Protein kinase C-mediated functional regulation of dopamine transporter isnot achieved by direct phosphorylation of the dopamine transporter protein

Dopaminergic neurotransmission is terminated by the action of the presynapt ic dopamine transporter (DAT). It mediates Na+/Cl- -dependent re-uptake of extracellular dopamine (DA) into the cell, and is regarded as a major regul atory mechanism for synaptic transmission. Previous works have documented t hat protein kinase C (PKC) activator or inhibitor alters DA uptake by DAT, suggesting that PKC phosphorylation plays an important regulatory mechanism in DAT function. Based on the existence of consensus amino acid sequences for PKC phosphorylation, it has been postulated that PKC regulation of DAT is mediated by the direct phosphorylation of DAT protein. In this study, we try to discover whether the functional regulation of DAT by PKC is due to direct phosphorylation of DAT. The PKC null mutant hDAT, where all putative PKC phosphorylation sites are eliminated, has been constructed by the repl acement of serine/threonine residues with glycines. The mutation itself sho wed no effect on the functional activities of DAT. The DA uptake activity o f PKC null mutant was equivalent to those of wild-type hDAT (80-110% of wil d-type). Phorbol ester activation of PKC inhibited DA uptake of wildtype hD AT by 35%, and staurosphorine blocked the effect of phorbol ester on DA upt ake. The same phenomena was observed in PKC null mutant DAT, although no si gnificant phosphorylation was observed by PKC activation. Confocal microsco pic analysis using EGFP-fused DAT revealed that the activation of PKC by ph orbol ester elicited fluorescent DAT to be internalized into the intracellu lar space both in wild-type and PKC null mutant DAT in a similar way. These results suggest that PKC-mediated regulation of DAT function is achieved i n an indirect manner, such as phosphorylation of a mediator protein or acti vation of a clathrin-mediated pathway.