The Na/Cl-dependent BGT1 transporter has osmoprotective functions by importing the small osmolyte betaine into the cytosol of renal medullary epithelial cells. We have demonstrated previously that the surface localization of the transporter in Madin-Darby canine kidney cells depends on its association with the LIN7 PDZ protein through a PDZ target sequence in the last 5 residues of the transporter (-KETHL). Here we describe a protein kinase C (PKC)-mediated mechanism regulating the association between BGT1 and LIN7. Reduced transport activity paralleled by the intracellular relocalization of the transporter was observed in response to the PKC activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. This activation caused clathrin-dependent internalization of the transporter and its targeting to a recycling compartment that contains the truncated transporter lacking the LIN7 binding motif (BGTDelta5) but not the LIN7 partner of BGT1. The decreased association between BGT1 and LIN7 was demonstrated further by coimmunoprecipitation studies and in vitro binding to recombinant LIN7 fusion protein. The TPA treatment induced phosphorylation of surface BGT1 on serine and threonine residues. However, a greater increase in phosphothreonines than phosphoserines was measured in the wild type transporter, whereas the opposite was true in the BGTSer mutant in which a serine replaced the threonine 612 in the LIN7 association motif (-KESHL). No similar increase in relative phosphoserines or phosphothreonines was found in the BGTDelta5 transporter. Moreover, phosphorylation of threonine 612 in a BGT COOH-terminal peptide impaired its association with recombinant LIN7. Taken together, these data demonstrate that the post-translational regulation of BGT1 surface density is a result of transporter phosphorylation and that threonine 612 is an essential residue in this PKC-mediated regulation.
|Titolo:||Protein kinase C-mediated phosphorylation of the BGT1 epithelial gamma-aminobutyric acid transporter regulates its association with LIN7 PDZ proteins - A post-translational mechanism regulating transporter surface density|
MASSARI, SILVIA (Primo)
PIETRINI, GRAZIA (Ultimo)
|Settore Scientifico Disciplinare:||Settore BIO/14 - Farmacologia|
|Data di pubblicazione:||2005|
|Digital Object Identifier (DOI):||10.1074/jbc.M412668200|
|Appare nelle tipologie:||01 - Articolo su periodico|