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Results: 5

1.
Figure 4

Figure 4. From: The Ubiquitin Conjugating Enzyme, UbcM2, Engages in Novel Interactions with Components of Cullin-3 Based E3 Ligases.

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), or recombinant, immobilized His6-S-UbcM2. Bead-associated proteins were separated by SDS–PAGE and visualized by either fluorography (35S-labeled proteins) or CBB staining (His6-S-tagged proteins). (B) Similar experiment to (A) using His6-S-CUL3 (1−417) on beads, and reaction mixtures were supplemented with 10 μM GST (lanes 1−12) or 10 μM GST-UbcM2 (lanes 13−18). For (A) and (B), the migration of molecular weight markers is indicated on the left. (C) Overexpressed RCBTB1 increases the binding of unmodified and neddylated CUL3 to UbcM2. HEK293T cells were transiently transfected with plasmids expressing myc-RCBTB1, HA3-CUL3, or myc-RCBTB1 and HA3-CUL3. Lysates derived from the transfected cells were combined with GSH-sepharose and either GST, GST-UbcM2, or GST-UbcH5b. Bead-associated proteins were analyzed by SDS–PAGE and Western blotting. Myc-RCBTB1 was detected with anti-RCBTB1, and HA3-CUL3 was detected with anti-HA 12CA5. The right panel shows the GST proteins stained with CBB. Asterisk represents the cross-reactivity of the anti-RCBTB1 antiserum with GST-UbcM2 (bottom blot, lanes 4−6).

Kendra S. Plafker, et al. Biochemistry. ;48(15):3527-3537.
2.
Figure 5

Figure 5. From: The Ubiquitin Conjugating Enzyme, UbcM2, Engages in Novel Interactions with Components of Cullin-3 Based E3 Ligases.

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 and either GST (lanes 8−13), GST-UbcM2 (lanes 14−19), or GST-UbcH5b (lanes 21−26). The bead-associated HA3-tagged cullins were analyzed by anti-HA 12CA5 Western blotting. Molecular weight (MW) markers were run in lanes 7 and 20, and inputs for each pulldown are shown in lanes 1−6. (B) Model of UbcM2 association with activated CUL3 ligases. In contrast to the Roc1/Rbx1-dependent recruitment of E2s to the C-terminal end of CUL3 complexes, we propose that UbcM2 can be recruited, in concert with substrate adaptors, to the N-terminal half of activated CUL3. The function(s) of UbcM2 in this context remains to be determined, but we speculate that the enzyme may ubiquitylate the cullin and/or the substrate adaptors. The small ball between the dimerized cullin represents Nedd8. The substrate adaptor is shown as a monomer but may be a dimer in many cases (49).

Kendra S. Plafker, et al. Biochemistry. ;48(15):3527-3537.
3.
Figure 2

Figure 2. From: The Ubiquitin Conjugating Enzyme, UbcM2, Engages in Novel Interactions with Components of Cullin-3 Based E3 Ligases.

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 RCBTB1 (Flag-RCBTB1) (lane 2). Lysates were combined with Flag-agarose, and the bead-associated and unbound proteins were analyzed by Western blotting with anti-Flag and anti-HA antibodies. (B) Deletion of the N-terminal 57 residues of CUL3 blocks RCBTB1 binding. A set of His6-S-tagged CUL3 proteins and RCBTB1 were expressed in separate TNT reactions. Each 35S-labeled CUL3 protein was then combined with an equal aliquot of 35S-labeled RCBTB1 and S-protein agarose beads. Bead-associated proteins were separated by SDS–PAGE and visualized by fluorography. The structural and functional integrity of the CUL3 truncations is demonstrated in Figure 3E. (C) 35S-labeled RCBTB1 was expressed in a TNT reaction and combined with S-protein agarose (–), or beads plus either recombinant wild type (WT) or (S53A, F54A) CUL3 from bacteria. Aliquots of bound (lanes 1−3) and unbound proteins (lanes 4−6) were resolved by SDS–PAGE and visualized by fluorography (35S-RCBTB1) or blotting with S-protein HRP (His6-S-CUL3). (D) Recombinant His6-S-CUL3 (1−417) was immobilized on S-protein beads and combined with the indicated 35S-labeled RCBTB1 proteins. The precipitated 35S-labeled proteins were resolved by SDS–PAGE and visualized by fluorography. Recombinant His6-S-CUL3 (1−417) was detected by CBB staining. Nonspecific binding of the 35S-labeled proteins to the S-protein agarose is shown in Supplemental Figure 1 in the Supporting Information.

Kendra S. Plafker, et al. Biochemistry. ;48(15):3527-3537.
4.
Figure 3

Figure 3. From: The Ubiquitin Conjugating Enzyme, UbcM2, Engages in Novel Interactions with Components of Cullin-3 Based E3 Ligases.

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 CUL3 was visualized by fluorography (top panel) and the GST proteins by CBB-staining (bottom panel). (B) Cartoon illustrating the architecture of a prototypical CUL3-based E3 ligase. CUL3 is bound at its N-terminal end to a BTB-containing substrate adaptor (BTB protein) and through its C-terminal domain to a RING-finger protein (RING). The BTB protein recruits substrates for ubiquitin modification and the RING-finger protein recruits an E2 into the complex. The E2 transfers ubiquitin (Ub) to the substrate. A conserved lysine in the C-terminal domain of CUL3 is modified with the ubiquitin-like modifier, Nedd8 (shown as a ball). (C) CUL3 can be precipitated by a select group of E2s. 35S-labeled CUL3 was combined with GST (lane 1) or the indicated GST-E2 fusion proteins (lanes 2−8) and GSH-sepharose. The class to which each E2 belongs is indicated below the lane numbers. (D) Same experiment as (C) except 35S-labeled CUL3 (1−417) was expressed in place of full length CUL3. (E) UbcM2 binding to CUL3 requires the N-terminal 57 residues of the cullin. A set of His6-S-tagged CUL3 proteins was expressed in individual TNT reactions and combined with GSH-sepharose and either GST (lanes 1−4), GST-UbcM2 (lanes 5−8), or GST-Roc1 (lanes 9−12). (F) Wild type or S53A, F54A CUL3 were expressed in TNT reactions and combined with GSH-sepharose and either GST (lanes 1, 2), GST-UbcM2 (lanes 3, 4), or GST-Roc1 (lanes 5, 6). For the data shown in A, C, D, E, and F, bead-associated proteins were separated by SDS–PAGE and visualized by either fluorography (35S-labeled proteins) or CBB staining (GST proteins).

Kendra S. Plafker, et al. Biochemistry. ;48(15):3527-3537.
5.
Figure 1

Figure 1. From: The Ubiquitin Conjugating Enzyme, UbcM2, Engages in Novel Interactions with Components of Cullin-3 Based E3 Ligases.

UbcM2 interacts with a C-terminal domain of RCBTB1 in a RING-finger protein-independent fashion. (A) Directed yeast two-hybrid results using wild type or catalytically inactive (C145S) UbcM2 as the bait and fragments from 4 putative E3 ligases as the prey. Top panel shows a photograph of a plate lacking leucine and tryptophan (LW) on which yeast were mated. Bottom panel is a photograph of a plate lacking leucine, tryptophan, and histidine but containing 10 mM 3-aminotriazole (LWH + 3-AT). The LWH + 3-AT plate was replica plated from the LW mating plate. (B) Schematic of RCBTB1. The ATS-like domain (black rectangle) spans residues 43 to 317 and has 39% homology with ATS1p, the alpha-tubulin suppressor protein from S. cerevisiae. Amino acids 359−465 comprise the BTB domain (patterned box). The numbers represent amino acid positions. (C) UbcM2 interacts with RCBTB1 independent of a bridging RING-finger protein. Parallel GST-fusion protein pulldowns of 35S-labeled RCBTB1 or 35S-labeled AO7 expressed by TNT. Fractions of bead-bound proteins (50% total) were resolved by SDS–PAGE. GST proteins were visualized by CBB-staining (bottom panels) and 35S-labeled RCBTB1 and AO7 by fluorography (top panels, lanes 1−5 and 6−10, respectively). Point mutants of GST-UbcM2 are denoted above lanes 3, 4, 5, 8, 9, and 10. (D) Directed yeast two-hybrid assay to demonstrate that the F122A and P155R, A156R mutants of UbcM2 are deficient in binding to a RING-finger domain from RNF5. The RING-finger domain of RNF5 resides between residues 22 and 87. As a positive control, all UbcM2 proteins interacted with the transport receptor importin-11. Diploid yeast were grown on LW plates and replica plated onto LWH + 3-AT plates to detect protein–protein interactions. (E) Recombinant Myc-UbcM2-His6 pulldown of the indicated 35S-labeled RCBTB1 proteins expressed in individual TNT reactions. Myc-UbcM2-His6 and any associated 35S-labeled proteins were precipitated with 9E10 monoclonal antibody and protein-A sepharose. Myc-UbcM2-His6 was visualized following SDS–PAGE by CBB staining (bottom panel, lanes 1−6) and the 35S-labeled RCBTB1 proteins were visualized by fluorography. Lanes 7−12 represent negative controls lacking the 9E10 monoclonal antibody. A schematic of each RCBTB1 construct is shown on the right with amino acid numbers. Binding was quantitated from 3 independent experiments, and band intensity was normalized to the number of methionines in each protein. % binding of each protein compared to wild type RCBTB1 is listed with standard deviations. Asterisk next to lane 6 marks a band representing residues 445−531 which contains only a single methionine.

Kendra S. Plafker, et al. Biochemistry. ;48(15):3527-3537.

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