Introduction The cell cycle is regulated by the activities of cyclin-dependent kinases (Cdks). In mammals, 11 different Cdks have been identified whereas in yeast only one major Cdk drives the cell cycle. Of the 11 mammalian Cdks, Cdk4 and Cdk6 promote entry and progression through G1, Cdk2 functions in entry and progression through S-phase, Cdc2 (Cdk1) regulates M-phase, and the other Cdks function in transcription or have unknown functions. The activities of Cdks are regulated by protein-protein interactions (with cyclins, inhibitors, and assembly factors), protein degradation, transcriptional control, subcellular localization, and multiple phosphorylations. Several functions for Cdk2 have been suggested, including entry into S-phase by Rb phosphorylation, initiation of DNA replication, exit from S-phase, and progression through G2 phase. We are investigating the functions of Cdk2 and other Cdks using an in vivo model system. - Aim 1: The in vivo functions of Cdk2. - Aim 2: Genetic and biochemical analysis of Cdk2 pathways. - Aim 3: Regulation of CDK6/KSHV-cyclin complexes.
Aim 1 : The in vivo functions of Cdk2. Cyclin-dependent kinases (Cdks) and their cyclin regulatory subunits control cell growth and division. Cdk2/cyclin E complexes are thought to be essential because they phosphorylate the retinoblastoma protein and drive cells through the G1/S transition into the S phase of the cell cycle. In addition, Cdk2 associates with cyclin A, which itself, is essential for cell proliferation during early embryonic development. In order to study the functions of Cdk2 in vivo, we generated Cdk2 knockout mice. Surprisingly, these mice are viable and therefore Cdk2 is not essential in the mouse. Cdk2, however, is essential for germ cell development since both male and female Cdk2-/- mice are sterile. Immunoprecipitates of cyclin E1 or A2 complexes from Cdk2-/- spleen extracts displayed no activity towards histone H1. Mouse embryonic fibroblasts from Cdk2-/- embryos entered delayed into S phase, indicating that Cdk2 function is important for cellular proliferation. In addition, spontaneous immortalization is delayed in Cdk2-/- MEFs suggesting a potential role for Cdk2 in progression of tumors. Our results demonstrate that Cdk2 is essential for meiosis but not mitosis in vivo. At this moment, we are following many leads from our Cdk2-/- work. Now that we have more Cdk2-/- mice available, we are studying a skin tumor model (TPA/DMAB), sterility in more detail, hearing loss, hematopoietic development, thymocyte development, and oligodendrocyte development. The Cdk2-/- MEFs are also being studied further and we are investigating the effect of MEK inhibitors (prevents cyclin D expression and therefore Cdk4 function), gamma irradiation, UV irradiation, and most notably the ablation of Cdc2 by siRNA. Many of these experiments could turn out to be very interesting.
Aim 2 : Genetic and biochemical analysis of Cdk2 pathways. Cdk2-/- mice did not display a sever phenotype. Therefore, we have to define the pathways in which Cdk2 is involved in more details. We are approaching these questions by mouse genetics and biochemistry. Double knockout mice of Cdk2 and Cdk4 or p27 are being generated at this moment. So far we have determined that Cdk2-/-p27-/- double mutants are viable and we are on the way to study these mice and MEFs derived from them. In contrast, we have not been able to generate Cdk2-/-Cdk4-/- mice, indicating that such double mutant mice die early during embryogenesis. Furthermore, we are also interested in the functions of Cdc2, since in the absence of Cdk2, Cdc2 might compensate for its functions. Therefore, we are generating conditional Cdc2 knockout mice and knocking-in Cdk2 into the Cdc2 locus. Currently, the targeting vectors are being generated and in the next months, we are expecting to finish them. These mice will be the basis for the biochemical analysis similar to the one that we are engaging for the analysis of the Cdk2-/- mice.
Aim 3 : Regulation of CDK6/KSHV-cyclin complexes. One step in the activation of Cdks is phosphorylation by the Cdk-activating kinase (CAK). We demonstrated the KSHV-cyclin could activate CDK6 in the absence of CAK phosphorylation in vitro and in vivo. A possible explanation is that CDK6 and/or KSHV-cyclin are phosphorylated at unknown sites. We have tried to identify such phosphorylation sites by sequencing and by mass spectrometry. Both methods have not yet produced to results we were looking for. In the mean time, we made mutations at potential phosphorylation sites. For KSHV-cyclin, we made more than 20 mutants and found in a first analysis that all of them are active in vitro. We are now analyzing these mutants in detail. Recently, we have focused on the functions of the KSHV-cyclin mutants when transfected into U2OS cells. We have obtained similar results than from the in vitro experiments. Using a GFP-CDK6 construct, we were able to determine the effect of the KSHV-cyclin mutants on the localization of CDK6. Some of the mutants of KSHV-cyclin mutants do not induce the intense nuclear staining of CDK6, which is the hallmark of wild-type KSHV-cyclin. We are following up on this and are also determining the phosphorylation state of the Retinoblastoma protein. These results should be published within the next year.
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| Kotoshiba, Shuhei; Gopinathan, Lakshmi; Pfeiffenberger, Elisabeth et al. (2014) p27 is regulated independently of Skp2 in the absence of Cdk2. Biochim Biophys Acta 1843:436-45 |
| Satyanarayana, Ande; Berthet, Cyril; Lopez-Molina, Javier et al. (2008) Genetic substitution of Cdk1 by Cdk2 leads to embryonic lethality and loss of meiotic function of Cdk2. Development 135:3389-400 |
| Berthet, Cyril; Rodriguez-Galan, Maria Cecilia; Hodge, Deborah L et al. (2007) Hematopoiesis and thymic apoptosis are not affected by the loss of Cdk2. Mol Cell Biol 27:5079-89 |
| Kaldis, Philipp (2007) Another piece of the p27Kip1 puzzle. Cell 128:241-4 |
| Rajareddy, Singareddy; Reddy, Pradeep; Du, Chun et al. (2007) p27kip1 (cyclin-dependent kinase inhibitor 1B) controls ovarian development by suppressing follicle endowment and activation and promoting follicle atresia in mice. Mol Endocrinol 21:2189-202 |
| Berthet, C; Kaldis, P (2007) Cell-specific responses to loss of cyclin-dependent kinases. Oncogene 26:4469-77 |
| Geng, Yan; Lee, Young-Mi; Welcker, Markus et al. (2007) Kinase-independent function of cyclin E. Mol Cell 25:127-39 |
| Mikule, Keith; Delaval, Benedicte; Kaldis, Philipp et al. (2007) Loss of centrosome integrity induces p38-p53-p21-dependent G1-S arrest. Nat Cell Biol 9:160-70 |
| Berthet, Cyril; Klarmann, Kimberly D; Hilton, Mary Beth et al. (2006) Combined loss of Cdk2 and Cdk4 results in embryonic lethality and Rb hypophosphorylation. Dev Cell 10:563-73 |
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