Regulation of amino acid/carnitine transporter B^0,+ (ATB^0,+) in astrocytes by protein kinase C: independent effects on raft and non‐raft transporter subpopulations

Abstract

Abbreviations used: Bis II, bisindolylmaleimide II; MbCD, methyl-bcyclodextrin; PBS, phosphate-buffered saline; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; SDS, sodium dodecyl sulfate. AbstractNeutral and basic amino acid transporter B 0,+ 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 B 0,+ 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, B 0,+ 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. B 0,+ 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 B 0,+ .Phorbol ester treatment results in trafficking of the transporter from the intracellular pool to non-raft microdomains and increased activity, while B 0,+ present in raft microdomains undergoes either internalization or is transferred laterally to non-raft domains.

Caveolin-1 - A Novel Interacting Partner of Organic Cation/Carnitine Transporter (Octn2): Effect of Protein Kinase C on This Interaction in Rat Astrocytes

“…Interestingly, clone PSER-7F12 reacted with the same proteins of M r of 130,000 and 46,000, as recognized by PSR-45. It should be emphasized that clone 4A9 was previously shown by our group to detect an increased phosphorylation of a low affinity carnitine transporter ATB 0,+ , both in astrocytes and HEK293 cells treated with PMA [33], [36]. It has to be added that neither a clone requiring proline or lysine C-terminal to phosphoserine (PSER-16B4) nor anti-phosphothreonine antibody (PTHR-4D11) gave any positive results (not shown).…”

“…1A), a process stimulated by 40% after pretreatment with PKC activator – PMA and reversed by Bis II. Carnitine transport in the presence of NaCl represents a sum of activity of all carnitine transporters, including a Na + /Cl – dependent amino acid/carnitine transporter ATB 0,+ [34], known to be present in astrocytes [33], [38]. Omission of chloride, i.e.…”

“…Astrocytes were homogenized and analysed by Western blotting, as given in [33] and the membranes were probed with either anti-Octn2 antibody (1∶100) or anti-caveolin-1 (1∶400), anti-flotillin-1 (1∶500) or anti-phosphoserine (1∶100) antibodies, as given in Figure legends. For two-dimentional electrophoresis, extracted proteins were precipitated with 25% trichloroacetic acid, washed with acetone and subsequently suspended in 8 M urea, 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 50 mM dithiothreitol (DTT), 0.2% ampholine, transferred onto polyacrylamide strips (pH 3–10), incubated for 1 h at 20°C, covered with paraffin oil and incubated for 16 h at 20°C.…”

“…The strips were transferred on 10% polyacrylamide gels with 5% stacking gels and subjected to electrophoresis and Western blot analysis with antibodies indicated in the Figure legend. For immunoprecipitation with Protein A Sepharose™ CL-4B astrocyte homogenates were subjected to preclearance, followed by immunoprecipitation with antibodies (4 µg) against indicated proteins [33]. HEK293 cells transfected with p3xFLAG-CMV14 alone or with plasmids carrying octn2 inserts were lysed in 150 mM NaCl, 50 mM Tris, pH 7.4 supplemented with 1% Nonidet P40, protease inhibitor cocktail and phosphatase inhibitors and after protein estimation subjected either to immunoblotting or to immunoprecipitation with anti-FLAG M2 affinity agarose gel.…”

“…Some transporters have been shown to directly interact with proteins present in cholesterol/sphingolipid-rich microdomains, as caveolin-1 [32]. In astrocytes internalization from rafts was proposed in case of an amino acid transporter ATB 0,+ [33], a protein transporting carnitine with a low affinity [34].…”