title: Protein phosphatase PP2A - a novel interacting partner of carnitine transporter OCTN2 (SLC22A5) in rat astrocytes.
abstract: l-Carnitine is essential for translocation of fatty acids for their mitochondrial β-oxidation, a process shown in the brain to take place in astrocytes. Organic cation and carnitine plasma membrane transporter OCTN2 (SLC22A5) is present in astrocytes. OCTN2 activity and localization were previously shown to be regulated by protein kinase C (PKC), although no phosphorylation of the transporter was detected. In this study, mass spectrometry was used to identify rOctn2-interacting partners in astrocytes: several cytoskeletal, ribosomal, mitochondrial, heat-shock proteins, as well as proteins involved in trafficking and signaling pathways. The analysis of signaling proteins shows that Octn2 co-precipitated with PP2A phosphatase catalytical (C) and structural (A) subunits, and with its regulatory B"' subunits - striatin, SG2NA, and zinedin. The Octn2/PP2A complex is mainly detected in endoplasmic reticulum. PKC activation increases both, carnitine transport and, as shown by immunofluorescence and surface biotinylation, transporter presence in plasma membrane. It also results in phosphorylation of SG2NA, zinedin, and catalytical subunit, although co-precipitation, immunocytochemistry, and proximity ligation assay experiments showed that only the amount of SG2NA decreased in the complex with Octn2. PP2A inhibition with okadaic acid does not lead to Octn2 phosphorylation; however, it abolishes observed effects of PKC activation. We postulate that PKC phosphorylates SG2NA, resulting in its dissociation from the complex and transfer of Octn2 to the plasma membrane, leading to increased transporter activity. The observed interaction could affect brain functioning in vivo, both in fatty acid metabolism and in control of carnitine homeostasis, known to change in certain brain pathologies.
authors.affiliation: Laboratory of Transport through Biomembranes, Department of Molecular and Cellular Neurobiology, Nencki Institute of Experimental Biology of Polish Academy of Sciences, Warsaw, Poland.
authors.forename: Barbara
authors.initials: B
authors.lastname: Juraszek
authors.affiliation: Laboratory of Transport through Biomembranes, Department of Molecular and Cellular Neurobiology, Nencki Institute of Experimental Biology of Polish Academy of Sciences, Warsaw, Poland.
authors.forename: Katarzyna A
authors.initials: KA
authors.lastname: Nałęcz
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D019319:Okadaic Acid / Q000494:pharmacology; D027701:Organic Cation Transport Proteins / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D054648:Protein Phosphatase 2 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D000074058:Solute Carrier Family 22 Member 5
chemical_list: D027701:Organic Cation Transport Proteins; C115441:Slc22a5 protein, rat; D000074058:Solute Carrier Family 22 Member 5; D019319:Okadaic Acid; D054648:Protein Phosphatase 2
keywords: Astrocytes; OCTN2; PP2A phosphatase; SG2NA; striatin; zinedin