Myc-enhanced expression of Cul1 promotes ubiquitin-dependent proteolysis and cell cycle progression - PubMed (original) (raw)

Myc-enhanced expression of Cul1 promotes ubiquitin-dependent proteolysis and cell cycle progression

R C O'Hagan et al. Genes Dev. 2000.

Abstract

The c-Myc oncoprotein plays an important role in the growth and proliferation of normal and neoplastic cells. To execute these actions, c-Myc is thought to regulate functionally diverse sets of genes that directly govern cellular mass and progression through critical cell cycle transitions. Here, we provide several lines of evidence that c-Myc promotes ubiquitin-dependent proteolysis by directly activating expression of the Cul1 gene, encoding a critical component of the ubiquitin ligase SCF(SKP2). The cell cycle inhibitor p27(kip1) is a known target of the SCF(SKP2) complex, and Myc-induced Cul1 expression matched well with the kinetics of declining p27(kip1) protein. Enforced Cul1 expression or antisense neutralization of p27(kip1) was capable of overcoming the slow-growth phenotype of c-Myc null primary mouse embryonic fibroblasts (MEFs). In reconstitution assays, the addition of in vitro translated Cul1 protein alone was able to restore p27(kip1) ubiquitination and degradation in lysates derived from c-myc(-/-) MEFs or density-arrested human fibroblasts. These functional and biochemical data provide a direct link between c-Myc transcriptional regulation and ubiquitin-mediated proteolysis and together support the view that c-Myc promotes G(1) exit in part via Cul1-dependent ubiquitination and degradation of the CDK inhibitor, p27(kip1).

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Figures

Figure 1

Figure 1

Cul1 is a direct transcriptional target of Myc and Mxi1. (A) Cul1 and Cul2 Northern blot of IMR90 Myc–ER cells untreated or treated with 1 μ

m

4-OHT. GAPDH serves as an internal loading control. In addition, no change in the expression of the SCFSKP2 complex components SKP2 or CDC34 was detected in genome-wide expression screens for c-MYC responsive gene targets or by Northern blot analysis (data not shown). (B) Cul1 immunoblot on IMR90 Myc–ER cells uninduced or treated with 1 μ

m

4-OHT. (C) Cul1 immunoblot on wild-type, or c-myc(−/−) MEFs. (D) Cul1 Northern blot of IMR90 cells expressing Mxi1–SR–ER or Mxi1–WR–ER after induction with 1 μ

m

4-OHT. GAPDH serves as an internal loading control. (E) Cul1 immunoblot on mxi1 (+/−), and mxi1(−/−) MEFs. (F) Schematic representation of the Cul1 promoter. The E-box, TATA-box and start sites for transcription (arrow) and translation (ATG) were determined by sequence analysis and are indicated. (G) Histogram representing the ability of Myc and Mxi1 to regulate the Cul1 promoter or a mutant in which the E-box CATGTG has been altered to CACTCA.Luciferase values were determined by luminometer and corrected for transfection efficiency by β-galactosidase assay. Values shown are the mean of three experiments plus and minus the standard deviation of the mean. Northern and immunoblot experiments were performed a minimum of two times with identical results.

Figure 2

Figure 2

Expression of Cul1 or inhibition of p27kip1 expression rescues proliferation in c-myc(−/−) MEFs. (A) Wild-type or c-myc(−/−) MEFS infected with retroviruses expressing c-Myc, Cul1, or antisense p27kip1 as indicated, were plated at 1 × 105 cells per well in replicate wells and cultured for the indicated number of days. Triplicate wells were trypsinized and counted. Values shown represent the mean of three experiments performed with independently derived MEF lines. (B) p27kip1 immunoblot on myc(−/−) MEFs infected with retrovirus expressing Myc, Cul1, or antisense p27kip1. Immunoblot for Max serves as a loading control. (C) Proliferation rate of wild-type and c-myc(−/−) MEFs infected with retroviruses expressing c-Myc, Cul1, or antisense p27kip1 as indicated. Proliferation was determined using a WST-1 cleavage assay (Boehringer Mannheim). Values shown represent the average of three independent experiments performed with independently derived MEF lines plus and minus the standard deviation of the mean. S-phase fractions were determined by FACS analysis of MEFs after staining with PI. Values shown represent the mean of three experiments with three independently derived MEF lines.

Figure 3

Figure 3

Myc enhances expression of Cul1 and stimulates degradation of p27kip1. (A) Immunoblot of Cul1 and p27kip1 expression on extracts from density arrested IMR90 MycER cells induced with 1 μ

m

4-OHT for the indicated times. Cul1 and p27kip1 panels were obtained from a single gel. Cell cycle profiles at each time point were determined as described in Materials and Methods. Blot illustrated is representative of three experiments. (B) p27kip1 protein was immunoprecipitated from density-arrested IMR90 cells containing MycER or empty vector that were metabolically labeled as described in Materials and Methods and then chased in the presence of 1 μ

m

4-OHT for the indicated times.

Figure 4

Figure 4

Cul1 is sufficient for Myc-dependent ubiquitination of p27kip1. (A) Ubiquitination of 35S-labeled p27kip1 was monitored in the presence of the indicated S100 cell extracts. Unlabeled Cul1 was added to the reactions in the lanes indicated. As a control, no purified ubiquitin was added to the reaction in lane 1. Following the in vitro ubiquitination reaction, p27kip1 was precipitated, resolved on SDS-PAGE, and detected by autoradiography. The three panels shown are from the same autoradiogram exposure of a single gel. Input p27kip1 is indicated by the arrow. (B) Ubiquitination of 35S-labeled p27kip1 in the presence of the indicated S100 extracts. K48R-ubiquitin was added where indicated to prevent polyubiquitination. Input p27kip1 is indicated by the arrow. (C) Ubiquitination of 35S-labeled p27kip1 in the presence of the indicated S100 extracts. Unlabeled Cul1 or Cul2 was added to the reactions in the lanes indicated. Input p27kip1 is indicated by the arrow. (D) Ubiquitination of 35S-labeled p53 in the presence of S100 extracts prepared from wild-type and c-myc(−/−) MEFs, and IMR90 MycER cells treated as indicated. Asterisk indicates input p53. Polyubiquitinated forms are indicated as (Ubq)n. All ubiquitination experiments were performed a minimum of three times with identical results.

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