Ser-322 is a critical site for PKC regulation of the MDCK cell taurine transporter (pNCT)

Previous studies have shown that the Madin-Darby canine kidney cell taurine transporter (pNCT) is downregulated by protein kinase C (PKC) activation. In this study, it is hypothesized that the highly conserved serine-322(Ser- 322) located in the fourth intracellular segment (S-4) may play an importan t role in the function of taurine transporter, which is modulated by PKC ph osphorylation. It is demonstrated that Ser-322 is the critical site of PKC phosphorylation, as determined by site-directed mutagenesis. When Ser-322 o f pNCT was changed to alanine (S322A) and this mutant was evaluated in an o ocyte expression system, taurine transport activity increased threefold com pared with control (wild-type pNCT). Activation of PKC by the active phorbo l ester 12-myristate 13-acetate did not influence taurine transport by muta nt S322A. Kinetic analysis showed that the mutation of Ser-322 essentially changed the V-max, rather than the K-m, of the transporter. Mutation of all other PKC consensus sites did not affect transporter activity when express ed in the oocyte system. Western blot analysis showed that expression of ta urine transporter protein was similar in oocytes injected with either wildt ype or mutant pNCT cRNA, indicating that the enhanced taurine transport act ivity by mutant S322A was not caused by a greater amount of transporter exp ressed in the oocyte. Furthermore, this study demonstrated that the taurine transporter was phosphorylated after PKC activation, and this effect was n ot observed in mutant S322A. In conclusion, Ser-322 is critical in PKC regu lation of taurine transporter activity. The steady-state taurine transporte r activity is tightly controlled by endogenous PKC phosphorylation of Ser-3 22, which is located in the fourth intracellular segment of the taurine tra nsporter.