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Max activity is affected by phosphorylation at two NH2-terminal sites
P J Koskinen1, I Västrik, T P Mäkelä
1Department of Virology, University of Helsinki, Finland.
Abstract:
Max is a nuclear phosphoprotein that has a dose-dependent role in regulation of Myc function. The DNA-binding activity of Max homodimers, but not of Myc/Max heterodimers, has been reported to be inhibited by NH2-terminal phosphorylation. (S. J. Berberich and M. D. Cole, Genes & Dev., 6: 166-176, 1992). Here, we have mapped the NH2-terminal in vivo phosphorylation sites of Max to Ser2 and Ser11 and show that the NH2 termini of the two major alternatively spliced forms of Max (p21max and p22max) are equally phosphorylated despite differences in their amino acid sequences following Ser11. A Max mutant deficient in the NH2-terminal phosphorylation was found to inhibit both basal and Myc-induced transcription of a reporter gene more efficiently than the wild-type protein. Similarly, the ability of Myc and Ras to induce transformation was more severely impaired by the mutant. These results indicate that the NH2-terminal phosphorylation diminishes the ability of Max to negatively interfere with Myc function. However, we found no evidence that Max phosphorylation would be regulated during cell growth or differentiation. Similarly, we observed no major cell cycle-dependent changes in the extent of phosphorylation between cell populations fractionated by centrifugal elutriation or by cell cycle inhibitors.
Insights
Max protein
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Max is a nuclear phosphoprotein regulating Myc function.
- NH2-terminal phosphorylation of Max inhibits DNA-binding activity of Max homodimers.
- Two major alternatively spliced forms of Max exist: p21max and p22max.
Purpose of the Study:
- To map in vivo phosphorylation sites of Max.
- To investigate the functional consequences of Max NH2-terminal phosphorylation on Myc function and cellular transformation.
- To determine if Max phosphorylation is regulated during cell growth, differentiation, or cell cycle.
Main Methods:
- In vivo phosphorylation site mapping using mutant analysis.
- Reporter gene assays to assess transcriptional regulation.
- Transformation assays with Myc and Ras in the presence of wild-type and mutant Max.
Main Results:
- Max NH2-terminal phosphorylation sites mapped to Ser2 and Ser11.
- NH2 termini of p21max and p22max are equally phosphorylated.
- A Max mutant deficient in NH2-terminal phosphorylation inhibited transcription and Myc/Ras-induced transformation more effectively than wild-type Max.
- Max NH2-terminal phosphorylation diminishes its ability to negatively regulate Myc function.
- No evidence of regulation of Max phosphorylation during cell growth, differentiation, or cell cycle.
Conclusions:
- NH2-terminal phosphorylation of Max plays a crucial role in modulating its interaction with Myc and its function as a transcriptional regulator.
- This phosphorylation event appears to reduce Max's inhibitory effect on Myc-driven processes.
- Max phosphorylation is not significantly regulated by cell growth, differentiation, or cell cycle status.