Cystic fibrosis is a disease of electrolyte transport abnormalities, which has, as its genetic basis, a mutation of the cystic fibrosis transmembrane regulator (CFTR), an apical secretory Cl channel. Correction of airway dysfunction in CF has centered on therapeutic approaches to activate apical Cl channels other than CFTR and genetic alteration of CFTR to correct abnormal Cl secretion and thus offset excess mucus accumulation. CFTR is regulated primarily by CAMP, however, the PI discovered that constitutive activity of PKC-epsilon is necessary for CAMP-dependent regulation of CFTR. Thus, regulation of CFTR function is more complicated than just cAMP-dependent phosphorylation of CFTR. New pilot studies indicate an association between PKC-epsilon and CFTR that might represent direct or indirect binding. In addition to CFTR, other tracheal epithelial proteins associate with PKC-epsilon, including scaffold proteins which might form a multiprotein complex that tethers PKC-epsilon proximal to its target. The hypothesis of this grant proposal is that PKC-epsilon regulates CFTR function through phosphorylation. This will be studied in detail in the following specific aims: 1) To determine whether activity of PKC- epsilon regulates its interaction with CFTR. Activity will be manipulated using PKC inhibitors or, to also decrease mass, with antisense oligonucleotides to PKC-epsilon. Enzyme activity will be correlated with co-purification of PKC-epsilon with CFTR and phosphorylation of wild type CFTR. Whether trafficking competent and incompetent mutant CFTR (G551D, deltaF508, respectively) alter CFTR interaction with PKC-epsilon and its phosphorylation of CFTR will be determined. 2) To determine whether activity of PKC-epsilon is regulated by association with a multiprotein complex. Binding of PKC-epsilon with recombinant or endogenous proteins (CFTR, RACK, actin) will be measured by direct binding or overlay assay and quantitated as a K-m for binding. Binding of activated and/or inactive enzyme and specificity for PKC isotype will be determined. Activity will be manipulated by omitting PKC activators or adding PKC inhibitor and by downregulating binding protein using an antisense approach. Whether rapid loss of CFTR function by PKC inhibitor is correlated to activity of serine/threonine protein phosphatase(s) and its association with PKC-epsilon finding partners will be determined. 3) To identify site(s) of interaction between PKC- epsilon and target/binding protein. Sequence motifs in specific domains of PKC-epsilon and/or binding will be predicted and tested using peptides to inhibit binding and cAMP-dependent activation of CFTR.
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| Liedtke, Carole M; Wang, Xiangyun (2006) The N-terminus of the WD5 repeat of human RACK1 binds to airway epithelial NHERF1. Biochemistry 45:10270-7 |
| Liedtke, Carole M (2004) Regulation of epithelial electrolyte transporters through protein-protein interactions. Adv Exp Med Biol 559:349-58 |
| Liedtke, Carole M; Raghuram, Viswanathan; Yun, C Chris et al. (2004) Role of a PDZ1 domain of NHERF1 in the binding of airway epithelial RACK1 to NHERF1. Am J Physiol Cell Physiol 286:C1037-44 |
| Liedtke, Carole M; Yun, C H Chris; Kyle, Nicole et al. (2002) Protein kinase C epsilon-dependent regulation of cystic fibrosis transmembrane regulator involves binding to a receptor for activated C kinase (RACK1) and RACK1 binding to Na+/H+ exchange regulatory factor. J Biol Chem 277:22925-33 |
