This project will test the hypothesis that some forms of CFTR are degraded in intracellular compartments. The initial experiments will be conducted in yeast, capitalizing on a series of mutants which halt processing of molecules at various stages in the endoplasmic reticulum (with or without vesicle formation), Golgi, or beyond. Wild type and mutant (deltaF508 and others) CFTR either in native form, containing a FLAG sequence for ease of immunologic identification, or fused to beta- galactosidase for ease of reporting and later genetic analysis will be followed along the processing sequence using sec, vps, erd, and kar2 mutants. The beta-galactosidase fusions will be utilized to facilitate screening for mutants which fail to process or degrade CFTR. The genes which are defective in the mutants will be cloned from yeast, and human homologs sought. The nature of the gene products will be determined and means of inhibiting them devised. These experiments will determine at which stage in protein processing CFTR fails to be properly processed and is degraded and will isolate the protein(s) responsible. If, as predicted by the work of Cheng and coworkers, a major cause of cystic fibrosis is the defective processing and degradation of mutant forms of CFTR, so that proper location at the plasma membrane is never achieved, these results might permit us to design interventions to permit processing and escape of some mutant protein to the membrane, where it may be possible to activate it and restore function.

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National Heart, Lung, and Blood Institute (NHLBI)
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Case Western Reserve University
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Liedtke, C M; Cody, D; Cole, T S (2001) Differential regulation of Cl- transport proteins by PKC in Calu-3 cells. Am J Physiol Lung Cell Mol Physiol 280:L739-47
Liedtke, C M; Cole, T S (2000) PKC signaling in CF/T43 cell line: regulation of NKCC1 by PKC-delta isotype. Biochim Biophys Acta 1495:24-33
Steagall, W K; Drumm, M L (1999) Stimulation of cystic fibrosis transmembrane conductance regulator-dependent short-circuit currents across DeltaF508 murine intestines. Gastroenterology 116:1379-88
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Kelley, T J; Drumm, M L (1998) Inducible nitric oxide synthase expression is reduced in cystic fibrosis murine and human airway epithelial cells. J Clin Invest 102:1200-7
Steagall, W K; Kelley, T J; Marsick, R J et al. (1998) Type II protein kinase A regulates CFTR in airway, pancreatic, and intestinal cells. Am J Physiol 274:C819-26
Liedtke, C M; Cole, T S (1998) Antisense oligonucleotide to PKC-epsilon alters cAMP-dependent stimulation of CFTR in Calu-3 cells. Am J Physiol 275:C1357-64
Liedtke, C M; Cole, T (1997) Antisense oligodeoxynucleotide to PKC-delta blocks alpha 1-adrenergic activation of Na-K-2Cl cotransport. Am J Physiol 273:C1632-40
Kelley, T J; Thomas, K; Milgram, L J et al. (1997) In vivo activation of the cystic fibrosis transmembrane conductance regulator mutant deltaF508 in murine nasal epithelium. Proc Natl Acad Sci U S A 94:2604-8

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