Abstract
(English)
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Part 1: A novel way of determining specifically the activity of c-Jun N-terminal kinases (JNKs) in whole cell extracts was established. Notably, the procedure is quantitative, does not require immunoprecipitation of the kinase and can be carried out with as little as 200 ng of total cell protein. The method consists of a colorimetric enzyme-linked immunosorbent assay (ELISA). It involves passive immobilization of the substrate GST-cJun on microtiter plate, selection of JNK protein kinases directly in substrate coated wells, kinase reaction, and detection of substrate phosphorylation by a phosphoepitope-specific antibody. The ability of this assay to selectively measure JNK activity relies on the high-affinity interaction between JNKs and c-Jun. Accordingly, we found that JNK protein kinases could be captured at the microtiter plate surface through binding to the immobilized GST-cJun. Moreover, JNKs retained the specificity of their interaction and phosphorylation of c-Jun with respect to the dependence on both intact docking domain and the dimerization state of c-Jun. This novel procedure represents a marked improvement on conventional radioactive assays in terms of sensitivity, accuracy of evaluation, low time consumption, high throughput, and amenability to automation. It is expected to be useful for the acceleration and facilitation of JNK activity measurement in cell extracts, in particular for large-scale screening of clinical samples.
Part 2: In a collaboration with the group of Dr. P. Jacquet, Mol, Belgium, we have determined the activities of Stress-Activated Protein Kinases (SAPKs) during the earliest phases of embryonic mouse development. The data are preliminary and require verification: The mouse oocytes, which are in cell cycle arrest, display little kinase activity. In meiosis, the kinases appear activated. Irradiation at 2.5 Gy seems to have no effect on the activation state of the kinases. Currently further assays are underway with the aim to reproduce the described data as well as to extend the data to later embryonic stages.
Part 3: We have established vascular endothelial cells in our laboratory as one of the somatic cell models to study radiation and growth factor effects. We have so far cloned and expressed canine vascular endothelial growth factors (VEGFs) and parts of their receptors and are comparing their biological activity with that of human VEGFs. Studies about the reactions of endothelial cells to ionising radiation are underway.
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