbcM2 is recruited to activated CUL3 ligases.
Lastly, we tested if UbcM2 might be a general cofactor for multiple cullins. We used CUL1, CUL3, and CUL4A for these experiments. Each cullin was overexpressed in HEK293T cells as either a full-length protein or as a fragment representing the N-terminal half of the cullin. Transfected-cell lysates were then precipitated with either GST, GST-UbcM2, or GST-UbcH5b. Neither GST nor GST-UbcH5b () precipitated the cullins to an appreciable extent. In contrast, GST-UbcM2 bound all three full-length cullins and their respective N-terminal halves (). These results bolster the notion that UbcM2 is a general cofactor for cullin-based E3 ligases.
Figure 5 UbcM2 binds to the N-terminal halves of multiple cullins. (A) HEK293T cells were transfected with plasmids encoding the indicated HA3-tagged cullins (full-length or N-terminal halves). Lysates from the transfected cells were combined with GSH-sepharose (more ...)