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Induction of p27 degradation by Myc through CyclinA/CDK1 complexes

AuthorsGarcía-Gutiérrez, Lucía ; Bretones, Gabriel ; Arechaga, Ignacio ; Santamaría, David; Barbacid, Mariano; León, Javier
KeywordsSymposia on Cell Proliferation and Genome Integrity
Issue Date2014
PublisherFundación Ramón Areces
CitationInternational Symposium Cell Proliferation and Genome Integrity (2014)
Abstractp27KIP1 (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 E-A/CDK2 and Cyclin D/CDK4-6 complexes. Upon mitogenic stimulation, expression of p27 is downregulated leading to cell cycle progression. The best characterized pathway leading to p27 downregulation involves phosphorylation at threonine 187, an event that targets p27 for SCFSKP2 mediated ubiquitination and degradation in proteasomes. The only kinase known so far to mediate this phosphorylation is the Cyclin E/CDK2 complex. Myc is known to antagonize the inhibitory effect of p27 on the cell cycle. Moreover, there is a correlation between high levels of Myc expression with low levels of p27 in many human tumors. However, the mechanisms by which Myc decreases p27 levels are not well understood. We have previously shown that Myc induces the expression of SKP2 as well as the phosphorylation of p27 at Thr187 (Bretones et al., J Biol Chem, 2011). To explore the mechanisms involved 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 (4HT). We first showed that induction of p27 in Kp27MER cell line inhibited less efficiently the kinase activity of CDK1 whereas it completely inhibited CDK2. Activation of Myc by 4HT leads to an increase of phospho-T187-p27 and the induction of Cyclin A. Also, Myc activation increases the in vitro kinase activity of immunocomplexes containing CDK1 and CDK2 Interestingly, CDK1 complexes from cells overexpressing p27 were able to phosphorylate p27 at T187 in vitro upon Myc activation, but CDK2 complexes were not. The result is consistent with the weak CDK1 inactivation exerted by p27 in these cells. Cyclin B/CDK1 has been reported to phosphorylate p27 in vitro, but the involvement of Cyclin A is unknown. As this cyclin is induced by Myc in our model, we asked for the role of Cyclin A/CDK1 in p27 phosphorylation. In vitro kinase assays performed with immunoprecipitated Cyclin A complexes showed the same p27 phosphorylation pattern as the observed with CDK1 complexes. Altogether these results suggest that Myc induction of p27-T187 phosphorylation can be carried out not only by Cyclin E/CDK2 as previously reported, but also by Cyclin A/CDK1. This phosphorylation of p27 is so far unreported. To confirm these results, we used three mouse embryonic fibroblast (MEF) derived cell lines lacking functional CDK genes: CDK2-/-, Cyclin E-/- and TKO (CDK2-/-; CDK4-/-; CDK6-/-). Overexpressing Myc-stable cell lines were generated for each cell line to study p27 phosphorylation. CDK1 and Cyclin A complexes from CDK2-/- MEFs subjected to in vitro kinase assays showed increased phospho-T187-p27 when Myc was overexpressed. Similarly, CDK1, CDK2 and Cyclin A complexes from Cyclin E-/- MEFs also showed increased phospho-T187-p27 when Myc was overexpressed. CDK1 complexes from TKO MEFs were unable to phosphorylate p27 in vitro while overexpression of Myc induced this phosphorylation. Purvalanol A (a CDK1 inhibitor) abolished the p27 phosphorylation in vitro but not CDK9 inhibitors. Consistent with the in vitro data, extracts from CDK2-/- and TKO MEFs showed higher levels of phospho-T187-p27 levels when Myc was activated and silencing of Myc in Myc-TKO MEFs decreased phospho-T187-p27, dismissing CDK2, CDK4 or CDK6 as responsible for this phosphorylation in this model. In conclusion, Myc promotes p27 degradation by inducing its phosphorylation at the Thr187, which is mediated not only by Cyclin E/CDK2 complexes, but also by Cyclin A/CDK1 complexes.
DescriptionResumen del póster presentado al International Symposium Cell Proliferation and Genome Integrity, celebrado en Santander (España) del 3 al 4 de abril de 2014.
Appears in Collections:(IBBTEC) Comunicaciones congresos
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