Research Interests
Contributions to Science
1. Detecting protein phosphorylation responses in intact epithelial cells stimulated to secrete chloride ions. Our group was the first to develop methods to resolve individual regulatory protein phosphorylation events during the neurohumoral regulation of trans-epithelial ion transport in any chloride secreting epithelium (1.1). These methods were used to discover a novel substrate of protein kinase C which other groups later characterized as MARCKS, a key effector for exocytosis (1.2). These methods also were used to discover that flavonols can regulate CFTR-mediated secretion (1.3); this work led others to perform clinical trials of a related molecule (genistein) as a potential treatment for CF. In work begun in 1987, our group used these methods to try to identify the gene causing CF (1.4).
1.1 Cohn JA. Vasoactive intestinal peptide stimulates protein phosphorylation in a colonic epithelial cell line. Am J Physiol 253:G420-G424, 1987.
1.2 Cohn JA. Protein kinase C mediates cholinergically regulated protein phosphorylation in chloride-secreting epithelium. Am J Physiol 258:C227-C233, 1990.
1.3 Nguyen TD, Canada AT, Heintz GG, Gettys TW, Cohn JA. Stimulation of secretion by the T84 colonic epithelial cell line with dietary flavonols. Biochem Pharmacol 41: 1879-1886, 1991.
1.4 Cohn JA, Kole J, Yankaskas JR. Protein phosphorylation responses in normal and cystic fibrosis airway epithelial cells. Am J Respir Cell Mol Biol 9:401-404, 1993.
2. Developing and validating antibodies to detect endogenous CFTR in human tissues. When the CFTR gene was discovered by positional cloning in 1989, there was an urgent need to determine the properties and localization of the gene’s protein product, CFTR. Among the antibodies developed by many research teams trying to detect CFTR, the antibodies we developed proved to be the most specific and sensitive. They had high affinity (2.1) and they provided clean signals for tissue staining, immunoprecipitation and Western blots (2.1, 2.2, 2.3). We identified the immunodetected protein as authentic CFTR based on tryptic fingerprints and amino acid sequencing (2.3). When we localized CFTR to the apical membrane of duct cells in the pancreas and biliary tract, this led to working models for the role of CFTR in these tissues (2.2, 2.4); these models provided the foundation for research by many others since then. For over a decade, the CFTR immunodetection methods developed by our group were widely recognized as the field’s reference standard.
2.1 Cohn J, Melhus O, Page LJ, Dittrich KL and Vigna SR. CFTR: Development of high-affinity antibodies and localization in sweat gland. Biochem Biophys Res Commun 181: 36-43, 1991.
2.2 Marino CR, Matovcik LM, Gorelick FS, Cohn JA. Localization of the cystic fibrosis transmembrane conductance regulator in pancreas. J Clin Invest 88: 712-716, 1991.
2.3 Cohn JA, Nairn AC, Marino CR, Melhus O, Kole J. Characterization of the CFTR in a colonocyte cell line. Proc Natl Acad Sci USA 89: 2340-2344, 1992.
2.4 Cohn JA, Strong TV, Picciotto MR, Nairn AC, Collins FS, Fitz JG. Localization of the CFTR in human bile duct epithelial cells. Gastroenterology 105:1857-1864, 1993.
3. Characterizing how cAMP-dependent protein kinase (PKA) regulates CFTR and related ion transport proteins. The immunodetection and tryptic mapping methods developed above (2.3) enabled us to identify which CFTR serine residues undergo phosphorylation when PKA stimulates CFTR-mediated ion transport (3.1). In further studies, we showed that PKA acts on CFTR to enable CFTR to control trans-epithelial sodium transport, a key mechanism for understanding the pathogenesis of CF lung disease (3.2). To further define the mechanism by which PKA acts on CFTR, we developed recombinant peptide models of a large cytoplasmic domain of CFTR (3.3). Using these models, we discovered that PKA acts on this CFTR domain to regulate both ATP binding and the interactions between this domain and another CFTR cytoplasmic domain (3.4). These findings have implications for the normal regulation of CFTR by PKA and for the impact of a disease-causing CFTR mutation (G551D) on this process.
3.1 Picciotto MR, Cohn JA, Bertuzzi G, Greengard P, Nairn AC. Phosphorylation of the cystic fibrosis transmembrane conductance regulator. J Biol Chem 267: 12742-12752, 1992.
3.2 Stutts MJ, Canessa C, Olsen JC, Hamrick M, Cohn JA, Rossier B, Boucher RC. CFTR as a cAMP-dependent regulator of sodium channels. Science 269: 847-850, 1995.
3.3 Howell LD, Borchardt RA, Cohn JA. ATP hydrolysis by a CFTR domain: Pharmacology and effects of the G551D mutation. Biochem Biophys Res Commun 271: 518-525, 2000.
3.4 Howell LD, Borchardt R, Kole J, Kaz AM, Randak C, Cohn JA. Protein kinase A regulates ATP hydrolysis and dimerization by a CFTR domain. Biochem J 378:151-159, 2004.
4. Defining how CF-causing mutations cause loss of CFTR function in epithelial cells. The CFTR detection methods developed in our lab led us to contribute to many significant discoveries concerning the cell biology of CF. One early collaboration identified submucosal glands as a prominent site of CFTR in human airways (4.1); this was unexpected and led to extensive research by many groups exploring the role of these glands in CF pathobiology. Another collaboration described how a chaperone protein interacted with F508del-CFTR to target it for non-lysosomal degradation (4.2); this was among the first examples of a chaperone protein recognizing a misfolded protein linked to a human disease. Another collaboration defined the impact of the 5T polymorphism on CFTR (4.3); we later developed a PCR-based assay for 5T and found this to be common among patients with pancreatitis (5.1). Finally, we collaborated on studies which helped define the mechanism by which F508del and other mutations cause CFTR to malfunction (4.2, 4.4); this was part of the work which led to the high throughput screening assays used by Vertex Pharmaceuticals to develop FDA-approved CFTR modulator drugs which are now widely used to treat CF.
4.1 Engelhardt JF, Yankaskas JR,. Ernst SA, Yang Y, Marino CR, Boucher RC, Cohn JA and Wilson JM. Submucosal glands are the predominant site of CFTR expression in the human bronchus. Nature Genet 2: 240-248, 1992.
4.2 Yang Y, Janich S, Cohn JA and Wilson JM. The common variant of cystic fibrosis transmembrane conductance regulator is recognized by hsp70 and degraded in a pre-Golgi nonlysosomal compartment. Proc Natl Acad Sci USA 90: 9480-9484, 1993.
4.3 Strong TV, Wilkinson DJ, Mansoura ML, Henze K, Yang Y, Wilson JM, Cohn JA, Dawson DC, Frizzell RA and Collins FS. Expression of an abundant alternatively spliced form of the CFTR gene is not associated with the appearance of a chloride conductance. Hum Molec Genet 2: 225-230, 1993.
4.4 Yang Y, Devor DC, Engelhardt JF, Ernst SA, Strong T, Collins FS, Cohn JA, Frizzell RA and Wilson JM. Molecular basis of defective anion transport in L cells expressing recombinant forms of CFTR. Hum Molec Genet 2: 1253-1261, 1993.
5. Defining the role of CFTR mutations in individuals who have chronic pancreatitis without CF lung disease. Until 1998, idiopathic chronic pancreatitis (ICP) was viewed as a sporadic condition with no evident genetic basis. Our group discovered that in many ICP patients, pancreatitis occurs as part of an inherited syndrome which also includes abnormal sweat secretion, male infertility and airway colonization with unusual pathogens (5.1, 5.3). ICP was arguably the first disease shown to exhibit non-Mendelian (complex) inheritance while being associated with mutations of a seemingly unrelated Mendelian disease gene (5.2); we found that highest disease risk was conferred by the combination of an abnormal CFTR genotype plus a polymorphism in a modifier gene (SPINK1, encoding a trypsin inhibitor which is expressed in a different part of the pancreas from CFTR). By contrast, CF carrier status also predisposes to ICP but its impact on disease risk is relatively low (5.4). These discoveries have implications for the pathogenesis of ICP, for genetic testing, and for therapy. By now, thousands of ICP patients have undergone genetic testing based on our work. Ongoing work in several labs is trying to develop new treatments for ICP based on these discoveries.
5.1 Cohn JA, Friedman KJ, Noone PG, Knowles MR, Silverman LM and Jowell PS. Relation between mutations in the cystic fibrosis gene and idiopathic pancreatitis. N Engl J Med 339: 653-658, 1998.
5.2 Noone PG, Zhou Z, Silverman LM, Jowell PS, Knowles MR and Cohn JA, Cystic fibrosis gene mutations and pancreatitis risk: Relation to epithelial ion transport and trypsin inhibitor gene mutations. Gastroenterology 121: 1310-1319, 2001.
5.3 Cohn JA, Noone PG and Jowell PS. Idiopathic pancreatitis related to CFTR: Complex inheritance and identification of a modifier gene. J Invest Med 50: 247S-255S, 2002.
5.4 Cohn JA, Neoptolemos JP, Feng J, Yan J, Jiang Z, Greenhalf W, McFaul C, Mountford R, and Sommer SS. Increased risk of idiopathic chronic pancreatitis in cystic fibrosis carriers. Hum Mutat 26: 303-307, 2005.