New Pathway of Vitamin A Action.

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DESCRIPTION (provided by applicant): Nutritional vitamin A depletion has been known for a century to cause a complex deficiency syndrome that affects multiple organs. Lack of retinoic acid (RA) leading to faulty transcription is the best studied mechanism. However, deficiencies in development, growth, reproduction and immunity were consequences of absence of vitamin A itself, since RA could not fully reverse these symptoms. We and others have described retinoid pathways that operate in the cytoplasm, independently of transcription. Further, we have identified serine/threonine kinases of the cRaf and PKC families as direct targets of vitamin A action. Vitamin A binds their regulatory domains and bound vitamin A sensitizes these kinases for redox-mediated activation. Redox activation refers to the alternative pathway operating through reactive oxygen species {ROS) as second messenger. The main objective is to prove the essential role that vitamin A plays in redox signaling, by establishing cause /effect relationships. Because ROS activates several kinases at once we propose to study the vitamin A dependence of two isoforms, alpha and theta, separately using a genetic approach. We will map the vitamin A binding site by scanning mutagenesis, already successful with cRaf (AIM #1). Using mutant PKCalpha and theta where retinolbinding sites are deleted, the biological function will be probed in vivo and in vitro with transgenic, as well as knock-in, cell lines and mice. IL-2 production and T cell proliferation are PKCtheta-dependent immunologically significant processes (AIM # 2). We hypothesize that ROS with help of vitamin A produces covalent modifications, leading to disassembly of the zinc-finger as prelude to kinase unfolding and activation. AIM # 3 is devoted to biochemical analyses of pertinent cysteine-modifications. Aim # 4 will focus on retinol as co-factor in mediating the release of zinc and conformation change in the zinc-finger consequent to redox activation, using intra-vital imaging. The results will establish a new mechanism of action of vitamin A, help understand vitamin A deficiency syndromes in reproduction, development and immunity, and open new approaches to prevention and treatment of cancer.