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Myc mediates the phosphorylation and degradation of p27 through activation of Cyclin A/CDK1

AutorGarcía-Gutiérrez, Lucía ; Bretones, Gabriel ; Arechaga, Ignacio ; Santamaría, David; Barbacid, Mariano; León, Javier
Fecha de publicación2014
Citación23 EACR (2014)
Resumen[Introduction]: p27KIP1 (p27 herein after), member of the KIP/CIP family of CDK inhibitors, accumulates in the nucleus of quiescent cells provoking cell cycle arrest at the G1 phase by inactivating Cyclin/CDK complexes. The best characterized pathway leading to p27 downregulation involves phosphorylation at threonine 187 targeting p27 for SCFSKP2-mediated ubiquitination and degradation. The only kinase known so far to mediate this phosphorylation is the Cyclin E/CDK2 complex. There is a correlation between high levels of Myc expression with low levels of p27 in many human tumors. We have previously shown that Myc induces the expression of SKP2 as well as the T187p27. [Material and Method]: We used the Kp27MER cell line, a K562 derivative cell line carrying a ZnSO4-inducible p27 construct and the chimerical MycER protein which can be activated by 4-hydroxy-tamoxifen, and three mouse embryonic fibroblast derived cell lines lacking functional CDK genes: CDK2−/−, Cyclin E−/− and TKO (CDK2−/−; CDK4−/−; CDK6−/−). Overexpressing Mycstable cell lines generated for the three mouse derived cell lines by lentiviral transduction. RT-qPCR and WB to study gene expression at mRNA and protein levels. In vitro kinase assays to study pT187p27 and phosphorylation detected by WB using phospho-specific antibodies. Purvalanol A and CAN508 (CDK1 and CDK9 inhibitors respectively) were tried in vitro to study the specificity of CDK1 kinase activity over pT187p27. [Results and Discussion]: Induction of p27 in Kp27MER cell line inhibits less efficiently the kinase activity of CDK1 whereas it completely inhibits CDK2. Myc activation leads to an increase of pT187p27 and Cyclin A induction. Besides, it increases the in vitro kinase activity of CDK1 and CDK2. Interestingly, CDK1 complexes from cells overexpressing p27 were able to phosphorylate p27 in vitro upon Myc activation, but CDK2 complexes were not. Cyclin B/CDK1 was reported to phosphorylate p27 in vitro, but the involvementof Cyclin A is unknown. As cyclin A is induced by Myc in our model, we asked for the role of Cyclin A/CDK1 in p27 phosphorylation. In vitro kinase assays with immunoprecipitated Cyclin A complexes showed the same p27 phosphorylation pattern as the observed with CDK1 complexes. CDK1 and Cyclin A complexes from CDK2−/− MEFs showed increased pT187p27 levels when Myc was overexpressed. Similarly, CDK1, CDK2 and Cyclin A complexes from Cyclin E−/− MEFs showed increased pT187p27 when Myc was overexpressed. CDK1 complexes from TKO MEFs were unable to phosphorylate p27 in vitro while overexpression of Myc induced it. Purvalanol A abolished pT187p27 but not CDK9 inhibitors. Consistent with the in vitro data, extracts from CDK2−/− and TKO MEFs showed higher levels of pT187p27 levels when Myc was activated. [Conclusion]: Myc promotes p27 degradation by inducing its phosphorylation at Thr187, which is mediated not only by Cyclin E/CDK2, but also by Cyclin A/CDK1.
DescripciónResumen del póster presentado en el 23rd Biennial Congress of the European Association for Cancer Research, celebrado del 5 al 8 de julio de 2014 en Munich (Alemania). Abstract publicado en: European Journal of Cancer 50(Suppl. 5): S72 (2014). ISSN: 0959-8049 DOI: 10.1016/S0959-8049(14)50270-5
URIhttp://hdl.handle.net/10261/130685
Aparece en las colecciones: (IBBTEC) Comunicaciones congresos
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