Function Of Cyclin A-Cdk2-dependent Rad9Phosphorylation In Apoptosis Progression

Apoptosis, also known as programmed cell death (PCD), is required for allmulti-cellular organisms to maintain homeostasis of their organ systems, and processcontrolled exact by gene. Recent studies show that uncontrolled apoptosis will lead toa large number of difficulty miscellaneous diseases, such as cancers, autoimmunediseases, neurodegenerative diseases etc. The inactivation of pro-apoptosis gene (suchas caspase, Bax, etc.) or the overexpression of anti-apoptosis gene (such as Bcl-2,Bcl-xL, etc.) are important reason to cause maligancy. Therefore, to clarify themechanism of signal transduction in tumor cell apoptosis, found apoptosis-relatedproteins, and effective apoptosis regulation could provide possible drug targets foranticancer drugs, the final effective treatment for cancer and other incurable diseasesof mankind.The process of apoptosis is regulated by a variety of cell signaling, according tothe signal source to the apoptosis pathway is divided into two categories: one isextrinsic pathway triggered by interactions between cytokines and death receptors,resulting inactivation of caspase-8; the other is intrinsic pathway can be initiated bymitochondrial membrane deporlarization that facilitates the release of cytochrome c,which is then associated with Apaf-1and caspase-9to promote caspase activation. Insome caspase-8mediated apoptosis，need the mitochondrial pathway to expandapoptosis signal, therefore mitochondrial is the control center for apoptosis.Cyclin-dependent kinases (Cdks) are a family of serine/threonine kinase, whichcould bind with cyclin form the activity cyclin-cdk complexes, and regulatesmammalian cell cycle progression by phosphorylating different substrates to promotecell mitosis. There is increasing evidence showed that cyclin A-Cdk2is not only thecell cycle regulatory proteins, but also plays an important role in apoptosis. Cyclin A-Cdk2activation has been observed selective up-regulated in the apoptosis ofginsenoside-Rh2(G-Rh2)-treated human hepatoma cells, panaxadiol-treatedSK-HEP-1cells, etoposide-treated HeLa cells, Transforming growthfactor-β1(TGF-β1)-treated human gastric cancer cells (SNU-16). Although thesestudies suggest an essential role of cyclin A-Cdk2in apoptosis, the precise molecularmechanism by which cyclin A-Cdk2regulates apoptotic pathways is still not clear.The BCL-2family members play a pivotal role in mitochondria-related apoptosis,according to the function in apoptosis Bcl-2family proteins are divided into twoclasses: the anti-apoptotic proteins (including Bcl-2, Bcl-xL, etc.) and pro-apoptoticproteins (such as Bax and Bak). Some of the pro-apoptotic proteins only have BH3domain called BH3-only protein, which could interact with anti-apoptotic proteins theability of the proteins to promote programmed cell death. Studies of apoptosis suggestthat following a death signal, the pro-apoptotic members that have been examined todate undergo a conformational change that enables them to active and target intomembranes, especially the mitochondrial outer membrane. Such as the pro-apoptoticmolecules Bax translocates to the mitochondria where it becomes an integralmembrane protein and cross-link-able as a homodimer, and then serve as obligatoryeffectors of cytochrome c release to induce apoptosis in response to diverse stimuli.Rad9is a checkpoint protein, can bind to Rad1and Hus1to form aheterotrimeric complex (the9-1-1complex), and it is believed to perform many of itssurveillance activities as part of this9-1-1heterotrimer, such as the maintenance ofgenome stability, the control of cell cycle checkpoints, the promotion of resistance toDNA damage. Later studies showed that the human Rad9contains a Bcl-2homology (BH-3)-like domain that is typical of BH3-only pro-apoptotic familymembers and that the overexpression of Rad9in a variety of human cell lines inducesapoptosis. Therefore, we hypothesized that the Rad9protein is a phosphorylationsubstrate of cyclin A-of Cdk2in apoptosis, then cyclin A-Cdk2participate in themitochondrial apoptotic pathway through the regulation of Rad9.In this study we draw some conclusions as follows:1. Rad9can be phosphorylated by cyclin A-Cdk2in vitro and in vivo, is a new phosphorylation substrate protein of cyclin A-Cdk2；2. Cyclin A-Cdk2phosphorylates Rad9at serine328during etoposide-inducedapoptosis in HeLa cells;3. Rad9translocates from nuclear to mitochondrial in etoposide induced HeLaapoptosis, and the phosphorylation of Rad9at serine328promotes thetranslocation;4. The up-regulation of cyclin A-Cdk2activity enhances Rad9-induced apoptosis byphosphorylating Rad9at serine328;5. The phosphorylation of Rad9at serine328is required for the interaction of Rad9with Bcl-xL.In this study we demonstrate that Rad9is a novel substrate of cyclin A-Cdk2andthat it is phosphorylated at serine328during etoposide-induced apoptosis in HeLacells. The phosphorylation of Rad9at serine328is important for Rad9’s functions,including its translocation from the nucleus to the mitochondria, its interaction withBcl-xL, and its consequent pro-apoptotic activity. Through this study we propose anew mechanism that the regulation of apoptosis process by cyclin A-Cdk2bemediated by phosphorylating Rad9at serine328. These results may clarify theapoptosis pathway and provide a new target for developing novel anticancer drugs.