1. Volume 147, Issue 6, Pages 1324-1339 (December 2011)
Loss of Tankyrase-Mediated Destruction of 3BP2 Is the Underlying Pathogenic Mechanism of Cherubism 
Noam Levaot, Oleksandr Voytyuk, Ioannis Dimitriou, Fabrice Sircoulomb, Arun Chandrakumar, Marcel Deckert, Paul M. Krzyzanowski, Andrew Scotter, Shengqing Gu, Salima Janmohamed, Feng Cong, Paul D. Simoncic, Yasuyoshi Ueki, Jose La Rose, Robert Rottapel 
Cell 
Volume 147, Issue 6, Pages (December 2011)
DOI: /j.cell
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2. Cell  , DOI: ( /j.cell )
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3. Figure 1 3BP2 Cherubism Mutant Protein Is Stabilized
(A) 3BP2 protein expression in macrophages derived from WT, Sh3bp2KI/+, or Sh3bp2KI/KI mice was determined by western blot analysis.
(B) Quantitative PCR of sh3bp2 mRNA derived from WT, Sh3bp2KI/+, or Sh3bp2KI/KI macrophages. Error bars represent SEM.
(C) Cycloheximide chase to ascertain 3BP2 protein stability in WT or Sh3bp2KI/KI bone marrow-derived macrophages (BMMs). BMMs obtained from either WT or Sh3bp2KI/KI mice were treated with cycloheximide for the indicated time intervals and lysed, and 3BP2 protein levels determined by western blot analysis. The percentages of 3BP2 protein levels were plotted as a function of time.
(D) 3BP2 and TNKS2 domain organization. 3BP2 is a PH and SH2 domain-containing adaptor protein. The RSSPDG sequence mutated in cherubism patients is shown. TNKS2 contains five repeat clusters (ARCs), a SAM domain, and the PARP domain.
(E) 3BP2 bind both TNKS and TNKS2. Myc-Tankyrase western blot of Flag-3BP2 immune complexes expressed in HEK293T cells.
(F) 3BP2 but not cherubism mutants bind to TNKS2. Tankyrase western blot of GST-3BP2 and GST-3BP2 cherubism mutant immune complexes.
(G) The 3BP2 targeting peptide (Hex) is sufficient to bind to TNKS2. Immobilized GST, GST-Hex(WT), or GST-Hex cherubism mutations R413Q, P416H, or G418R were incubated with lysates from HEK293T cells overexpressing Myc-TNKS2 and probed for Myc-TNKS2 by western blot. The levels of the recombinant GST-Hex peptide were evaluated by Coomassie staining.
See also Figure S1.
Cell  , DOI: ( /j.cell )
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4. Figure 2 3BP2 Is ADP-Ribosylated by TNKS2 In Vitro and In Vivo
(A) Ribosylation of 3BP2 and TNKS2 in vitro require the SAM and PARP domains of TNKS2. Flag-3BP2 immune complexes were subjected to an in vitro PARP reaction using [32P]NAD+. Proteins were separated by SDS-PAGE and visualized by autoradiography.
(B) The PARP inhibitor PJ-34 inhibits TNKS2-dependent ribosylation of 3BP2. Flag-3BP2 immune complexes were subjected to an in vitro PARP reaction in the presence of increasing concentration of PJ-34. Lower panel, quantified inhibition of TNKS2 PARP activity by PJ-34, as evaluated by Myc-TNKS2 autoribosylation.
(C) 3BP2 but not cherubism mutants of 3BP2 are ribosylated by TNKS2. Flag-3BP2 or Flag-3BP2 cherubism mutants were subjected to an in vitro PARP reaction. Ribosylation was measured by autoradiography.
(D) Ribosylated TNKS2 is present in 3BP2 but not cherubism mutant protein complexes. Flag-3BP2 and Myc-TNKS2 were coexpressed in HEK293T cells. Flag-3BP2 protein complexes were dissociated in 1% SDS heated to 68°C then subjected to reprecipitation with an anti-Myc antibody and probed with an anti- PAR antibody.
(E) Endogenous 3BP2 but not a cherubism mutant is ribosylated in primary osteoclasts. Western blots of 3BP2 immune complexes derived from WT or Sh3bp2KI/KI bone marrow-derived osteoclasts were probed with anti-PAR-specific antibodies. See also Figure S2.
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5. Figure 3
TNKS2 Controls 3BP2 Protein Levels through Ubiquitin-Mediated Proteolysis
(A) Proteasome inhibition by MG132 and LLnL reverses TNKS2-dependent suppression of 3BP2 protein levels.
(B) Ubiquitylation of 3BP2 is stimulated by TNKS2 and requires PARP activity. HEK293T cells were transfected with TNKS2 and HA-ubiquitin and incubated with proteosome or PARP inhibitors as indicated. Endogenous 3BP2 was immunoprecipitated and probed for HA-ubiquitin.
(C) TNKS2 stimulates ubiquitin-K48 modification of 3BP2. GST-3BP2 or GST-3BP2R413Q was expressed in HEK293T in the absence or presence of TNKS2. GST-3BP2 proteins were precipitated and probed with anti-UbK48-specific antibodies.
(D) The E3-ubiquitin ligase RNF146 binds to 3BP2 but not a cherubism mutant. Flag-3BP2 or Flag-3BP2R413Q immune complexes were probed for HA-RNF146 or Myc-TNKS2. The levels of protein expression for each condition are indicated in the panels below.
(E) Binding of RNF146 to 3BP2 is ADP-ribosylation dependent. Flag-3BP2 or Flag-3BP2R413Q immune complexes derived from cells cultured in the presence of Tankyrase inhibitor XAV-939 were probed for HA-RNF146 or Myc-TNKS2.
(F) Ubiquitylation of 3BP2 requires the ring finger and the WWE domain of RNF146. HA-RNF146 but not HA-RNF146ΔWWE or HA-RNF146ΔRF mutants induce 3BP2 ubiquitylation. HA-RNF146, HA-RNF146ΔWWE, or HA-RNF146ΔRF were coexpressed with Flag-3BP2 and Myc-TNKS2. Flag-3BP2 immune complexes were probed with anti-UbK48-specific antibodies.
(G) Depletion of RNF146 stabilizes 3BP2 protein levels. HEK293T cells were transfected with one of three individual shRNAs targeting RNF146 (shRNF1–3) or two nonspecific shRNAs. Endogenous 3BP2 and actin proteins were probed by western blot.
See also Figure S3.
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6. Figure 4
Elevated Levels of Wild-Type 3BP2 Protein Are Sufficient to Induce Enhanced Osteoclast Formation and TNF-α Secretion by Macrophages
(A) Lysates from RAW264.7 cell infected with retroviruses expressing WT Flag-3BP2, Flag-3BP2P416R, or empty vector were probed with anti-Flag antibodies.
(B) TRAP-positive multinucleated osteoclast cells derived from RAW264.7 infected cells as in (A). Images obtained using a Leica inverted microscope with 5× objectives.
(C) TNF-α levels in supernatants from RAW264.7 infected cells as in (A) from four independent experiments are shown. Error bars represent SEM.
(D) SRC, SYK, and VAV are hyperphosphorylated in Sh3bp2KI/KI osteoclasts. Lysates from osteoclasts derived from WT or Sh3bp2KI/KI mice were probed with phosphospecific antibodies against SRC, SYK, or VAV (left panel). The total protein levels are shown respectively on the right panel.
(E) SRC kinase activity is elevated in Sh3bp2KI/KI osteoclasts. Bone marrow-derived osteoclasts from either WT (lane 1) or Sh3bp2KI/KI (lane 2) were lysed and subjected to a SRC-specific immune complex in vitro kinase reaction. The specific kinase activity was calculated as the ratio of the autophosphorylation signal to the level of SRC expression.
(F) SYK immune complex in vitro kinase reaction performed as in (E).
(G) RAC-GTP levels are elevated in Sh3bp2KI/KI osteoclasts. RAC-GTP levels in osteoclast-derived WT or Sh3bp2KI/KI mice were measured using a PAK GST-PBD pull-down assay. RAC-GTP levels in the WT osteoclasts were normalized to 1, and RAC-GTP levels in Sh3bp2KI/KI osteoclasts expressed as fold difference compared to WT values.
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7. Figure 5
Primary Bone Marrow-Derived Macrophages Depleted of TNKS/TNKS2 Undergo Accelerated Osteoclastogenesis
(A) TNKS/TNKS2-deficient osteoclasts phenocopy cherubism osteoclasts. BMMs from WT, Sh3bp2KI/KI, or Tnks2−/− mice were infected with scrambled shRNA retrovirus (first three rows). BMMs derived from Tnks2−/− mice were infected with tnks-specific shRNA retrovirus (fourth row). Cells were then grown in CSF-1 (30 ng/ml) and RANKL (5 ng/ml, 10 ng/ml, or 40 ng/ml) for 3 days to initiate osteoclastogenesis, then TRAP stained. Experiments were performed in triplicate. Scale bar: 50 μm.
(B) Both tnks and tnks2 mRNA are expressed in osteoclasts. Tankyrase mRNA levels in BMMs cultured in RANKL and CSF-1 as determined by qPCR at day 0 and day 5. ∗p < 0.05; NS, no statistical significance. Error bars represent SEM.
(C) tnks mRNA levels in Tnks2−/− osteoclasts infected with scrambled or tnks-specific shRNA retrovirus as determined by qPCR. Error bars represent SEM.
(D and E) TNKS/TNKS2-depleted osteoclasts express elevated levels of phosphorylated SRC, SYK, and VAV and increased levels of 3BP2 protein. Lysates from primary osteoclasts in (A) were probed with phosphospecific antibodies against SRC, SYK, or VAV (D). The total protein levels as determined by western blot are shown (E).
(F) RNF146-deficient osteoclasts phenocopy cherubism osteoclasts. BMMs from WT or Sh3bp2−/− mice infected with scrambled shRNA or RNF146-specific shRNA were grown in CSF-1 (30 ng/ml) and RANKL (5 ng/ml) for 3 days then TRAP stained. Cultured osteoclasts from Sh3bp2KI/KI mice are shown for comparison. Experiments were performed in triplicate. Scale bars: 50 μm.
(G) RNF146-depleted osteoclasts express elevated levels of 3BP2. Lysates from primary osteoclasts expressing two different RNF146-targeting shRNAs (shrnf146-1 and shrnf146-2) or a scrambled shRNA (shscm) were probed by western blot for 3BP2 protein expression.
See also Figure S4.
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8. Figure 6
Tankyrase Inhibition Enhances Osteoclastogenesis in a 3BP2-Dependent Manner
(A) BMMs harvested from WT or Sh3bp2−/− mice were grown under osteoclastogenic conditions in the presence of IWR-1 (2 μM) or PJ34 (2 μM) and then TRAP stained. Scale bars: 50 μm.
(B and C) Osteoclasts grown in the presence of IWR-1 or PJ-34 express elevated levels of activated SRC, SYK, and VAV and increased protein levels of 3BP2. Lysates from cells treated as in (A) were blotted with phosphospecific antibodies against SRC, SYK, or VAV, as in Figure 5D. (C) SRC, SYK, VAV, and 3BP2 protein levels as determined by western blot are shown.
(D) TNF-α levels in the supernatant of primary macrophages incubated for the indicated times with CSF-1 in the presence or absence of IWR-1 (2 μM). Error bars represent SEM.
(E) BMMs harvested from WT and Sh3bp2KI/KI mice were cultured under osteoclastogenic conditions in the presence of the SRC inhibitors AZD0530 (0.5 μM) or PP1 (5 μM) then TRAP stained. Scale bars: 50 μm.
(F) WT BMMs were cultured under osteoclastogenic condition in the presence of IWR-1 and either AZD0530 (0.5 μM) or PP1 (5 μM) then TRAP-stained. Scale bars: 50 μm.
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9. Figure 7
Radiation Chimeric Mice, which Lack Tankyrase, Phenocopy Features of Cherubism
(A) 106 Tnks2−/− lineage-depleted bone marrow cells (C57Bl/6, CD45.2) were infected with a lentivirus harboring a Tnks-specific shRNA and then injected together with 105 CD45.1 WT bone marrow cells into lethally irradiated, syngeneic (CD45.1) recipients (TNKS/2 chimera). WT control chimeric mice were generated from CD45.2-derived bone marrow cells infected with a lentivirus expressing a scrambled shRNA hairpin (WT chimera).
(B) Fifteen weeks after transplantation, engraftment was measured by the percentage of CD45.2-positive leukocytes in the peripheral blood.
(C) Percentage of donor monocytes (CD45.2+CD11b+) in chimeric mice is shown. WT, n = 4; TNKS/2, n = 5; ∗p < 0.16.
(D) μCT reconstruction of the trabecular bone of WT or TNKS/2 chimeric mice 15 weeks after injection (left). μCT-derived measurements of the trabecular bone volume fraction (BV/TV) are shown (right). WT, n = 4; TNKS/2, n = 5; ∗p < 0.05.
(E) TRAP staining of tibias of WT and TNKS/2 chimeric mice.
(F and G) Histomorphometric analysis of (F) osteoclast surface to bone surface (OcS/BS) and (G) osteoclast number to bone perimeter (N.Oc/BS) in the tibias of WT and TNKS/2 chimeric mice 15 weeks after transplantation of donor cells. WT, n = 3; TNKS/2, n = 5; ∗p < Error bars represent SEM.
(H) BMMs harvested from WT or TNKS/2 chimeric mice were grown under osteoclastogenic conditions and TRAP stained. Scale bars: 50 μm.
Error bars represent SEM. See also Figure S5.
Cell  , DOI: ( /j.cell )
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10. Figure S1
Characterization of TNKS2 Binding to 3BP2, Related to Figure 1
3BP2 binds to the ankyrin repeat region of TNKS2. GST (lane 2) or GST-tagged 3BP2 (lanes 3–7) were coexpressed with Flag-TNKS2 (lanes 2, 3), Flag-TNKS2ΔN-1 lacking first half of the ankyrin region (lane 4), Flag-TNKS2ΔN-2 lacking the second half of the ankyrin region including the SAM domain (lane 5), Flag-TNKS2ΔANK lacking the entire ankyrin region (lane 6), or Flag-TNKS2ΔSAM lacking the SAM domain (lane 7). 3BP2 was precipitated from cell lysates and probed for TNKS2 by western blot. Glutathione beads served as a control (lane 1). The levels of 3BP2 and TNKS2 are shown below in the lower two panels.
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11. Figure S2
The N Terminus and C Terminus of 3BP2 Contain ADP-Ribosylated Sites and 3BP2 ADP-Ribosylation, and Destabilization Is Sensitive to the Position of the Tankyrase-Binding Sequence, Related to Figure 2
(A) An N-terminal fragment (Flag-3BP2 1–420) and two distinct C-terminal fragments (Flag-3BP2 372–stop, Flag-3BP2 372-stop) of 3BP2 are ADP-ribosylated by TNKS2 in HEK293T cells. A scheme of the ectopic expressed constructs is shown below (circles = Flag tag, PH = Pleckstrin homology domain, triangle = WT degron sequence, SH2 = Src homology 2 domain).
(B) The cherubism mutant chimeric protein 3BP2R423Q-WT degron harbouring the WT Tankyrase binding sequence at its C-terminal restores Tankyrase binding but is insensitive to Tankyrase-induced destabilization. Controls include the cherubism mutant Flag-3BP2R423Q and the chimeric protein Flag-3BP2R423Q-Mut degron with the cherubism mutant degron fused to the C terminus. Neither were able to bind Tankyrase. A scheme of the ectopic expressed constructs is shown below (circle = Flag tag, PH = Pleckstrin homology domain, X = cherubism mutant degron sequence, SH2 = Src homology 2 domain, triangle = WT degron sequence).
(C and D) The Flag-3BP2R423Q-WT degron protein with restored Tankyrase binding was none-the-less unable to be efficiently PARsylated by either TNKS (C) or TNKS2 (D).
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12. Figure S3
TNKS2 Induces 3BP2 Destabilization and Ubiquitylation, Related to Figure 3
(A) Wild-type but not cherubism mutant 3BP2 protein levels are sensitive to TNKS2 expression. GST-3BP2 (lanes 1–4) or the cherubism mutant GST-3BP2-R413Q (lanes 5–8) was coexpressed with increasing amounts of the TNKS2 expression plasmid in HEK293T cells. Lysates were probed for expression of GST-3BP2 (row 1) or TNKS2 (row 2). Actin loading controls are shown (row 3).
(B) 3BP2 protein levels are insensitive to expression of TNKS2ΔPARP. GST-3BP2 was coexpressed with increasing amounts of the PARP domain-deleted TNKS2 mutant (TNKS2ΔPARP) plasmid in HEK293T cells. Lysates were probed for expression of GST-3BP2 (row 1) or TNKS2ΔPARP (row 2). GST3BP2 was precipitated and probed for TNKS2ΔPARP (row 3). The amount of GST3BP2 immunoprecipitated is shown (row 4). Tubulin loading controls are shown (row 5).
(C) 3BP2 is directly ubiquitylated in the presence of TNKS2. HEK293T cells were cotransfected with HA-Ub, Flag-3BP2, and either Myc-tag peptide (Myc) (lane 1) or Myc-TNKS2 (lane 2) in the presence of the proteasome inhibitors LLNL and MG132. After lysis, 3BP2 was precipitated using an anti-Flag-specific antibody. The protein complexes were then dissociated in 1% SDS heated to 68°C and after dilution with lysis buffer, ubiquitylated proteins were reprecipitated with an HA-specific antibody. The protein complex was resolved by SDS-PAGE and probed for 3BP2 with the anti-Flag antibody by western blotting. The levels of the TNKS2, 3BP2 and Actin are shown in the lower three panels.
(D) rnf146-specific shRNAs effectively deplete rnf146 transcript levels in HEK293T cells. rnf146 mRNA levels in HEK293T transfected with one of three rnf146-specific shRNAs (shrnf1-3) or controls shRNAs (shgfp and shrfp) as determined by qPCR. Error bars represent SEM.
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13. Figure S4
3BP2 ADP-Ribosylation Is Stimulated by TNKS, Related to Figure 5
(A) Flag-3BP2 but not the cherubism mutant Flag-3BP2R413Q is ADP-ribosylated by TNKS. HEK293T cells were cotransfected with Myc-TNKS (lanes 2 and 4) and either Flag-3BP2R413Q (lanes 2) or Flag-3BP2 (lanes 4). Controls include the Myc-tag peptide coexpressed with Flag-3BP2R413Q (lane 1) or Flag-3BP2 (lane 3). After lysis, Flag-3BP2 was immunoprecipitated and probed with anti-PAR-specific antibodies. The level of precipitated 3BP2 protein is shown in the second panel. The expression of TNKS, 3BP2, and actin in lysates are shown in the lower three panels.
(B) rnf146-specific shRNAs effectively deplete rnf146 transcript levels in osteoclasts. rnf146 mRNA levels in WT or Sh3bp2−/− osteoclasts infected with a retrovirus expressing either a scrambled or rnf146-specific shRNA as determined by qPCR. Error bars represent SEM.
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14. Figure S5
Analysis of Trabecular Bone Volume and Serum TNF-α Levels in Tnks2−/−, Related to Figure 7
(A) μCT reconstruction of the trabecular region below the distal femurs growth plate of 5-month-old WT and Tnks2−/− mice.
(B) μCT-derived measurements of the trabecular bone volume fraction (BV/TV), n = 4.
(C) Serum TNF-α levels in 22-week-old WT and Tnks2−/− mice as determined by ELISA. n = 4. Error bars represent SEM.
Cell  , DOI: ( /j.cell )
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