左旋肉碱是必需的易位脂肪酸的线粒体β-氧化,大脑中显示的星形胶质细胞发生的过程。有机阳离子和肉碱质膜转运蛋白OCTN2(SLC22A5)存在于星形胶质细胞中。OCTN2活性和定位先前显示受蛋白激酶C(PKC)调节,尽管没有检测到转运蛋白的磷酸化。在这项研究中,质谱用于鉴定星形胶质细胞中与rOctn2相互作用的伴侣:几种细胞骨架,核糖体,线粒体,热休克蛋白,以及参与贩运和信号通路的蛋白质。信号蛋白的分析表明,Octn2与PP2A磷酸酶催化(C)和结构(A)亚基共沉淀,及其监管B亚基-纹状体,SG2NA,还有Zinedin.Octn2/PP2A复合物主要在内质网中检测到。PKC激活同时增加,肉碱运输和,如免疫荧光和表面生物素化所示,转运体存在于质膜中。它还导致SG2NA的磷酸化,zinedin,和催化亚基,虽然共沉淀,免疫细胞化学,和邻近连接测定实验表明,在与Octn2的复合物中,只有SG2NA的量减少。用冈田酸抑制PP2A不会导致Octn2磷酸化;然而,它消除了观察到的PKC活化作用。我们假设PKC磷酸化SG2NA,导致其从复合物中解离并将Octn2转移到质膜上,导致运输活动增加。观察到的相互作用可能会影响体内的大脑功能,在脂肪酸代谢和肉碱稳态的控制中,已知在某些脑部病变中发生变化。
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.