Screen And Functional Study Of Cyclin A-Cdk2 Substrates In Apoptosis Process

Apoptosis, or programmed cell death, plays a central role in the development and homeostasis of all multi-cellular organisms. Apoptosis defined exact process by gene regulation, and uncontrolled apoptosis will lead to a large number of difficulty miscellaneous diseases, such as cancers, autoimmune diseases, neurodegenerative diseases etc. Therefore, to investigate the signal transduction mechanism and to achieve effective control of apoptosis become the important research goal in modern Medicine and Pharmacy.Into the 1990s, apoptosis research has become a hot topic of the biological field. The mechanisms of apoptosis have been more understood at the present time, and there are two main apoptosis pathways summarized: First, through caspase-8 of the uptake of apoptosis surface activate pathway; the other one is the release of Cytochrome c and Samc from mitochondria. Cytochrome c and the cytoplasm Apaf-1, caspase-9 precursor, ATP / dATP formated apoptotic bodies, trigger caspases cascade, lead to cell death. Cell-surface receptor-mediated apoptosis need to be amplified through mitochondrial pathway in some cells where caspase-8 activity can not sufficiently trigger apoptisis. Therefore, mitochondrial is the control center for apoptosis. A series of results showed that the Bcl-2 family members regulating the mitochondrial Cytochrome c release directly or indirectly. However, how the complex cell apoptosis stimuli (such as DNA damage, the loss of growth factors, ROS, etc.) pass to the Bcl-2 family and mitochondria is still not entirely clear. Cyclin-dependent kinase (Cdks) is a serine / threonine protein kinase, play a central role in cell cycle. In the different cell cycle phases different Cdk is activated. Then phosphorylate their specific substrate to drive the cell cycle. However, recent studies show that a series of specific Cdks (Cdk2, Cdc2, and Cdk4) involved in the regulation of apoptosis. The regulation effect of Cdk2 in apoptosis has been commonly recognized. For example, etoposide-induced apoptosis in human leukemia cells, loss of growth factors caused apoptosis in HUVEC cells, UV irradiation induced apoptosis in mesangial cells, on these processes Cdk2 are specifically activated. And the enzyme activity is apoptosis required. In particular, scientists found that inhibition of Cdk2 activity specifically by small molecule inhibitors can effectively block chemotherapy-induced apoptosis of hair follicle epithelial cells in animal models.Even other research teams have found that the regulatory role of Cdk2 in apoptosiss. It is not yet been clarified that how activation of Cdk2 related to cell apoptosis pass the signal into the cell on the apoptosis pathway. Using different means (high-level expression of endogenous Cdk2 inhibitory protein p21CIP1, p27KIP1, a non-active point mutation Cdk2 (Cdk2-dn), or the small molecule inhibitors for Cdk2) to inhibit Cdk2 activity, the cells death were prevented. Therefore, we believe that the role of Cdk2 regulation on apoptosis may be achieved through phosphorylating certain substrate proteins; and these substrates are different from those substrates by enzyme-driven in cell cycle. There should be the Cyclin A-Cdk2 specific substrate in apoptosis. Discovery apoptotic Cdk2 substrate would be a key step for studying the mechanism of enzyme regulating apoptosis.In this paper we draw some conlusions as follows:1. We provide evidence for the first time, that Cyclin A-Cdk2 activity was upregulated and this event required for the progression of apoptosis of HeLa cells induced by treatment with Paclitaxel;2. Cyclin A-Cdk2 plays an important role in the mitochondrial membrane potential depolarization and leading to the release of Cytochrome c, we supposed Cyclin A-Cdk2 could affect the mitochondrial pathway to carry out regulation of apoptosis through regulation of mitochondrial membrane permeability; 3. We found a apoptotic Cyclin A-Cdk2 substrate, Bad. And indentified its phosphorylation site, serine 91;4. The phosphorylation of Bad enhances its pro-apoptotic activity after phosphorylation at serine 91 by Cyclin A-Cdk2.In this study we demonstrate the essential role of Cdk2 activity in paclitaxel-induced HeLa cell apoptosis process, and provide evidence for the possible signal transduction mechanism of Cdk2 in the apotosis regulation pathway. In addition, we found a new Cyclin A-Cdk2 substrate, Bad, and indentified its phosphorylation site. Through further functional study we propose that in some extent the regulation of cyclin A-Cdk2 in apoptosis process may be mediated by phosphorylting. These results may provide a new target for developing novel cancer reagents.