Antigen receptor signaling induces MAP kinase-mediated phosphorylation and degradation of the BCL-6 transcription factor

Abstract

The bcl-6 proto-oncogene encodes a POZ/zinc finger transcriptional repressor expressed in germinal center (GC) B and T cells and required for GC formation and antibody affinity maturation. Deregulation of bcl-6 expression by chromosomal rearrangements and point mutations of the bcl-6 promoter region are implicated in the pathogenesis of B-cell lymphoma. The signals regulating bcl-6 expression are not known. Here we show that antigen receptor activation leads to BCL-6 phosphorylation by mitogen-activated protein kinase (MAPK). Phosphorylation, in turn, targets BCL-6 for rapid degradation by the ubiquitin/proteasome pathway. These findings indicate that BCL-6 expression is directly controlled by the antigen receptor via MAPK activation. This signaling pathway may be crucial for the control of B-cell differentiation and antibody response and has implications for the regulation of other POZ/zinc finger transcription factors in other tissues.

Figure 1

Phosphorylation of BCL-6 by ERK2 in vitro: (A) Schematic representation of wild-type and mutant GST–BCL-6 fusion proteins. (*) Serines within MAPK phosphorylation sites (PXSP). (ZF) Zinc finger domain. (B) ERK2 kinase assays using GST–BCL-6 wild-type and deletion mutants as substrates in the presence of [γ-³²P]ATP. (C) (Top) ERK2 kinase assay for wild-type (WT) or mutant (Ala333; Ala333,343) GST-BCL6ΔZF proteins. (Bottom) Coomassie blue staining of the gel shown at top demonstrating comparable amounts of proteins loaded. Molecular mass markers are shown at left.

Figure 1

Phosphorylation of BCL-6 by ERK2 in vitro: (A) Schematic representation of wild-type and mutant GST–BCL-6 fusion proteins. (*) Serines within MAPK phosphorylation sites (PXSP). (ZF) Zinc finger domain. (B) ERK2 kinase assays using GST–BCL-6 wild-type and deletion mutants as substrates in the presence of [γ-³²P]ATP. (C) (Top) ERK2 kinase assay for wild-type (WT) or mutant (Ala333; Ala333,343) GST-BCL6ΔZF proteins. (Bottom) Coomassie blue staining of the gel shown at top demonstrating comparable amounts of proteins loaded. Molecular mass markers are shown at left.

Figure 1

Phosphorylation of BCL-6 by ERK2 in vitro: (A) Schematic representation of wild-type and mutant GST–BCL-6 fusion proteins. (*) Serines within MAPK phosphorylation sites (PXSP). (ZF) Zinc finger domain. (B) ERK2 kinase assays using GST–BCL-6 wild-type and deletion mutants as substrates in the presence of [γ-³²P]ATP. (C) (Top) ERK2 kinase assay for wild-type (WT) or mutant (Ala333; Ala333,343) GST-BCL6ΔZF proteins. (Bottom) Coomassie blue staining of the gel shown at top demonstrating comparable amounts of proteins loaded. Molecular mass markers are shown at left.

Figure 2

BCL-6 protein degradation induced by overexpression of MEK-2E in 293T cells. (A) Western blot analysis of 293T cells transfected with 5 μg of BCL-6 (lanes 1,3) and 5 μg of MEK-2E (lanes 2,3) using anti-BCL-6 (N-70-6; top) or anti-ERK2 (C-14; middle) antibodies. (Bottom) The results of solid-phase ERK2 kinase assay performed on cell extract from the same transfectants used in the top. (B) Western blot (top) and Northern blot (bottom) analysis of BCL-6 in 293T cells transfected with pMT2T–BCL-6 (BCL-6) (5 μg) (lanes 1–7) and various amounts of MEK-2E–CMV (MEK-2E) (lanes 2–4) or MEK–CMV (MEK) (5, 10, or 15 μg) (lanes 5–7) as indicated. (C) Western blot analysis of 293T cell extracts transfected with wild-type BCL-6 (lanes 1–3) or BCL-6_Ala333,343 (lanes 4–6) in the absence (lanes 1,4) or presence (lanes 2,3,5,6) of cotransfected MEK-2E. (Bottom) The results of densitometric scanning of the autoradiography; analogous results were obtained in three independent experiments. (D) Analysis of BCL-6 transrepression activity in the presence of active MEK. 293T cells were transfected with 2.0 pmole of B6BS–TK–Luc, 0.04 pmole of pMT2T–BCL-6 (lanes 2–8) or pMT2T–BCL-6_Ala333,343 (lanes 11–14) and increasing amounts (0.1, 0.2, 0.4, 0.4 pmole) of MEK-2E (lanes 3–5,9,12–14) or MEK (lanes 6–8,10) as indicated. Cells were harvested 48 hr after transfection and luciferase activities were measured by a luminometer.

Figure 2

BCL-6 protein degradation induced by overexpression of MEK-2E in 293T cells. (A) Western blot analysis of 293T cells transfected with 5 μg of BCL-6 (lanes 1,3) and 5 μg of MEK-2E (lanes 2,3) using anti-BCL-6 (N-70-6; top) or anti-ERK2 (C-14; middle) antibodies. (Bottom) The results of solid-phase ERK2 kinase assay performed on cell extract from the same transfectants used in the top. (B) Western blot (top) and Northern blot (bottom) analysis of BCL-6 in 293T cells transfected with pMT2T–BCL-6 (BCL-6) (5 μg) (lanes 1–7) and various amounts of MEK-2E–CMV (MEK-2E) (lanes 2–4) or MEK–CMV (MEK) (5, 10, or 15 μg) (lanes 5–7) as indicated. (C) Western blot analysis of 293T cell extracts transfected with wild-type BCL-6 (lanes 1–3) or BCL-6_Ala333,343 (lanes 4–6) in the absence (lanes 1,4) or presence (lanes 2,3,5,6) of cotransfected MEK-2E. (Bottom) The results of densitometric scanning of the autoradiography; analogous results were obtained in three independent experiments. (D) Analysis of BCL-6 transrepression activity in the presence of active MEK. 293T cells were transfected with 2.0 pmole of B6BS–TK–Luc, 0.04 pmole of pMT2T–BCL-6 (lanes 2–8) or pMT2T–BCL-6_Ala333,343 (lanes 11–14) and increasing amounts (0.1, 0.2, 0.4, 0.4 pmole) of MEK-2E (lanes 3–5,9,12–14) or MEK (lanes 6–8,10) as indicated. Cells were harvested 48 hr after transfection and luciferase activities were measured by a luminometer.

Figure 2

BCL-6 protein degradation induced by overexpression of MEK-2E in 293T cells. (A) Western blot analysis of 293T cells transfected with 5 μg of BCL-6 (lanes 1,3) and 5 μg of MEK-2E (lanes 2,3) using anti-BCL-6 (N-70-6; top) or anti-ERK2 (C-14; middle) antibodies. (Bottom) The results of solid-phase ERK2 kinase assay performed on cell extract from the same transfectants used in the top. (B) Western blot (top) and Northern blot (bottom) analysis of BCL-6 in 293T cells transfected with pMT2T–BCL-6 (BCL-6) (5 μg) (lanes 1–7) and various amounts of MEK-2E–CMV (MEK-2E) (lanes 2–4) or MEK–CMV (MEK) (5, 10, or 15 μg) (lanes 5–7) as indicated. (C) Western blot analysis of 293T cell extracts transfected with wild-type BCL-6 (lanes 1–3) or BCL-6_Ala333,343 (lanes 4–6) in the absence (lanes 1,4) or presence (lanes 2,3,5,6) of cotransfected MEK-2E. (Bottom) The results of densitometric scanning of the autoradiography; analogous results were obtained in three independent experiments. (D) Analysis of BCL-6 transrepression activity in the presence of active MEK. 293T cells were transfected with 2.0 pmole of B6BS–TK–Luc, 0.04 pmole of pMT2T–BCL-6 (lanes 2–8) or pMT2T–BCL-6_Ala333,343 (lanes 11–14) and increasing amounts (0.1, 0.2, 0.4, 0.4 pmole) of MEK-2E (lanes 3–5,9,12–14) or MEK (lanes 6–8,10) as indicated. Cells were harvested 48 hr after transfection and luciferase activities were measured by a luminometer.

Figure 2

BCL-6 protein degradation induced by overexpression of MEK-2E in 293T cells. (A) Western blot analysis of 293T cells transfected with 5 μg of BCL-6 (lanes 1,3) and 5 μg of MEK-2E (lanes 2,3) using anti-BCL-6 (N-70-6; top) or anti-ERK2 (C-14; middle) antibodies. (Bottom) The results of solid-phase ERK2 kinase assay performed on cell extract from the same transfectants used in the top. (B) Western blot (top) and Northern blot (bottom) analysis of BCL-6 in 293T cells transfected with pMT2T–BCL-6 (BCL-6) (5 μg) (lanes 1–7) and various amounts of MEK-2E–CMV (MEK-2E) (lanes 2–4) or MEK–CMV (MEK) (5, 10, or 15 μg) (lanes 5–7) as indicated. (C) Western blot analysis of 293T cell extracts transfected with wild-type BCL-6 (lanes 1–3) or BCL-6_Ala333,343 (lanes 4–6) in the absence (lanes 1,4) or presence (lanes 2,3,5,6) of cotransfected MEK-2E. (Bottom) The results of densitometric scanning of the autoradiography; analogous results were obtained in three independent experiments. (D) Analysis of BCL-6 transrepression activity in the presence of active MEK. 293T cells were transfected with 2.0 pmole of B6BS–TK–Luc, 0.04 pmole of pMT2T–BCL-6 (lanes 2–8) or pMT2T–BCL-6_Ala333,343 (lanes 11–14) and increasing amounts (0.1, 0.2, 0.4, 0.4 pmole) of MEK-2E (lanes 3–5,9,12–14) or MEK (lanes 6–8,10) as indicated. Cells were harvested 48 hr after transfection and luciferase activities were measured by a luminometer.

Figure 3

BCL-6 contains PEST sequences that are required for phosphorylation-induced degradation. (A) Schematic representation of HA-tagged BCL-6 proteins. PEST sequences were identified by the PEST-FIND program. PEST1: AA336–AA351 (KSDCQPNSPTESCSSK), score 9.4; PEST2: AA365–AA371(KSPTDPK), score 5.0; PEST3: AA406–AA430 (RAYTAPPACQPPMEPENLDLQSPTK), score 2.6. (B) 293T cells were transfected with 5 μg of pMT2T vectors expressing HA–BCL-6 (lanes 1–3), HA–BCL-6Δ (300–417) (lanes 4–6), or HA–BCL-6ZF (lanes 7–9) in the absence of MEK-2E (lanes 1,4,7) or in the presence of increasing amount (5, 10 μg) of MEK-2E (lanes 2,5,8,3,6,9). Forty-eight hours after transfection, equal amounts of cell lysates were analyzed (after normalization for transfection efficency based on β-galactosidase activity of cotransfected plasmids) by 8% SDS-PAGE and Western blot using anti-HA (12CA5) antibodies.

Figure 3

BCL-6 contains PEST sequences that are required for phosphorylation-induced degradation. (A) Schematic representation of HA-tagged BCL-6 proteins. PEST sequences were identified by the PEST-FIND program. PEST1: AA336–AA351 (KSDCQPNSPTESCSSK), score 9.4; PEST2: AA365–AA371(KSPTDPK), score 5.0; PEST3: AA406–AA430 (RAYTAPPACQPPMEPENLDLQSPTK), score 2.6. (B) 293T cells were transfected with 5 μg of pMT2T vectors expressing HA–BCL-6 (lanes 1–3), HA–BCL-6Δ (300–417) (lanes 4–6), or HA–BCL-6ZF (lanes 7–9) in the absence of MEK-2E (lanes 1,4,7) or in the presence of increasing amount (5, 10 μg) of MEK-2E (lanes 2,5,8,3,6,9). Forty-eight hours after transfection, equal amounts of cell lysates were analyzed (after normalization for transfection efficency based on β-galactosidase activity of cotransfected plasmids) by 8% SDS-PAGE and Western blot using anti-HA (12CA5) antibodies.

Figure 4

MAPK-induced BCL-6 degradation is mediated by the ubiquitin/proteasome pathway. (A) Western blot analysis of BCL-6 proteins in 293T cells transfected with BCL-6 in the absence or presence of cotransfected MEK-2E treated with 0.2% DMSO (lanes 1–3), 50 μm Calpain inhibitor II (lanes 4–6), or 50 μm MG132 (lanes 7–9) (added 8 hr after transfection). (B) 293T cells were transfected with BCL-6, His₆–Ub, and MEK-2E in the absence (lanes 1–4) or presence of MG132 (lanes 5–8). Cell lysates were immunoprecipitated with anti-BCL-6 antibodies (N-70-6) and the immunoprecipitants were analyzed by 6% SDS-PAGE followed by Western blot analysis using anti-ubiquitin antibodies.

Figure 4

MAPK-induced BCL-6 degradation is mediated by the ubiquitin/proteasome pathway. (A) Western blot analysis of BCL-6 proteins in 293T cells transfected with BCL-6 in the absence or presence of cotransfected MEK-2E treated with 0.2% DMSO (lanes 1–3), 50 μm Calpain inhibitor II (lanes 4–6), or 50 μm MG132 (lanes 7–9) (added 8 hr after transfection). (B) 293T cells were transfected with BCL-6, His₆–Ub, and MEK-2E in the absence (lanes 1–4) or presence of MG132 (lanes 5–8). Cell lysates were immunoprecipitated with anti-BCL-6 antibodies (N-70-6) and the immunoprecipitants were analyzed by 6% SDS-PAGE followed by Western blot analysis using anti-ubiquitin antibodies.

Figure 5

BCL-6 is phosphorylated and degraded by antigen-receptor signaling in B cells. Ramos cells (1 × 10⁶/ml) were treated with anti-IgM (10 μg/ml) and harvested at different time points after treatment as indicated. (A) (Top three panels) Equal amounts of cell extracts were used for Western blot analysis using anti-BCL-6 (top), or anti-ERK2 (middle) antibodies, and for solid-phase ERK2 kinase assays (MBP, bottom). Equal amounts of RNAs (10 μg) were used for Northern blot analysis with BCL-6 or GAPDH probes (bottom). (B) Hyperphosphorylated BCL-6 proteins are more unstable. Ramos cells were pulse labeled for 1 hr with [³⁵S]methionine and [³⁵S]cysteine, and then treated with anti-IgM (10 μg/ml) for 30 min and subsequently incubated in the presence of an excess of nonradioactive methionine and cysteine for the indicated times (chase). Cell extracts were immunoprecipitated with anti-BCL-6 antibodies and analyzed by SDS-PAGE followed by autoradiography. (C) Anti-IgM induced BCL-6 phosphorylation and degradation is prevented by a specific MEK inhibitor. Western blot analysis of BCL-6 in Ramos cells treated with anti-IgM in the presence of 0.2% DMSO or 50 μm PD098059 (added 30 min before anti-IgM treatment). (D) Anti-IgM induced BCL-6 degradation is prevented by a specific proteasome inhibitor. Western blot analysis of BCL-6 in Ramos cells treated with anti-IgM in the presence of 0.2% DMSO (lanes 2–4), 50 μm Calpain inhibitor II (lanes 5–7), and 50 μm MG132 (lanes 8–10) (added 1 hr before the treatment). (E) Mutant BCL-6 proteins are resistant to anti-IgM-induced degradation. Ramos cells stably transfected with pHeBo–MT–HA–BCL6, pHeBo–MT–HA–BCL-6_Ala333,343 and pHeBo–MT–HA–BCL6ZF were treated with 1 μm CdCl₂ for 6 hr to induce exogenous BCL-6 expression. Cells were then treated wth anti-IgM (10 μg/ml) and harvested at different time points as indicated. Equal amounts of cell extracts were loaded on 7% (HA–BCL–6 or HA–BCL–6_Ala333,343) or 10% SDS-PAGE (HA–BCL-6ZF) and the amount of exogenous BCL-6 proteins were analyzed by Western blot using anti-HA antibodies (12CA5).

Figure 5

BCL-6 is phosphorylated and degraded by antigen-receptor signaling in B cells. Ramos cells (1 × 10⁶/ml) were treated with anti-IgM (10 μg/ml) and harvested at different time points after treatment as indicated. (A) (Top three panels) Equal amounts of cell extracts were used for Western blot analysis using anti-BCL-6 (top), or anti-ERK2 (middle) antibodies, and for solid-phase ERK2 kinase assays (MBP, bottom). Equal amounts of RNAs (10 μg) were used for Northern blot analysis with BCL-6 or GAPDH probes (bottom). (B) Hyperphosphorylated BCL-6 proteins are more unstable. Ramos cells were pulse labeled for 1 hr with [³⁵S]methionine and [³⁵S]cysteine, and then treated with anti-IgM (10 μg/ml) for 30 min and subsequently incubated in the presence of an excess of nonradioactive methionine and cysteine for the indicated times (chase). Cell extracts were immunoprecipitated with anti-BCL-6 antibodies and analyzed by SDS-PAGE followed by autoradiography. (C) Anti-IgM induced BCL-6 phosphorylation and degradation is prevented by a specific MEK inhibitor. Western blot analysis of BCL-6 in Ramos cells treated with anti-IgM in the presence of 0.2% DMSO or 50 μm PD098059 (added 30 min before anti-IgM treatment). (D) Anti-IgM induced BCL-6 degradation is prevented by a specific proteasome inhibitor. Western blot analysis of BCL-6 in Ramos cells treated with anti-IgM in the presence of 0.2% DMSO (lanes 2–4), 50 μm Calpain inhibitor II (lanes 5–7), and 50 μm MG132 (lanes 8–10) (added 1 hr before the treatment). (E) Mutant BCL-6 proteins are resistant to anti-IgM-induced degradation. Ramos cells stably transfected with pHeBo–MT–HA–BCL6, pHeBo–MT–HA–BCL-6_Ala333,343 and pHeBo–MT–HA–BCL6ZF were treated with 1 μm CdCl₂ for 6 hr to induce exogenous BCL-6 expression. Cells were then treated wth anti-IgM (10 μg/ml) and harvested at different time points as indicated. Equal amounts of cell extracts were loaded on 7% (HA–BCL–6 or HA–BCL–6_Ala333,343) or 10% SDS-PAGE (HA–BCL-6ZF) and the amount of exogenous BCL-6 proteins were analyzed by Western blot using anti-HA antibodies (12CA5).

Figure 5

BCL-6 is phosphorylated and degraded by antigen-receptor signaling in B cells. Ramos cells (1 × 10⁶/ml) were treated with anti-IgM (10 μg/ml) and harvested at different time points after treatment as indicated. (A) (Top three panels) Equal amounts of cell extracts were used for Western blot analysis using anti-BCL-6 (top), or anti-ERK2 (middle) antibodies, and for solid-phase ERK2 kinase assays (MBP, bottom). Equal amounts of RNAs (10 μg) were used for Northern blot analysis with BCL-6 or GAPDH probes (bottom). (B) Hyperphosphorylated BCL-6 proteins are more unstable. Ramos cells were pulse labeled for 1 hr with [³⁵S]methionine and [³⁵S]cysteine, and then treated with anti-IgM (10 μg/ml) for 30 min and subsequently incubated in the presence of an excess of nonradioactive methionine and cysteine for the indicated times (chase). Cell extracts were immunoprecipitated with anti-BCL-6 antibodies and analyzed by SDS-PAGE followed by autoradiography. (C) Anti-IgM induced BCL-6 phosphorylation and degradation is prevented by a specific MEK inhibitor. Western blot analysis of BCL-6 in Ramos cells treated with anti-IgM in the presence of 0.2% DMSO or 50 μm PD098059 (added 30 min before anti-IgM treatment). (D) Anti-IgM induced BCL-6 degradation is prevented by a specific proteasome inhibitor. Western blot analysis of BCL-6 in Ramos cells treated with anti-IgM in the presence of 0.2% DMSO (lanes 2–4), 50 μm Calpain inhibitor II (lanes 5–7), and 50 μm MG132 (lanes 8–10) (added 1 hr before the treatment). (E) Mutant BCL-6 proteins are resistant to anti-IgM-induced degradation. Ramos cells stably transfected with pHeBo–MT–HA–BCL6, pHeBo–MT–HA–BCL-6_Ala333,343 and pHeBo–MT–HA–BCL6ZF were treated with 1 μm CdCl₂ for 6 hr to induce exogenous BCL-6 expression. Cells were then treated wth anti-IgM (10 μg/ml) and harvested at different time points as indicated. Equal amounts of cell extracts were loaded on 7% (HA–BCL–6 or HA–BCL–6_Ala333,343) or 10% SDS-PAGE (HA–BCL-6ZF) and the amount of exogenous BCL-6 proteins were analyzed by Western blot using anti-HA antibodies (12CA5).

Figure 5

BCL-6 is phosphorylated and degraded by antigen-receptor signaling in B cells. Ramos cells (1 × 10⁶/ml) were treated with anti-IgM (10 μg/ml) and harvested at different time points after treatment as indicated. (A) (Top three panels) Equal amounts of cell extracts were used for Western blot analysis using anti-BCL-6 (top), or anti-ERK2 (middle) antibodies, and for solid-phase ERK2 kinase assays (MBP, bottom). Equal amounts of RNAs (10 μg) were used for Northern blot analysis with BCL-6 or GAPDH probes (bottom). (B) Hyperphosphorylated BCL-6 proteins are more unstable. Ramos cells were pulse labeled for 1 hr with [³⁵S]methionine and [³⁵S]cysteine, and then treated with anti-IgM (10 μg/ml) for 30 min and subsequently incubated in the presence of an excess of nonradioactive methionine and cysteine for the indicated times (chase). Cell extracts were immunoprecipitated with anti-BCL-6 antibodies and analyzed by SDS-PAGE followed by autoradiography. (C) Anti-IgM induced BCL-6 phosphorylation and degradation is prevented by a specific MEK inhibitor. Western blot analysis of BCL-6 in Ramos cells treated with anti-IgM in the presence of 0.2% DMSO or 50 μm PD098059 (added 30 min before anti-IgM treatment). (D) Anti-IgM induced BCL-6 degradation is prevented by a specific proteasome inhibitor. Western blot analysis of BCL-6 in Ramos cells treated with anti-IgM in the presence of 0.2% DMSO (lanes 2–4), 50 μm Calpain inhibitor II (lanes 5–7), and 50 μm MG132 (lanes 8–10) (added 1 hr before the treatment). (E) Mutant BCL-6 proteins are resistant to anti-IgM-induced degradation. Ramos cells stably transfected with pHeBo–MT–HA–BCL6, pHeBo–MT–HA–BCL-6_Ala333,343 and pHeBo–MT–HA–BCL6ZF were treated with 1 μm CdCl₂ for 6 hr to induce exogenous BCL-6 expression. Cells were then treated wth anti-IgM (10 μg/ml) and harvested at different time points as indicated. Equal amounts of cell extracts were loaded on 7% (HA–BCL–6 or HA–BCL–6_Ala333,343) or 10% SDS-PAGE (HA–BCL-6ZF) and the amount of exogenous BCL-6 proteins were analyzed by Western blot using anti-HA antibodies (12CA5).

Figure 5

BCL-6 is phosphorylated and degraded by antigen-receptor signaling in B cells. Ramos cells (1 × 10⁶/ml) were treated with anti-IgM (10 μg/ml) and harvested at different time points after treatment as indicated. (A) (Top three panels) Equal amounts of cell extracts were used for Western blot analysis using anti-BCL-6 (top), or anti-ERK2 (middle) antibodies, and for solid-phase ERK2 kinase assays (MBP, bottom). Equal amounts of RNAs (10 μg) were used for Northern blot analysis with BCL-6 or GAPDH probes (bottom). (B) Hyperphosphorylated BCL-6 proteins are more unstable. Ramos cells were pulse labeled for 1 hr with [³⁵S]methionine and [³⁵S]cysteine, and then treated with anti-IgM (10 μg/ml) for 30 min and subsequently incubated in the presence of an excess of nonradioactive methionine and cysteine for the indicated times (chase). Cell extracts were immunoprecipitated with anti-BCL-6 antibodies and analyzed by SDS-PAGE followed by autoradiography. (C) Anti-IgM induced BCL-6 phosphorylation and degradation is prevented by a specific MEK inhibitor. Western blot analysis of BCL-6 in Ramos cells treated with anti-IgM in the presence of 0.2% DMSO or 50 μm PD098059 (added 30 min before anti-IgM treatment). (D) Anti-IgM induced BCL-6 degradation is prevented by a specific proteasome inhibitor. Western blot analysis of BCL-6 in Ramos cells treated with anti-IgM in the presence of 0.2% DMSO (lanes 2–4), 50 μm Calpain inhibitor II (lanes 5–7), and 50 μm MG132 (lanes 8–10) (added 1 hr before the treatment). (E) Mutant BCL-6 proteins are resistant to anti-IgM-induced degradation. Ramos cells stably transfected with pHeBo–MT–HA–BCL6, pHeBo–MT–HA–BCL-6_Ala333,343 and pHeBo–MT–HA–BCL6ZF were treated with 1 μm CdCl₂ for 6 hr to induce exogenous BCL-6 expression. Cells were then treated wth anti-IgM (10 μg/ml) and harvested at different time points as indicated. Equal amounts of cell extracts were loaded on 7% (HA–BCL–6 or HA–BCL–6_Ala333,343) or 10% SDS-PAGE (HA–BCL-6ZF) and the amount of exogenous BCL-6 proteins were analyzed by Western blot using anti-HA antibodies (12CA5).