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. 2000 Apr;20(7):2423-35.
doi: 10.1128/MCB.20.7.2423-2435.2000.

c-Myc proteolysis by the ubiquitin-proteasome pathway: stabilization of c-Myc in Burkitt's lymphoma cells

M A Gregory  1 , S R Hann
Affiliations

c-Myc proteolysis by the ubiquitin-proteasome pathway: stabilization of c-Myc in Burkitt's lymphoma cells

M A Gregory et al. Mol Cell Biol. 2000 Apr.

Abstract

The c-Myc oncoprotein is a transcription factor which is a critical regulator of cellular proliferation. Deregulated expression of c-Myc is associated with many human cancers, including Burkitt's lymphoma. The c-Myc protein is normally degraded very rapidly with a half-life of 20 to 30 min. Here we demonstrate that proteolysis of c-Myc in vivo is mediated by the ubiquitin-proteasome pathway. Inhibition of proteasome activity blocks c-Myc degradation, and c-Myc is a substrate for ubiquitination in vivo. Furthermore, an increase in c-Myc stability occurs in mitotic cells and is associated with inhibited c-Myc ubiquitination. Deletion analysis was used to identify regions of the c-Myc protein which are required for rapid proteolysis. We found that a centrally located PEST sequence, amino acids 226 to 270, is necessary for rapid c-Myc degradation, but not for ubiquitination. Also, N-terminal sequences, located within the first 158 amino acids of c-Myc, are necessary for both efficient c-Myc ubiquitination and subsequent degradation. We found that c-Myc is significantly stabilized (two- to sixfold) in many Burkitt's lymphoma-derived cell lines, suggesting that aberrant c-Myc proteolysis may play a role in the pathogenesis of Burkitt's lymphoma. Finally, mutation of Thr-58, a major phosphorylation site in c-Myc and a mutational hot spot in Burkitt's lymphoma, increases c-Myc stability; however, mutation of c-Myc is not essential for stabilization in Burkitt's lymphoma cells.

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Figures

FIG. 1
FIG. 1
Inhibition of proteasome activity inhibits c-Myc turnover. (A) COS-7 cells were treated with either DMSO or ALLN (100 μM) for 2 h and pulse-chased in the presence of DMSO or ALLN. The cells were labeled with [35S]methionine/cysteine for 10 min (pulse) and incubated in unlabeled complete medium for the indicated times (chase). Cell lysates were prepared, equalized for total labeled protein, and c-Myc was immunoprecipitated with anti-MycFL, followed by analysis by SDS-PAGE and autoradiography. (B) COLO320 cells were treated with DMSO, ALLN (100 μM), ALLM (100 μM), E-64 (50 μM), or lactacystin (50 μM) for 2 h, pulse-chased in the presence of the respective compound, and analyzed as described for panel A. Lactacystin was not included in the chase medium because it is an irreversible inhibitor of the proteasome.
FIG. 2
FIG. 2
c-Myc is ubiquitinated in vivo. (A) Bk3A cells were left untreated or treated with ALLN (100 μM) for 2, 6, or 24 h as indicated. Cell lysates were prepared and analyzed by Western blotting using anti-av-Myc12C. (B) COS-7 cells were transiently transfected with expression plasmids encoding murine c-Myc (CMV–c-Myc2 [2 μg]) and HA-tagged ubiquitin (CMV-HA-Ub [4 μg]) as indicated. Forty-six hours later, cells were left untreated or treated with ALLN (100 μM) for 2 h as indicated. Cell lysates were subjected to immunoprecipitation (IP) with anti-av-Myc12C and subsequently analyzed by Western blotting with anti-HA antibody 12CA5 to detect c-Myc–HA–ubiquitin conjugates. The blot was stripped and reprobed with the monoclonal antibody C33 to detect c-Myc expression (lower panel). (C) COLO320 cells were transiently transfected with CMV-HA-Ub (4 μg) as indicated. Forty-six hours later, cells were left untreated or treated with ALLN (100 μM) for 2 h as indicated. Cell lysates were subjected to immunoprecipitation with anti-MycFL, followed by Western blot analysis with 12CA5 to detect c-Myc–HA–ubiquitin conjugates.
FIG. 3
FIG. 3
Deletion analysis of c-Myc protein turnover. COS-7 cells were transiently transfected with 2 μg of an expression plasmid encoding wild-type human (WT hu) c-Myc (pSV-myc) or c-Myc with the indicated deletion mutation. Forty-eight hours after transfection, cells were subjected to pulse-chase analysis as described in the legend to Fig. 1A, and c-Myc was immunoprecipitated with anti-MycFL.
FIG. 4
FIG. 4
Deletion of a PEST sequence stabilizes c-Myc. COS-7 cells were transiently transfected with 2 μg of an expression plasmid encoding wild-type (WT) murine c-Myc (CMV–c-Myc2) or c-Myc with the indicated deletion mutation. Forty-eight hours after transfection, the cells were subjected to pulse-chase analysis as described in the legend to Fig. 1A followed by immunoprecipitation of exogenous c-Myc proteins with anti-av-Myc12C. The data obtained from densitometric analysis of the experiment shown above were expressed as the relative percentage of the amount of c-Myc protein at the zero time point and plotted as a function of time.
FIG. 5
FIG. 5
PEST sequence of c-Myc is not required for ubiquitination. (A) COS-7 cells were transiently transfected with 4 μg of a plasmid encoding HA-tagged ubiquitin (CMV-HA-Ub) and 2 μg of a plasmid encoding wild-type murine c-Myc or the c-Myc PEST deletion mutant d226–270 as indicated. Forty-eight hours after transfection, cell lysates were prepared. Exogenous c-Myc proteins were immunoprecipitated with anti-av-Myc12C and subsequently analyzed by Western blotting using anti-HA antibody 12CA5 to detect c-Myc–HA–ubiquitin conjugates. (B) The blot shown in panel A was stripped and reprobed with anti-av-Myc12C to examine c-Myc expression.
FIG. 6
FIG. 6
Deletion of N-terminal sequences stabilizes c-Myc. (A) COS-7 cells were transiently transfected with 2 μg of an expression plasmid encoding wild-type (WT) murine c-Myc or the indicated c-Myc deletion mutant. Forty-eight hours after transfection, the cells were subjected to pulse-chase analysis as described in the legend to Fig. 1A, followed by immunoprecipitation of exogenous c-Myc proteins with anti-av-Myc12C. The arrow indicates a nonspecific background band. The data obtained from densitometric analysis of the experiment shown above were expressed as the relative percentage of the amount of c-Myc protein at the zero time point and plotted as a function of time. (B) COS-7 cells were transiently transfected with 2 μg of an expression plasmid encoding wild-type murine c-MycS or the indicated c-MycS deletion mutant. Pulse-chase analysis, immunoprecipitation of c-MycS, and densitometric plotting were carried out as described for panel A.
FIG. 7
FIG. 7
Deletion of N-terminal sequences inhibits c-Myc ubiquitination. (A) COS-7 cells were transiently transfected with 4 μg of a plasmid encoding HA-tagged ubiquitin (CMV-HA-Ub) and 2 μg of a plasmid encoding wild-type murine c-MycS or the indicated c-MycS deletion mutant. Forty-eight hours after transfection, cell lysates were prepared. Exogenous c-MycS proteins were immunoprecipitated with anti-av-Myc12C and subsequently analyzed by Western blotting with anti-HA antibody 12CA5 to detect c-MycS–HA–ubiquitin conjugates. (B) The blot shown in panel A was stripped and reprobed with anti-av-Myc12C to examine c-MycS expression.
FIG. 8
FIG. 8
Degradation and ubiquitination of c-Myc are inhibited in mitotic cells. (A) Bk3A cells were blocked in mitosis by treatment with nocodazole for 24 h. To examine the rate of c-Myc turnover, unsynchronized, or nocodazole-treated cells were treated with cycloheximide (chx) for the indicated amount of time, followed by Western blot analysis with anti-av-Myc12C to detect endogenous c-Myc. (B) Unsynchronized or mitosis-arrested Bk3A cells were treated the proteasome inhibitor ALLN (100 μM) for the indicated amount of time and analyzed by Western blotting with anti-av-Myc12C.
FIG. 9
FIG. 9
c-Myc is stabilized in Burkitt's lymphoma cell lines. EBV-immortalized B cells or the indicated Burkitt's lymphoma cell line were subjected to pulse-chase analysis as described in the legend to Fig. 1A, followed by immunoprecipitation of c-Myc with anti-MycFL. The half-lives were determined by a logarithmic analysis of the data obtained from densitometric scanning and the values shown are an average from two or more independent experiments. A representative pulse-chase experiment is shown.
FIG. 10
FIG. 10
Mutation of Thr-58 increases c-Myc stability. (A) NIH 3T3 cells were transiently transfected with 2 μg of a plasmid encoding wild-type (WT) murine c-Myc or c-Myc with a Thr-58-to-Ala mutation (T58A). Forty-eight hours after transfection, the cells were subjected to pulse-chase analysis as described in the legend to Fig. 1A followed by immunoprecipitation of exogenous c-Myc proteins with anti-av-Myc12C. The half-life values were determined by a logarithmic analysis of the data obtained from densitometric scanning and the values shown are an average from two independent experiments. (B) CA46 cells were stably transfected with a plasmid encoding wild-type murine c-Myc as described in Materials and Methods (CA46/mycWT). c-Myc expression in untransfected CA46 and CA46/mycWT was analyzed by immunoprecipitation (IP) with either anti-(α)-MycFL or anti-av-Myc12C. (C) CA46 or CA46/mycWT cells were subjected to pulse-chase analysis followed by immunoprecipitation with either anti-MycFL (for CA46) or anti-av-Myc12C (for CA46/mycWT). endog., endogenous. The half-life values were determined as described for panel A.
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