The Fbw7 tumor suppressor regulates glycogen synthase kinase 3 phosphorylation-dependent c-Myc protein degradation

Abstract

Myc proteins regulate cell growth and division and are implicated in a wide range of human cancers. We show here that Fbw7, a component of the SCF(Fbw7) ubiquitin ligase and a tumor suppressor, promotes proteasome-dependent c-Myc turnover in vivo and c-Myc ubiquitination in vitro. Phosphorylation of c-Myc on threonine-58 (T58) by glycogen synthase kinase 3 regulates the binding of Fbw7 to c-Myc as well as Fbw7-mediated c-Myc degradation and ubiquitination. T58 is the most frequent site of c-myc mutations in lymphoma cells, and our findings suggest that c-Myc activation is one of the key oncogenic consequences of Fbw7 loss in cancer. Because Fbw7 mediates the degradation of cyclin E, Notch, and c-Jun, as well as c-Myc, the loss of Fbw7 is likely to elicit profound effects on cell proliferation during tumorigenesis.

Fig. 1.

Fbw7 negatively regulates c-Myc abundance and function. (A) 293 cells were cotransfected with c-Myc (lanes 2-8) and FLAG-Fbw7 vectors as shown. Lysates were immunoblotted for c-Myc (C-33) or Fbw7 (FLAG); asterisk indicates a background band. (B) 293 cells were cotransfected with c-Myc, Max, and FLAG-Fbw7 as indicated. Lysates were blotted for c-Myc-N262 or Fbw7 (FLAG). (C) 293 cells were transfected as shown with c-Myc and Fbw7 and blotted for Fbw7 (FLAG) and c-Myc-N262 expression. MG indicates cells treated with MG-132 for2h(+) or untreated controls (-). (D) Pulse-chase analysis of 293 cells transfected with c-Myc and either Fbw7γ or empty vector (see Methods). Lane 1, untransfected cells. (E) Cells were cotransfected with c-Myc, a c-Myc reporter plasmid, and Fbw7γ as indicated. Transcriptional activation is shown (see text).

Fig. 3.

The regulation of c-Myc by Fbw7 requires both GSK-3 activity and c-Myc T58. (A) Sequence alignment of c-Myc T58 with cyclin E T380 and the Cdc4 phosphodegron (CPD) consensus. <KR> indicates where basic residues are unfavorable. (B) 293 cells were transfected as indicated, and blotted for c-Myc-N262 and Fbw7 (FLAG). (C) 293 cells were cotransfected with the indicated c-Myc constructs and either dnFbw7^WD or vector. Fbw7 and c-Myc abundance in anti-FLAG immunoprecipitates and total lysates is shown. (D) In vitro translated Fbw7 or Fbw6 were bound to an immobilized c-Myc peptide (residues 51-69) when unphosphorylated, or when T58, S62, or both are phosphorylated. (E) 293 cells were transfected with c-Myc (lanes 2-6), and Fbw7γ. Cells in lanes 4 and 5 were cotransfected with the axin GSK-interacting domain (GID), or an inactive GID mutant (GID^*). Cells in lane 6 were treated with LiCl.

Fig. 2.

The physical interaction of Fbw7 and c-Myc is sensitive to proteasomal function. (A) 293 cells were transfected as indicated; vec, vector alone. Upper two gels, c-Myc and F-box protein abundance in lysates. Lower two gels, c-Myc and F-box protein abundance in anti-F-box protein (anti-FLAG) immunoprecipitates (IP). MG-132 treatment is indicated. (B) HeLa cells were transduced with retroviruses expressing FLAG-Fbw7α or FLAG-Fbw7γ and treated with MG-132 as shown, and endogenous c-Myc and Fbw7 abundance in lysates and anti-Fbw7 (FLAG) immunoprecipitates is shown. Asterisk indicates a background band.

Fig. 4.

Endogenous c-Myc abundance is regulated by endogenous Fbw7. (A) U2OS cells, HFF, and HeLa cells were transduced with retroviral vectors encoding control (C) siRNA or two different Fbw7 siRNAs (U2OS and HFF cells, Fbw7-1 siRNA; HeLa cells, Fbw7-2 siRNA). Lysates were blotted for c-Myc, tubulin, GSK-3, Cdk2, and Hsp90. (B) Pulse-chase analysis of endogenous c-Myc stability in HeLa cells transduced with either si-Fbw7-2 or control retroviruses. (C) 293 cells were transfected with increasing amounts of dnFbw7^WD and the amount of endogenous c-Myc protein is shown. (D) 293 cells were transfected with vector or dnFbw7^WD and lysates were analyzed for total c-Myc (9E10) or pT58-c-Myc (p-Myc) protein after treatment with cycloheximide (CHX) for the indicated times (min).

Fig. 5.

Fbw7 catalyzes phosphorylation-dependent c-Myc ubiquitination in vitro.(A) c-Myc or c-MycT58A was translated in vitro by using reticulocyte extracts in the presence of GSK-3 and subjected to in vitro ubiquitination reactions (see text). Reticulocyte extract containing Fbw7 was added as indicated. (B) In vitro ubiquitination of c-Myc in reticulocyte-based extracts as described above except that GSK-3 and Erk were added as indicated, and all samples contained Fbw7. (C) c-Myc or c-MycT58A was translated in wheat germ extracts containing either Fbw2 or Fbw7 as indicated, and in vitro ubiquitination reactions were performed.