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. ? ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA089362-07
Application #
7417904
Study Section
Cellular and Molecular Immunology - B (CMI)
Program Officer
Lees, Robert G
Project Start
2001-04-01
Project End
2010-05-31
Budget Start
2008-06-01
Budget End
2010-05-31
Support Year
7
Fiscal Year
2008
Total Cost
$334,697
Indirect Cost
Name
Sloan-Kettering Institute for Cancer Research
Department
Type
DUNS #
064931884
City
New York
State
NY
Country
United States
Zip Code
10065
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Acin-Perez, Rebeca; Hoyos, Beatrice; Zhao, Feng et al. (2010) Control of oxidative phosphorylation by vitamin A illuminates a fundamental role in mitochondrial energy homoeostasis. FASEB J 24:627-36
Chiu, Haw-Jyh; Fischman, Donald A; Hammerling, Ulrich (2008) Vitamin A depletion causes oxidative stress, mitochondrial dysfunction, and PARP-1-dependent energy deprivation. FASEB J 22:3878-87
Hoyos, Beatrice; Jiang, Sulin; Hammerling, Ulrich (2005) Location and functional significance of retinol-binding sites on the serine/threonine kinase, c-Raf. J Biol Chem 280:6872-8
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Korichneva, Irina; Hoyos, Beatrice; Chua, Ramon et al. (2002) Zinc release from protein kinase C as the common event during activation by lipid second messenger or reactive oxygen. J Biol Chem 277:44327-31
Hoyos, Beatrice; Imam, Asiya; Korichneva, Irina et al. (2002) Activation of c-Raf kinase by ultraviolet light. Regulation by retinoids. J Biol Chem 277:23949-57