cAMP-independent activation of CFTR Cl channels by the tyrosine kinase inhibitor genistein.
Genistein, a protein tyrosine kinase inhibitor, activates the cystic fibrosis transmembrane conductance regulator (CFTR) in transfected NIH-3T3 fibroblasts that express the CFTR (3T3-CFTR). CFTR activity was assayed by 125I efflux and by patch clamping in the cell-attached mode. Both forskolin and genistein stimulated 125I efflux and activated a 9-10 pS anion channel in 3T3-CFTR cells but failed to activate 125I efflux in mock-transfected NIH-3T3 cells. Genistein, unlike forskolin and 3-isobutyl-1-methylxanthine, did not increase intracellular adenosine 3',5'-cyclic monophosphate (cAMP) above control levels. This demonstrates that genistein-dependent activation does not involve inhibition of phosphodiesterase activity and suggests that stimulation does not involve a direct activation of protein kinase A. Genistein stimulated 125I efflux to approximately 50% of the maximal rate with forskolin. Genistein did not increase 125I efflux at saturating forskolin but decreased the concentration of forskolin required for half-maximal stimulation. Orthovanadate (VO4), a phosphotyrosine phosphatase inhibitor, inhibited genistein-induced channel activation with an inhibition constant of approximately 20 microM. These effects suggest that, in addition to activation by protein kinase A, the CFTR is regulated by a tyrosine kinase-dependent pathway.
Illek
B
B
Department of Molecular and Cell Biology, University of California, Berkeley 94720, USA.
Fischer
H
H
Santos
G F
GF
Widdicombe
J H
JH
Machen
T E
TE
Reenstra
W W
WW
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D016475:3T3 Cells; D000818:Animals; D002118:Calcium / Q000378:metabolism; D018118:Chloride Channels / Q000187:drug effects / Q000378:metabolism*; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000502:physiology*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004594:Electrophysiology; D019833:Genistein; D007425:Intracellular Membranes / Q000378:metabolism; D007529:Isoflavones / Q000494:pharmacology*; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*; D014638:Vanadates / Q000494:pharmacology
D018118:Chloride Channels; D007529:Isoflavones; D008565:Membrane Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; D014638:Vanadates; D019833:Genistein; D000242:Cyclic AMP; D011505:Protein-Tyrosine Kinases; D002118:Calcium; D015056:1-Methyl-3-isobutylxanthine