Two apical membrane proteins in intestinal epithelial cells are important effectors of cAMP-mediated signaling that result in secretory diarrheas: NHE-3 and CFTR. Both are attached via PDZ interactions to NHERF proteins, which, in turn, are attached to ezrin and actin. The integrity of this scaffold is essential for PKA/cAMP signaling on CFTR and may play a role as an apical retention signal for polarization. Previous published work in our lab has highlighted the importance of intermediate filaments (IPs) in the organization of the apical domain in simple epithelial cells, and suggest that IPs play a role in the organization of the apical actin-based scaffold in these cells. Furthermore, IPs mediate the assembly of ezrin into the above-mentioned scaffold, and PKCiota or alpha may be responsible for the activation of ezrin. The hypothesis is that binding of ezrin to apical intermediate filaments initiates the apical localization of ezrin in intestinal cells and provides a microdomain-restricted site for its activation by PKCi. The assembly of this ezrin-based scaffold is essential for the function of the effectors of secretory diarrheas (NHE-3 and CFTR). To test this hypothesis I propose to: 1) Identify the molecular interactions between ezrin and keratins and their role to localize ezrin to the apical domain of enterocytes, using T567D / T567A ezrin mutants in vitro binding assays, or GFP-ezrin constructs in vivo. 2) Identify the physiological activator of ezrin in the brushborder and the molecular interactions between PKCi or a and keratins, using recombinant PKCisoforms, expression of anti- PKCi shRNA, dominant negative PKCisoforms, pharmacological blockers, stable TET-inducible CACO-2 cell lines that knock down keratin 8, and K8 null mice. And 3) Test the functional consequences of apical ezrin complex assembly on cAMP-dependent CI""""""""secretion, PKA localization and activation, signaling downstream of ezrin and CFTR localization, using the experimental variables and reagents created and analyzed in the previous aims in Ussing chamber experiments. The long- term goal of this project is to identify the molecular interactions responsible for the assembly of the apical ezrin- actin scaffold in intestinal cells that can be manipulated to interferewith the function of ion channels in diarrheal disorders. Lay statement: Secretory diarrheas are a severe health problem in the U.S. and worldwide. All of them operate through common effectors in the intestine that enable the secretion of chloride (and water). The machinery that supports these membrane proteins is based on cytoskeletal components and is assembled around a protein known as ezrin. This project seeks to test the hypothesisthat ezrin (and its associated chloride secreting proteins) require spatial cues from a division of the cytoskeleton,the intermediate filaments, to become assembled in the precise location to secrete water to the lumen of the intestine. The study of protein-protein interactions that result in the precise and adequate localization of ezrin and its associated proteins will provide potential points for molecular intervention on the function of this molecular complex.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK076652-04
Application #
7746453
Study Section
Gastrointestinal Cell and Molecular Biology Study Section (GCMB)
Program Officer
May, Michael K
Project Start
2007-01-01
Project End
2011-12-31
Budget Start
2010-01-01
Budget End
2010-12-31
Support Year
4
Fiscal Year
2010
Total Cost
$302,266
Indirect Cost
Name
University of Miami School of Medicine
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
052780918
City
Coral Gables
State
FL
Country
United States
Zip Code
33146
Forteza, Radia; Figueroa, Yolanda; Mashukova, Anastasia et al. (2016) Conditional knockout of polarity complex (atypical) PKC? reveals an anti-inflammatory function mediated by NF-?B. Mol Biol Cell 27:2186-97
Salas, Pedro J; Forteza, Radia; Mashukova, Anastasia (2016) Multiple roles for keratin intermediate filaments in the regulation of epithelial barrier function and apico-basal polarity. Tissue Barriers 4:e1178368
Mashukova, Anastasia; Forteza, Radia; Salas, Pedro J (2016) Functional Analysis of Keratin-Associated Proteins in Intestinal Epithelia: Heat-Shock Protein Chaperoning and Kinase Rescue. Methods Enzymol 569:139-54
Hyun, Jinhee; Romero, Laura; Riveron, Reldy et al. (2015) Human intestinal epithelial cells express interleukin-10 through Toll-like receptor 4-mediated epithelial-macrophage crosstalk. J Innate Immun 7:87-101
Mashukova, Anastasia; Kozhekbaeva, Zhanna; Forteza, Radia et al. (2014) The BAG-1 isoform BAG-1M regulates keratin-associated Hsp70 chaperoning of aPKC in intestinal cells during activation of inflammatory signaling. J Cell Sci 127:3568-77
Kravtsov, Dmitri; Mashukova, Anastasia; Forteza, Radia et al. (2014) Myosin 5b loss of function leads to defects in polarized signaling: implication for microvillus inclusion disease pathogenesis and treatment. Am J Physiol Gastrointest Liver Physiol 307:G992-G1001
Forteza, Radia; Wald, Flavia A; Mashukova, Anastasia et al. (2013) Par-complex aPKC and Par3 cross-talk with innate immunity NF-?B pathway in epithelial cells. Biol Open 2:1264-9
Mashukova, Anastasia; Forteza, Radia; Wald, Flavia A et al. (2012) PDK1 in apical signaling endosomes participates in the rescue of the polarity complex atypical PKC by intermediate filaments in intestinal epithelia. Mol Biol Cell 23:1664-74
Mashukova, Anastasia; Wald, Flavia A; Salas, Pedro J (2011) Tumor necrosis factor alpha and inflammation disrupt the polarity complex in intestinal epithelial cells by a posttranslational mechanism. Mol Cell Biol 31:756-65
Wald, Flavia A; Forteza, Radia; Diwadkar-Watkins, Runa et al. (2011) Aberrant expression of the polarity complex atypical PKC and non-muscle myosin IIA in active and inactive inflammatory bowel disease. Virchows Arch 459:331-8

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