Research objectives and content
This project is designed to investigate the events induced by the CaMKII phosphorylation cascade in order to activate the ubiquitin-dependent cyclin B degradation during meiosis. The project focus his attention to answer two main questions: 1) Is CaMKII involved in the formation of cyclin B-ubiquitin intermediates? 2) Does CaMKII phosphorylate the APC complex?
1) Role of CaMKII on the formation of cyclin B-ubiquitin complexes. Xenopus CSF extracts will be preincubated with 125I-labeled ubiquitin plus human cyclin B1 during 5 min. Subsequently, Ca2+ will be added to the extracts in order to activate cyclin degradation. The results will be checked by SDS-PAGE electrophoresis followed by autoradiography as a function of time elapsed from Ca2+ addition.
2) Role of CaMKII in the phosphorylation of subunits of the APC complex APC complex will be purified from Xenopus CSF extracts as previously described. Once purified, endogenous phosphorylation assays will be performed either in the presence of purified normal CaMKII enzyme or of constitutively active CaMKII (1-290) peptide together with 32P-yATP. The phosphorylated pattern will be visualized by SDS-PAGE electrophoresis and autoradiography. Localization of the different proteins of APC complex will be checked by coomassie blue staining and western blot assays with specific antibodies.
Training content (objective, benefit and expected impact)
During the last four years I have centred my interest on two main subjects, one of them related with cell cycle control and the other one with the study of nuclear calmodulin functions. Dr. Dorie was the first that described an important role of calmodulin in the cell cycle control, and moreover, his studies were developed in Xenopus oocytes, one of the most useful models to study cell cycle control. This two facts make me interest in the possibility to develop a postdoctoral stay in Dr. Dorie's laboratory. This stay would allow me by one hand to get fresh knowledge on Xenopus oocytes model and on the other hand to integrate the two fields which I have been involved with. Moreover, the results of the present project could considerably contribute to the understanding of cell cycle control either in physiologic or in the pathologic states as cancer. Links with industry/industrial relevance (22)