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Results: 4

Figure 2

Figure 2. RING105 binds TSSC5 by co-immunoprecipitation. From: Tumor suppressor candidate TSSC5 is regulated by UbcH6and a novel ubiquitin ligase RING105.

(a) Schematic representation of TSSC5 protein and TSSC5 plasmid constructs used for testing ubiquitin targeting. TSSC5 is predicted to be an integral membrane protein with 10 transmembrane domains. (The orientation of TSSC5 with respect to its insertion into cytoplasmic membranes such as ER has not been determined.) Potential ubiquitin acceptor lysine residues are presented as circles. (b) Ectopically expressed TSSC5 was associated with cytoplasmic membranes. HeLa cells were transfected with HA-tagged TSSC5F (full length) and prepared for immunofluorescence with anti-HA antibody. DNA was stained with DAPI (upper panel). (c) Limited colocalization of GFP-RING105 and HA-TSSC5F. GFP-RING105 and HA-TSSC5F (full length) were cotransfected to HeLa cells. At 14 h after transfection, cells were treated with MG132 for 2 h to prevent RING105 degradation, and fixed for localization analysis. Panels represent DAPI (purple), GFP-RING105 (green) and HA-TSSC5F (red) and GFP-HA overlay. (d) HA-tagged TSSC5 co-immunoprecipitated with GFP-tagged RING105. HeLa cells were transfected with HA-TSSC5F (full length) and GFP or GFP-RING105. After 18 h, cells were treated with MG132 for 2 h and then extracted. The extracts were immunoprecipitated with anti-HA and anti-GFP. Samples from the whole extract (input) and the immunoprecipitate (IP) were probed with anti-HA and anti-GFP.

HY Yamada, et al. Oncogene. ;25(9):1330-1339.
Figure 3

Figure 3. TSSC5 ubiquitylation. From: Tumor suppressor candidate TSSC5 is regulated by UbcH6and a novel ubiquitin ligase RING105.

(a) TSSC5 C-terminus is polyubiquitylated in vivo. 293T cells were transfected with plasmid encoding HA-tagged TSSC5 C-terminus (TSSC5C). After 18 h, cells were incubated with or without 10 µm MG132 for 2 h. Cell extracts were denatured to break up protein complexes and then HA-tagged proteins were immunoprecipitated with anti-HA antibody. The immunoprecipitates were blotted with anti-HA or antiubiquitin. (b) TSSC5 6th loop is the smallest fragment that is polyubiquitylated efficiently in vivo. Plasmid encoding HA-tagged TSSC5 6th loop was transfected into 293T cells. At 18 h after transfection, cells were treated with MG132 for 2 h and then extracts were prepared for immunoprecipitation with anti-HA antibody in denaturing conditions. Immunoprecipitates were blotted with anti-HA and anti-ubiquitin. Only fragments of TSSC5 containing the 6th loop exhibited polyubiquitylation, while other regions containing lysines (such as the TSSC5 10th loop) did not exhibit polyubiquitylaion (negative data, not shown). (c) Only the combination of UbcH6 and RING105 facilitated polyubiquitylation of TSSC5C in vitro. Immunoprecipitated GFP- or GFP-RING105 was incubated with E1, the indicated E2, GST-ubiquitin, ATP and S-tagged TSSC5C for 2 h at 30°C. Reactions were terminated with 1% SDS and S-tagged protein was collected with S-protein beads. Ubiquitylation was probed with anti-GST. Although several of the E2’s result in the accumulation of high molecular weight bands on the S-tag-precipitated protein, only UbcH6 results in bands that are generated only in the presence of RING105. (d) More detailed examination of the UbcH6/RING105-mediated ubiquitylation reaction shows that high molecular weight bands contain both TSSC5 (labeled with anti-S tag) and GST-ubiquitin (labeled with anti-GST), demonstrating that the high molecular weight bands are poly(GST)ubiquitylated forms of TSSC5C. Note that in the presence of RING105, majority of TSSC5 was polyubiquitylated. In contrast, without RING105 polyubiquitin chain was rarely observed.

HY Yamada, et al. Oncogene. ;25(9):1330-1339.
Figure 4

Figure 4. RING105 expression delays the G1-to-S transition in HeLa cells. From: Tumor suppressor candidate TSSC5 is regulated by UbcH6and a novel ubiquitin ligase RING105.

(a) RING105 transfection to cycling HeLa cells. HeLa cells were transfected with plasmids expressing RING105-GFP constructs (see Figure 1b) and the effect on the cell cycle was analysed by flow cytometry 24–40 h after transfection. Experiments were repeated at least twice and typical results from GFP-positive cells are shown. Numbers indicate estimated G0/G1 percentage. Cells transfected with plasmids expressing full-length (+1) or the C-terminal region containing the RING-finger domain (+4) showed a substantial accumulation of G1 cells. Point mutations of the RING-finger domain in full-length TSSC5 (+2, +3) or in the C-terminal construct (+5, +6) significantly reduced accumulation of cells in G1. (b) RING105 transfection and DNA-damaging agent VM26 (teniposide) treatment. Transfected HeLa cells were treated with the topoisomerase II inhibitor VM26 (0.2 µg/ml, 12 h) leading to DNA damage. This treatment led to an accumulation of cells in S phase/G2 phase. In cells transfected with plasmids expressing full-length (+1.Full) and C-terminus RING-finger domain (+4.C) of RING105, the S/G2 peaks were reduced, suggesting that the transfection causes delay in the cell cycle progression before S phase. (c) RING105 transfection and spindle inhibitor nocodazole treatment. HeLa cells were transfected with empty plasmid (vector) or with plasmids encoding full-length RING105 (+1.Full), N-terminus (+7.N) and C-terminus (+4.C) of RING105 in the absence (left column) or presence (right column) of nocodazole (100 ng/ml, 16 h). Nocodazole induces mitotic-checkpoint-dependent G2/M arrest. Populations transfected with empty vector or with plasmids expressing the N-terminus showed the normal increase in the G2/M peak. Populations expressing the full-length RING105-GFP (+1) showed a diminished degree of accumulation, while cells expressing the C-terminus RING-finger domain (+4.C) showed almost no accumulation in G2/M (arrows).

HY Yamada, et al. Oncogene. ;25(9):1330-1339.
Figure 1

Figure 1. RING105 is a RING-finger-containing ubiquitin ligase. From: Tumor suppressor candidate TSSC5 is regulated by UbcH6and a novel ubiquitin ligase RING105.

(a) RING105 protein domain structure. RING105 protein is 350 amino acids and contains a PA (protease-associated) domain, a RING-finger domain and a PEST domain. Two TM (transmembrane domain) are also highlighted. (b) Plasmid constructs. RING105 was truncated, or point mutations (represented as black bars) were introduced in conserved positions within the RING-finger domain. Mutations at corresponding sites were previously shown to inactivate ubiquitylation activity in the chfr protein (Kang et al., 2002). The protein sequence alignment comparing RING-finger domains for RING105, Apc11 and chfr is also shown. The residues that were mutated to inactivate the RING finger are indicated by arrows. The cysteine and histidine residues that form zinc-chelating ‘fingers’ are shown in bold. (c) RING105 is degraded by the proteasome in a manner that is dependent on its own RING-finger domain. 293T cells were transfected with plasmids encoding the indicated GFP-tagged RING105 constructs and cell extracts were prepared for immunoblotting with anti-GFP. In the lower blot, the cells were treated with proteasome inhibitor MG132 (10 µm) 2 h before preparation of cell extracts. Note that mutants containing point mutations within the RING finger are stable (compare lane 3 with lanes 4 and 5 in the upper blot). Note also that MG132 treatment results in stabilization of the full-length RING105 protein (compare lane 3 (asterisk) in the upper and lower blots). (d) RING105 has autoubiquitylation activity in vitro when tested with UbcH5a and UbcH6. Immunoprecipitated GFP- or GFP-tagged RING105 were incubated with indicated E2 (ubiquitin-conjugating enzyme), GST-ubiquitin, E1 and ATP for 60 min at 30°C. After the reaction, the beads were rinsed extensively, then treated with SDS sample buffer, separated by PAGE and transferred to membranes. They were then probed with anti-GFP (left panel) and anti-GST (ubiquitin) (right panel). The high molecular weight bands appearing above the GFP-RING105 doublet (black arrowheads) when RING105 is incubated in conjunction with UbcH5a and UbcH6 reflect polyubiquitylated proteins. (e) GFP-tagged RING105 associated with cytoplasmic membranes. HeLa cells were transfected with GFP-RING105 (full) (construct 1). After 18 h, cells were treated with 10 µm MG132 for 2 h and observed. (f) RING105 is enriched in the liver and kidney. Extracts from human tissues were probed with anti-RING105 antibody. Further description of the antibody is shown in Supplementary Information 1.

HY Yamada, et al. Oncogene. ;25(9):1330-1339.

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