title: Role of tyrosine phosphorylation in potassium channel activation. Functional association with prolactin receptor and JAK2 tyrosine kinase.
abstract: Chinese hamster ovary (CHO) cells, stably transfected with the long form of the prolactin (PRL) receptor (PRL-R) cDNA, were used for PRL-R signal transduction studies. Patch-clamp technique in whole cell and cell-free configurations were employed. Exposure of transfected CHO cells to 5 nM PRL led to the increase of Ca(2+)- and voltage-dependent K+ channel (KCa) activity. The effect was direct as it was observed also in excised patch experiments. A series of tyrosine kinase inhibitors was studied to investigate the possible involvement of protein tyrosine kinases in KCa functioning and its stimulation by PRL. Genistein, lavendustin A, and herbimycin A decreased in a concentration and time-dependent manner the amplitude of the KCa current in whole cell and the open probability of KCa channels in cell-free experiments. The subsequent application of PRL was ineffective. The protein tyrosine phosphatase inhibitor orthovanadate (1 mM) stimulated KCa channel activity in excised patches, indicating that channels can be modulated in opposite directions by protein tyrosine kinase and protein tyrosine phosphatase. Moreover, in whole cell experiments as well as in excised patch recordings, anti-JAK2 tyrosine kinase antibody decreased the KCa conductance and the open probability of the KCa channels. Subsequent application of PRL was no longer able to stimulate KCa conductance. Immunoblotting studies using the same anti-JAK2 antibody, revealed the constitutive association of JAK2 kinase with PRL-R. Preincubation of anti-JAK2 antibody with the JAK2 Immunizing Peptide abolished the effects observed using anti-JAK2 antibody alone in both electrophysiological and immunoblotting studies. We conclude from these findings that these KCa channels are regulated through tyrosine phosphorylation/dephosphorylation; JAK2 tyrosine kinase, constitutively associated with PRL-R, is implicated in PRL stimulation of KCa channels.
authors.affiliation: Laboratory of Neurophysiology, University of Bordeaux II, CNRS URA 1200, France.
authors.forename: N B
authors.initials: NB
authors.lastname: Prevarskaya
authors.forename: R N
authors.initials: RN
authors.lastname: Skryma
authors.forename: P
authors.initials: P
authors.lastname: Vacher
authors.forename: N
authors.initials: N
authors.lastname: Daniel
authors.forename: J
authors.initials: J
authors.lastname: Djiane
authors.forename: B
authors.initials: B
authors.lastname: Dufy
mesh_terms: D000818:Animals; D016227:Benzoquinones; D016466:CHO Cells; D006224:Cricetinae; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019833:Genistein; D015640:Ion Channel Gating / Q000187:drug effects; D007529:Isoflavones / Q000494:pharmacology; D053614:Janus Kinase 2; D007700:Kinetics; D047029:Lactams, Macrocyclic; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D018408:Patch-Clamp Techniques; D010636:Phenols / Q000494:pharmacology; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D015221:Potassium Channels / Q000187:drug effects / Q000502:physiology*; D011388:Prolactin / Q000494:pharmacology*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism*; D011518:Proto-Oncogene Proteins; D011809:Quinones / Q000494:pharmacology; D011981:Receptors, Prolactin / Q000096:biosynthesis / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D017828:Rifabutin / Q000031:analogs & derivatives; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection
chemical_list: D016227:Benzoquinones; D004791:Enzyme Inhibitors; D007529:Isoflavones; D047029:Lactams, Macrocyclic; D010636:Phenols; D015221:Potassium Channels; D011518:Proto-Oncogene Proteins; D011809:Quinones; D011981:Receptors, Prolactin; D011994:Recombinant Proteins; D017828:Rifabutin; D019000:Phosphotyrosine; C062898:lavendustin A; C020754:herbimycin; D011388:Prolactin; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D053614:Janus Kinase 2