Difference: UlrichHammerling (3 vs. 4)
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| Abstract Grant Number: 2R01CA089362-04A2 PI Name: HAMMERLING, ULRICH G. PI Email: u-hammerling@ski.mskcc.org PI Title: MEMBER Project Title: NOVEL RECEPTORS OF VITAMIN A IN THE CYTOPLASM Abstract: DESCRIPTION (provided by applicant): Our inquiries into the mechanism of the immunodeficiency caused by nutritional vitamin A depletion have uncovered an unprecedented role for retinoids and zinc in signal transduction. When activated by the second messenger-like reactive oxygen species, serine/threonine kinases required bound vitamin A as co-factor. Our objectives have progressed from the definition of the receptor sites on Protein Kinase C to inquiries on the biological significance, and to the function of the retinoid binding domains themselves. These coincide with zinc-coordinated structures, named zinc-fingers, that are prevalent in a multitude of signalling molecules. Despite their appearance as rigid structures we believe their true purpose is to serve as reversible hinges. This "linchpin" hypothesis, central to the proposal, is based on the findings that activation of PKC by reactive oxygen entails the release of Zn2+ ions. Unexpectedly, but with unifying logic, the classic 2nd messenger, diacylglycerol, and the tumor promoter, phorbol ester, also cause zinc mobilization. Evidence raised in vitro and in vivo suggest a causal relationship between zinc release, dissolution of the coordination center, and unfolding of PKC as a prelude for catalytic competence. Using a combination of structural biological, biochemical, genetic and imaging approaches we propose to investigate the role of zinc-fingers as hinge-like elements involved in the unfolding and activation of PKC. Four interrelated approaches will be applied to define: Structural changes of zinc-fingers by NMR (Aim 1), Covalent biochemical changes that lead to zinc-finger disassembly by Mass Spectrometry (Aim 2), Relationship of zinc-fingers to other domains of PKC by mutagenesis (Aim 3), Conformation change of zinc-finger hinge in vivo by FRET imaging. These studies will also uncover the mechanism underlying the 2nd messenger function of reactive oxygen. The prospects are high for a fundamental paradigm shift on the role of retinol and zinc ions in signal transduction and how zinc-fingers are to be viewed. Furthermore, our work on the function of vitamin A and zinc is of immediate relevance to nutrition, the immune system and cancer. Thesaurus Terms: cytoplasm, enzyme activity, protein kinase C, retinoid, retinoid binding protein biological signal transduction, cysteine, intermolecular interaction, oxidative stress, zinc nutrition related tag, tissue /cell culture Institution: SLOAN-KETTERING INSTITUTE FOR CANCER RES 1275 YORK AVE NEW YORK, NY 100216007 Fiscal Year: 2005 Department: Project Start: 01-APR-2001 Project End: 31-MAY-2009 ICD: NATIONAL CANCER INSTITUTE IRG: CMI | ||||||||
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| > > | Grant Number: 5R01DK069348-04 Project Title: New Pathway of Vitamin A Action. PI Information: Name Email Title HAMMERLING, ULRICH G. u-hammerling@ski.mskcc.org MEMBER Abstract: 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. Public Health Relevance: This Public Health Relevance is not available. Thesaurus Terms: binding site, biological signal transduction, oxidation reduction reaction, protein kinase C, retinoid, vitamin A deficiency cofactor, free radical oxygen, isozyme, phosphorylation, protein structure RNA interference, cell line, fluorescence microscopy, gel mobility shift assay, genetically modified animal, green fluorescent protein, immunoprecipitation, laboratory mouse, mass spectrometry, matrix assisted laser desorption ionization, site directed mutagenesis Institution: SLOAN-KETTERING INSTITUTE FOR CANCER RES 1275 YORK AVE NEW YORK, NY 10065 Fiscal Year: 2008 Department: Project Start: 15-SEP-2005 Project End: 30-JUN-2010 ICD: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES IRG: INMP | |||||||
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