Regulation of amino acid/carnitine transporter B0,+ (ATB0,+) in astrocytes by protein kinase C: independent effects on raft and non-raft transporter subpopulations

P>Neutral and basic amino acid transporter B0,+ belongs to a Na,Cl-dependent superfamily of proteins transporting neurotransmitters, amino acids and osmolytes, known to be regulated by protein kinase C (PKC). The present study demonstrates an increased phosphorylation of B0,+ on serine moiety after treatment of rat astrocytes with phorbol 12-myristate 13-acetate, a process correlated with an augmented activity of l-leucine transport and an enhanced presence of the transporter at the cell surface. After solubilization with Triton X-100 and sucrose gradient centrifugation, B0,+ was detected in non-raft as well as in detergent-resistant raft fractions under control conditions, while phorbol 12-myristate 13-acetate treatment resulted in a complete disappearance of the transporter from the raft fraction. B0,+ was observed to interact with caveolin-1 and flotillin-1 (reggie-2) proteins, the markers of detergent-resistant microdomains of plasma membrane. As verified in immunocytochemistry and immunoprecipitation experiments, modification of PKC activity did not affect these interactions. It is proposed that PKC reveals different effects on raft and non-raft subpopulations of B0,+. Phorbol ester treatment results in trafficking of the transporter from the intracellular pool to non-raft microdomains and increased activity, while B0,+ present in raft microdomains undergoes either internalization or is transferred laterally to non-raft domains.