Role of conserved peptide in taurine transporter inactivation modulated by protein kinase C.
Activation of protein kinase C (PKC) by the active phorbol ester 12-myristate 13-acetate (PMA, 100 nM) or phorbol-12, 13-dibutyrate (100 nM) reduced taurine uptake by 80% in oocytes given injections with cRNA from and expressing the Madin-Darby canine kidney cell taurine transporter pNCT. Inhibition of PKC by calphostin C or staurosporine increased taurine uptake by 20% and 400%, respectively. The inhibitory effect of PMA on taurine uptake was blocked by calphostin C, a specific inhibitor of PKC. Modulation by PMA mainly affected the apparent affinity K(m) (from 5.6 microM to 18.1 microM) with minimal effect on the maximal velocity (25% decrease) of the transporter. A polyclonal antibody (AbS4) directed against a conserved intracellular segment (S4) of the Madin-Darby canine kidney cell taurine transporter enhanced taurine uptake by pNCT cRNA-treated oocytes. The effect of AbS4 was blocked by incubation with the corresponding peptide antigen. Preimmune IgG and peptide antigen had no effect on taurine transporter activity expressed in oocytes. Modulation seemed to occur through phosphorylation of a consensus PKC site located on segment S4 of the transporter, because downregulation of the transporter by PMA (100 nM) was abolished by preinjection of AbS4 (12 ng/ oocyte). In contrast, downregulation of the transporter by PMA could not be restored by AbS4 when pNCT-expressing oocytes were pretreated with PMA (50 nM). In conclusion, the peptide segment recognized by this antibody appears to participate directly in taurine transporter inactivation that is modulated by PKC phosphorylation.
Department of Pediatrics, University of Tennessee, Memphis, LeBonheur Children's Medical Center 38103, USA.
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Han
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Budreau
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Chesney
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D002460:Cell Line / Q000378:metabolism; D017124:Conserved Sequence; D004285:Dogs; D004789:Enzyme Activation; D005260:Female; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D026901:Membrane Transport Proteins; D009865:Oocytes / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014982:Xenopus laevis; D000099247:Taurine Transporters
D015124:8-Bromo Cyclic Adenosine Monophosphate; D000906:Antibodies; D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D000099247:Taurine Transporters