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Mnt-Max to Myc-Max complex switching regulates cell cycle entry.

Walker W, Zhou ZQ, Ota S, Wynshaw-Boris A, Hurlin PJ - J. Cell Biol. (2005)

Bottom Line: Here, we show that c-Myc induction during cell cycle entry leads to a transient decrease in Mnt-Max complexes and a transient switch in the ratio of Mnt-Max to c-Myc-Max on shared target genes.Mnt overexpression suppressed cell cycle entry and cell proliferation, suggesting that the ratio of Mnt-Max to c-Myc-Max is critical for cell cycle entry.These results demonstrate that Mnt-Myc antagonism plays a fundamental role in regulating cell cycle entry and proliferation.

View Article: PubMed Central - PubMed

Affiliation: Shriners Hospitals for Children, Portland, OR 97201, USA.

ABSTRACT
The c-Myc oncoprotein is strongly induced during the G0 to S-phase transition and is an important regulator of cell cycle entry. In contrast to c-Myc, the putative Myc antagonist Mnt is maintained at a constant level during cell cycle entry. Mnt and Myc require interaction with Max for specific DNA binding at E-box sites, but have opposing transcriptional activities. Here, we show that c-Myc induction during cell cycle entry leads to a transient decrease in Mnt-Max complexes and a transient switch in the ratio of Mnt-Max to c-Myc-Max on shared target genes. Mnt overexpression suppressed cell cycle entry and cell proliferation, suggesting that the ratio of Mnt-Max to c-Myc-Max is critical for cell cycle entry. Furthermore, simultaneous Cre-Lox mediated deletion of Mnt and c-Myc in mouse embryo fibroblasts rescued the cell cycle entry and proliferative block caused by c-Myc ablation alone. These results demonstrate that Mnt-Myc antagonism plays a fundamental role in regulating cell cycle entry and proliferation.

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Complex switching between Mnt–Max and c-Myc–Max during cell cycle entry. (a) Western blot showing Mnt and c-Myc levels at 0, 4, 8, and 24 h after serum stimulation of quiescent MEFs. (b) Levels of Mnt and c-Myc found in low stringency anti-Max immunoprecipitations during cell cycle entry. Note reduction in Mnt (and Mnt–Max complexes) at times of high c-Myc levels. (c) Pulse-chase analysis of Mnt turnover when complexed to Max. Cells were metabolically labeled with medium containing [35S]methionine (pulse) then “chased” with medium containing unlabeled methionine. Mnt was immunoprecipitated under high stringency conditions (representing total Mnt) or from low stringency Max immunoprecipitated material at 0, 1, 2, and 4 h, as indicated, during the chase period. B, immunogen blocked antibody.
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fig1: Complex switching between Mnt–Max and c-Myc–Max during cell cycle entry. (a) Western blot showing Mnt and c-Myc levels at 0, 4, 8, and 24 h after serum stimulation of quiescent MEFs. (b) Levels of Mnt and c-Myc found in low stringency anti-Max immunoprecipitations during cell cycle entry. Note reduction in Mnt (and Mnt–Max complexes) at times of high c-Myc levels. (c) Pulse-chase analysis of Mnt turnover when complexed to Max. Cells were metabolically labeled with medium containing [35S]methionine (pulse) then “chased” with medium containing unlabeled methionine. Mnt was immunoprecipitated under high stringency conditions (representing total Mnt) or from low stringency Max immunoprecipitated material at 0, 1, 2, and 4 h, as indicated, during the chase period. B, immunogen blocked antibody.

Mentions: The accelerated G0 to S-phase transition exhibited by Mnt MEFs (Hurlin et al., 2003) suggested that the balance between Mnt–Max and c-Myc–Max complexes may regulate proper cell cycle entry. Therefore we examined the relationship between Mnt and c-Myc steady-state levels and complex formation with Max during cell cycle entry of primary human fibroblasts. Fibroblasts were driven into quiescence by a combination of confluence arrest and serum starvation, then stimulated to enter the cell cycle by the addition of 10% FCS. Tritiated thymidine incorporation was used to confirm entry into S-phase (unpublished data). Consistent with our previous results using primary MEFs, Mnt levels were maintained at a near constant level during cell cycle entry (Fig. 1 a). As expected, c-Myc levels were initially present at extremely low levels in quiescent cells and rapidly, but transiently, induced (Fig. 1 a). To examine Mnt–Max and c-Myc–Max complex formation during cell cycle entry, low stringency anti-Max immunoprecipitations were performed. Low stringency anti-Max immunoprecipitations were treated with high stringency buffer to elute bound proteins and secondary immunoprecipitations were performed on the eluates with anti–c-Myc and Mnt antibodies (Hurlin et al., 1997a). Whereas Max remained at near constant levels during cell cycle entry, its interaction with Mnt and c-Myc fluctuated as a function of c-Myc levels (Fig. 1 b). Although c-Myc and c-Myc–Max levels increased from a very low level in unstimulated cells to high levels at 4 h, a corresponding drop in Mnt–Max levels occurred, despite near constant Mnt levels (Fig. 1 a). As c-Myc and c-Myc–Max levels subsequently declined at 8 and 24 h after serum stimulation, levels of Mnt–Max increased (Fig. 1 b). These results suggest that induction of c-Myc to high levels during the G0 to S-phase transition sets up a competition between c-Myc and Mnt for Max, that results in a limited supply of Max.


Mnt-Max to Myc-Max complex switching regulates cell cycle entry.

Walker W, Zhou ZQ, Ota S, Wynshaw-Boris A, Hurlin PJ - J. Cell Biol. (2005)

Complex switching between Mnt–Max and c-Myc–Max during cell cycle entry. (a) Western blot showing Mnt and c-Myc levels at 0, 4, 8, and 24 h after serum stimulation of quiescent MEFs. (b) Levels of Mnt and c-Myc found in low stringency anti-Max immunoprecipitations during cell cycle entry. Note reduction in Mnt (and Mnt–Max complexes) at times of high c-Myc levels. (c) Pulse-chase analysis of Mnt turnover when complexed to Max. Cells were metabolically labeled with medium containing [35S]methionine (pulse) then “chased” with medium containing unlabeled methionine. Mnt was immunoprecipitated under high stringency conditions (representing total Mnt) or from low stringency Max immunoprecipitated material at 0, 1, 2, and 4 h, as indicated, during the chase period. B, immunogen blocked antibody.
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Related In: Results  -  Collection

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fig1: Complex switching between Mnt–Max and c-Myc–Max during cell cycle entry. (a) Western blot showing Mnt and c-Myc levels at 0, 4, 8, and 24 h after serum stimulation of quiescent MEFs. (b) Levels of Mnt and c-Myc found in low stringency anti-Max immunoprecipitations during cell cycle entry. Note reduction in Mnt (and Mnt–Max complexes) at times of high c-Myc levels. (c) Pulse-chase analysis of Mnt turnover when complexed to Max. Cells were metabolically labeled with medium containing [35S]methionine (pulse) then “chased” with medium containing unlabeled methionine. Mnt was immunoprecipitated under high stringency conditions (representing total Mnt) or from low stringency Max immunoprecipitated material at 0, 1, 2, and 4 h, as indicated, during the chase period. B, immunogen blocked antibody.
Mentions: The accelerated G0 to S-phase transition exhibited by Mnt MEFs (Hurlin et al., 2003) suggested that the balance between Mnt–Max and c-Myc–Max complexes may regulate proper cell cycle entry. Therefore we examined the relationship between Mnt and c-Myc steady-state levels and complex formation with Max during cell cycle entry of primary human fibroblasts. Fibroblasts were driven into quiescence by a combination of confluence arrest and serum starvation, then stimulated to enter the cell cycle by the addition of 10% FCS. Tritiated thymidine incorporation was used to confirm entry into S-phase (unpublished data). Consistent with our previous results using primary MEFs, Mnt levels were maintained at a near constant level during cell cycle entry (Fig. 1 a). As expected, c-Myc levels were initially present at extremely low levels in quiescent cells and rapidly, but transiently, induced (Fig. 1 a). To examine Mnt–Max and c-Myc–Max complex formation during cell cycle entry, low stringency anti-Max immunoprecipitations were performed. Low stringency anti-Max immunoprecipitations were treated with high stringency buffer to elute bound proteins and secondary immunoprecipitations were performed on the eluates with anti–c-Myc and Mnt antibodies (Hurlin et al., 1997a). Whereas Max remained at near constant levels during cell cycle entry, its interaction with Mnt and c-Myc fluctuated as a function of c-Myc levels (Fig. 1 b). Although c-Myc and c-Myc–Max levels increased from a very low level in unstimulated cells to high levels at 4 h, a corresponding drop in Mnt–Max levels occurred, despite near constant Mnt levels (Fig. 1 a). As c-Myc and c-Myc–Max levels subsequently declined at 8 and 24 h after serum stimulation, levels of Mnt–Max increased (Fig. 1 b). These results suggest that induction of c-Myc to high levels during the G0 to S-phase transition sets up a competition between c-Myc and Mnt for Max, that results in a limited supply of Max.

Bottom Line: Here, we show that c-Myc induction during cell cycle entry leads to a transient decrease in Mnt-Max complexes and a transient switch in the ratio of Mnt-Max to c-Myc-Max on shared target genes.Mnt overexpression suppressed cell cycle entry and cell proliferation, suggesting that the ratio of Mnt-Max to c-Myc-Max is critical for cell cycle entry.These results demonstrate that Mnt-Myc antagonism plays a fundamental role in regulating cell cycle entry and proliferation.

View Article: PubMed Central - PubMed

Affiliation: Shriners Hospitals for Children, Portland, OR 97201, USA.

ABSTRACT
The c-Myc oncoprotein is strongly induced during the G0 to S-phase transition and is an important regulator of cell cycle entry. In contrast to c-Myc, the putative Myc antagonist Mnt is maintained at a constant level during cell cycle entry. Mnt and Myc require interaction with Max for specific DNA binding at E-box sites, but have opposing transcriptional activities. Here, we show that c-Myc induction during cell cycle entry leads to a transient decrease in Mnt-Max complexes and a transient switch in the ratio of Mnt-Max to c-Myc-Max on shared target genes. Mnt overexpression suppressed cell cycle entry and cell proliferation, suggesting that the ratio of Mnt-Max to c-Myc-Max is critical for cell cycle entry. Furthermore, simultaneous Cre-Lox mediated deletion of Mnt and c-Myc in mouse embryo fibroblasts rescued the cell cycle entry and proliferative block caused by c-Myc ablation alone. These results demonstrate that Mnt-Myc antagonism plays a fundamental role in regulating cell cycle entry and proliferation.

Show MeSH
Related in: MedlinePlus