LMP1 inhibits Siah-1 expression on the transcriptional level. Total RNA purified from LMP1-transfected DG75 cells was subjected to RT-PCR (Left). The level of Siah-1 transcripts was quantified by real-time RT-PCR (Right). Siah-1 expression in each LMP1-transfected sample is shown in term of relative qualification compared with expression of GAPDH, which has been ascribed an arbitrary value of 1. (Error bars = SD, n = 2 in each group.)

Working model of ubiquitin-dependent proteasome pathways for β-catenin in B lymphoma cells. Ubiquitin/proteasome machinery for β-catenin destruction is highly effective in BL cells. At least two ubiquitin-dependent proteasome pathways for β-catenin degradation are active in these cells. One pathway is the classical degradation of β-catenin through the SCF^β-TrCP ubiquitin ligase complex that depends on phosphorylation of β-catenin by GSK3β kinase. Another pathway is through ubiquitination of β-catenin by the Siah-1^SIP-Skp1-Ebi complex. The EBV oncogene LMP1 can up-regulate β-catenin in B lymphoma cells by inhibition of the latter: Siah-1-dependent ubiquitination.

LMP1 is involved in GSK3β-independent activation of β-catenin. (a) The level of β-catenin in DG75 cells after transfection with either wild-type (WT) or mutant form (S37A) of β-catenin (1 μg) for 48 h in the presence or absence of LMP1 (1 μg) was compared. For lanes 5 and 6, cells were treated with 20 mM LiCl for 24 h. (b) Tcf/Lef transcriptional activity in DG75 cells transfected with either WT or mutant form of β-catenin in the presence or absence of LMP1. The data indicate the average fold of TOPFlash/FOPFlash from two independent experiments prepared in triplicate. (c) LMP1 (0, 1, and 3 μg) was cotransfected with luciferase reporter into DG75 cells in the presence or absence of 20 mM LiCl, and luciferase assay was performed as described in Fig. 3b. In each experiment, the total amount of DNA was kept at 10 μg.

LMP1 reduces ubiquitination of β-catenin in B lymphoma cells. (a) DG75 cells were either mock-treated (lanes 1 and 2) or treated with 10 μM MG101 (lanes 3 and 4) in the presence (lanes 2 and 4) or absence (lanes 1 and 3) of LMP1 for 4 h, and the level of β-catenin was determined. (b) Increasing amounts of pLMP1 (0, 1, and 3 μg, respectively) were transfected along with pHA-ubiquitin (3 μg) into DG75 cells for 48 h. The total amount of DNA was kept at 10 μg. Ubiquitin-conjugated products were immunoprecipitated with HA antibody and pulled down with A/G PLUS-Agarose. The level of ubiquitin-attached β-catenin was detected by Western blotting with β-catenin antibody. As a loading control, the amount of IgG in the precipitates and the level of tubulin in the total lysates are shown. Each band was quantified as described in Fig. 1a. (b Right) The correlation between total and ubiquitinated β-catenin.

LMP1 up-regulates β-catenin in B-cell lines. (a) Correlation of β-catenin and LMP1 protein levels in EBV-positive and -negative B cell lines. Total cell lysates were probed with β-catenin and LMP1 antibodies. Each band was quantified with the use of bioproril bio 1d image analysis software (Vilber Lourmat, Marne-la-Vallée, France). (b) LMP1 expression increases β-catenin levels in EBV-negative B lymphoma cells. Increasing amounts of LMP1-expressing vector (pcLMP1) were transfected into DG75 for 48 h, and protein levels of β-catenin and LMP1 were detected with indicated antibodies. (c) Increase of β-catenin/Tcf transcriptional activity by LMP1. pcLMP1 (1 μg) was cotransfected with either wild-type (TOPFlash) or mutant (FOPFlash) reporter plasmids into DG75 cells. The total amount of DNA was kept at 10 μg. After 48 h of transfection, luciferase activity was determined. The data represent two independent experiments prepared in triplicate.

Inhibition of Siah-1 ubiquitin ligase up-regulates β-catenin in B lymphoma cells. (a) LMP1-dependent reduction of Siah-1 expression. DG75 cells were transfected with 0, 1, and 3 μg of pcLMP1 (lanes 1–3). After 48 h of transfection, the protein levels of LMP1, β-catenin and Siah-1 were determined with the indicated antibodies. In addition, genetically identical BL cell lines, Sav I and III, were used (lanes 4 and 5). (b) Functional inhibition of Siah-1 ubiquitin-ligase increasesβ-catenin/Tcf transcriptional activity. One microgram of Myc-tagged Siah-1 dominant-negative mutant-expressing plasmid (pSiah-1DN) was transfected into DG75 in the presence or absence of pcLMP1 (1 μg). The data indicate the average fold of TOPFlash/FOPFlash from two independent experiments prepared in triplicate. (c) Functional inhibition of Siah-1 ubiquitin-ligase increases β-catenin protein stabilization. One microgram of pSiah-1DN was transfected into DG75 cells in the presence or absence of pcLMP1 (1 μg). In each experiment, the total amount of DNA was kept at 10 μg. The expression of Siah-1DN was detected with an anti-c-Myc antibody. (d) Inhibition of endogenous Siah-1 expression stabilizes β-catenin. Siah-1 siRNA was transfected at the indicated concentration in the absence (lanes 1, 3, and 5) or presence (lanes 2, 4, and 6) of pcLMP1 (1 μg). The final concentration of siRNA in each transfection was made up to 50 nM by supplementation with a control siRNA. Protein levels of β-catenin, Siah-1, and LMP1 were detected with indicated antibodies.