Role of protein phosphatases in the activation of CFTR (ABCC7) by genistein and bromotetramisole
| Autor: | Jiexin Luo, Mary D. Pato, Tang Zhu, Alexandra Evagelidis, John W. Hanrahan |
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| Rok vydání: | 2000 |
| Předmět: |
Physiology
Phosphatase Tetramisole Genistein Cystic Fibrosis Transmembrane Conductance Regulator CHO Cells Biology Cystic fibrosis chemistry.chemical_compound Cricetinae medicine Phosphoprotein Phosphatases Animals Patch clamp Enzyme Inhibitors Phosphorylation Chinese hamster ovary cell Cell Biology medicine.disease Precipitin Tests Cystic fibrosis transmembrane conductance regulator Cell biology Isoenzymes chemistry Biochemistry Chloride channel biology.protein Subcellular Fractions |
| Zdroj: | American journal of physiology. Cell physiology. 279(1) |
| ISSN: | 0363-6143 |
| Popis: | Genistein and bromotetramisole (Br-t) strongly activate cystic fibrosis transmembrane conductance regulator (CFTR; ABCC7) chloride channels on Chinese hamster ovary cells and human airway epithelial cells. We have examined the possible role of phosphatases in stimulation by these drugs using patch-clamp and biochemical methods. Genistein inhibited the spontaneous rundown of channel activity that occurs after membrane patches are excised from cAMP-stimulated cells but had no effect on purified protein phosphatase type 1 (PP1), PP2A, PP2B, PP2C, or endogenous phosphatases when assayed as [32P]PO4release from prelabeled casein, recombinant GST-R domain fusion protein, or immunoprecipitated full-length CFTR. Br-t also slowed rundown of CFTR channels, but, in marked contrast to genistein, it did inhibit all four protein phosphatases tested. Half-maximal inhibition of PP2A and PP2C was observed with 0.5 and 1.5 mM Br-t, respectively. Protein phosphatases were also sensitive to (+)- p-Br-t, a stereoisomer of Br-t that does not inhibit alkaline phosphatases. Br-t appeared to act exclusively through phosphatases since it did not affect CFTR channels in patches that had low apparent endogenous phosphatase activity (i.e., those lacking spontaneous rundown). We conclude that genistein and Br-t act through different mechanisms. Genistein stimulates CFTR without inhibiting phosphatases, whereas Br-t acts by inhibiting a membrane-associated protein phosphatase (probably PP2C) that presumably allows basal phosphorylation to accumulate. |
| Databáze: | OpenAIRE |
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