par Pita, Jaime Miguel 
;Coulonval, Katia ;Roger, P. P. ;Raspé, Eric
Référence 27th Télévie’s Researchers Seminar (Brussels)
Publication Non publié, 2015-02-11
;Coulonval, Katia ;Roger, P. P. ;Raspé, Eric Référence 27th Télévie’s Researchers Seminar (Brussels)
Publication Non publié, 2015-02-11
Poster de conférence
| Résumé : | - Introduction :Eukaryotic cell cycle depends on the sequential formation and activation of different cyclin-dependent kinase (CDK) complexes. Cell cycle commitment at the G1 phase restriction point depends on inactivating phosphorylations of the central cell cycle/tumor suppressor pRb by CDK4/CDK6, whose activity requires its binding to D-type cyclins (CCND1-3). As the master integrator of G1 phase regulations, CDK4 is the first cell cycle CDK to be activated in response to mitogenic/oncogenic signalling cascades. In pRb-proficient cancers, CDK4 activation and activity might be the most crucial direct target of oncogenic perversions of signalling pathways. CDK4 inhibitory compounds are currently tested in a growing number of phase II/III clinical trials against various pRb-proficient chemotherapy-resistant cancers. Our group has identified the T172-phosphorylation of CDK4 as the determining step of its activation and has described critical regulation of this event in various cell systems, including human breast cancers (1,2). Moreover, our data indicates that kinase(s) different from the only known CDK-activating kinase (CAK; formed by the nuclear cyclin H-CDK7-Mat1 complex) may be responsible for T172-phosphorylation of CDK4 (3).- Aims :To develop an assay that allows the identification of CDK4-interacting proteins, including the activating kinase(s), and the screening of compounds altering the CDK4 activation.- Methods :We have developed a tetracycline inducible system for expression of CDK4 alone and D-type cyclins-CDK4 chimeras, which are fused to the AviTag, allowing for biotinylation by coexpressed BirA enzyme. Lentiviral transductions were performed in MCF7 breast cancer cell line and in colocarcinoma HCT116 cells engineered to express an analog-sensitive CDK7 (CDK7as/as), which can be selectively inhibited by bulky adenine analogs like 4-amino-1-tert-butyl-3-(1’-naphthylmethyl)pyrazolo[3,4-d]pyrimidine (1-NMPP1).- Results :Transgenes were specifically induced and biotinylated upon doxycycline and biotin addition. The biotin tag allowed the separation and purification of the recombinant CDK4 proteins by using streptavidin-coated beads. These purified proteins were able to phosphorylate a pRb fragment in vitro and were associated to p21 and p27 proteins, showing that immunoprecipitated fusions retained the kinase activity and interacting partners. T172A mutation of the CDK4 moiety demonstrated that this activity depended on CDK4 phosphorylation. Furthermore, we demonstrated that activity modulation was conserved. Purified fusions extracted from serum-deprived and -stimulated cells had decreased and increased kinase activity, respectively. Transposition of the assay to the DELFIA time-resolved fluorescence technology allowed to miniaturize the assay to the 96-well format.- Conclusions and perspectives : The developed system has been validated and is suitable for the identification of proteins interacting with the CDK4 complexes. It is also suitable to screen small molecules including potential cancer drugs and kinase siRNAs.- References : 1. Bockstaele, L., Kooken, H., Libert, F., Paternot, S., Dumont, J. E., de Launoit, Y., Roger, P. P., and Coulonval, K. (2006). Regulated activating Thr172 phosphorylation of cyclin-dependent kinase 4(CDK4): its relationship with cyclins and CDK "inhibitors". Mol.Cell Biol. 26, 5070-5085.2. Paternot, S., Bockstaele, L., Bisteau, X., Kooken, H., Coulonval, K., and Roger, P. P. (2010). Rb inactivation in cell cycle and cancer: The puzzle of highly regulated activating phosphorylation of CDK4 versus constitutively active CDK-activating kinase. Cell Cycle 9, 689-699.3. Bisteau X., Paternot S., Colleoni B., Ecker K., Coulonval K., De Groote P., Declercq W., Hengst L., Roger P. P. (2013). CDK4 T172 phosphorylation is central in a CDK7-dependent bidirectional CDK4/CDK2 interplay mediated by p21 phosphorylation at the restriction point. PLoS Genet. 9(5):e1003546. doi: 10.1371/journal.pgen.1003546. |
