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Abstract
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 (BGTΔ5) 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 BGTΔ5 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.
Highlights
The Na/Cl-dependent BGT1 transporter has osmoprotective functions by importing the small osmolyte betaine into the cytosol of renal medullary epithelial cells
protein kinase C (PKC) Activation Decreases BGT1 Activity and Relocalizes the Transporter in Endocytotic Compartments—To verify whether serine/threonine kinase proteins regulate BGT1, we measured its activity in response to the activation of PKC and protein kinase A (PKA) in a Madin-Darby canine kidney (MDCK)-mycBGT1 clone by evaluating [3H]GABA uptake, because BGT1 transports GABA with even higher affinity than betaine
The reduction in GABA transport activity in response to TPA treatment was accompanied by the intracellular relocalization of BGT1 (Fig. 1C, compare the lateral surface staining in a with the intracellular staining in b), which appeared to be completely prevented by the PKC inhibitor GF109203X (c)
Summary
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). 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 clathrindependent internalization of the transporter and its targeting to a recycling compartment that contains the truncated transporter lacking the LIN7 binding motif (BGT⌬5) but not the LIN7 partner of BGT1. 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. Our findings demonstrate a novel mechanism for the regulation of transporter activity based on the PKC-mediated modulation of PDZ protein interactions with target sequences
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