The response of eukaryotic cells to ionizing radiation (IR) includes cell cycle arrest, activation of DNA repair mechanisms and, under certain conditions, lethality. The IR induced signaling cascades responsible for the regulation of these events, however, remain unclear. Our previous work has focused on the transcriptional activation of early response genes, particularly c-jun and EGR-1, in irradiated cells. Analysis of the cis- acting elements that are activated by IR exposure has demonstrated involvement of the Jun/AP-1 site in the c-jun/AP-1 site in the c-jun promoter and CArG elements in the EGR-1 promoter. These findings provided the basis for demonstrating IR-induced activation of the cytoplasmic MAP and pp90rsk serine/threonine protein kinases which have been shown to act as upstream signals in the induction of these early response genes. The proposed work will extend our previous and ongoing efforts by studying involvement of the Src-like protein tyrosine kinase (PTK) p56/p53lyn in IR- induced signaling events. Our preliminary findings support activation of Lyn in the nucleus of irradiated cells. The association of nuclear lyn with c-Abl or a c-Abl related kinase and a protein tyrosine phosphatase (PTP) will be studied as a possible mechanism for IR-induced Lyn activation. The finding that Lyn binds to the cell cycle regulatory protein p34cdc2 following IR treatment will also be studied as an event responsible for regulating the DNA-damage-dependent premitotic checkpoint. cAbl is an upstream effector of the stress activated protein (SAP) kinases which phosphorylate c-Jun and induce c-jun gene transcription. Since the preliminary data indicate that IR also stimulates both c-Abl and SAP kinase activity, our hypothesis is that IR-induced activation of c-Abl is responsible at least in part for our previous finding that c-jun expression is induced in irradiated cells. These issues will be addressed in the proposed work.
The Specific Aims are: 1) To study the involvement of the protein tyrosine kinase Lyn in IR- induced signaling. 2) To identify mechanisms responsible for activation of nuclear Lyn in irradiated cells. 3) To study functional interactions between Lyn and Cdc2 in irradiated cells. 4) To define IR-induced nuclear signals responsible for activation of c- jun transcription.
