Krantz D E, Peter D, Liu Y, Edwards R H
Department of Neurology, University of California School of Medicine, San Francisco, California 94143-0435, USA.
J Biol Chem. 1997 Mar 7;272(10):6752-9. doi: 10.1074/jbc.272.10.6752.
The vesicular monoamine transporters (VMATs) package monoamine neurotransmitters into secretory vesicles for regulated exocytotic release. One isoform occurs in the adrenal gland (VMAT1) and another in the brain (VMAT2). To assess their potential for regulation, we have investigated the phosphorylation of the VMATs. Using heterologous expression in Chinese hamster ovary, PC12, and COS cells, we find that rat VMAT2, but not VMAT1, is constitutively phosphorylated. Phosphoamino acid analysis indicates that this phosphorylation occurs on serine residues, and the analysis of VMAT1-VMAT2 chimeras and site-directed mutagenesis localize the phosphorylation sites to serines 512 and 514 at the carboxyl terminus of VMAT2. Since these residues occur in an acidic region, we tested the ability of the acidotropic kinases casein kinase I (CKI) and casein kinase II (CKII) to phosphorylate bacterial fusion proteins containing the carboxyl terminus of VMAT2. Purified CKI and CKII phosphorylate the wild-type carboxyl terminus of VMAT2, but not a double mutant with both serines 512 and 514 replaced by alanine. The protein kinase inhibitor CKI-7 and unlabeled GTP both block in vitro phosphorylation by cell homogenates, indicating a role for CKII and possibly CKI in vivo. Both kinases phosphorylate the VMAT2 fusion protein to a much greater extent than a similar fusion protein containing the carboxyl terminus of VMAT1, consistent with differential phosphorylation of the two transporters observed in intact cells. These results provide the first demonstration of phosphorylation of a vesicular neurotransmitter transporter and a potential mechanism for differential regulation of the two VMATs.
囊泡单胺转运体（VMATs）将单胺类神经递质包装到分泌囊泡中，以便进行调节性胞吐释放。一种异构体存在于肾上腺中（VMAT1），另一种存在于大脑中（VMAT2）。为了评估它们的调节潜力，我们研究了VMATs的磷酸化情况。通过在中国仓鼠卵巢细胞、PC12细胞和COS细胞中进行异源表达，我们发现大鼠VMAT2（而非VMAT1）会发生组成型磷酸化。磷酸氨基酸分析表明这种磷酸化发生在丝氨酸残基上，对VMAT1-VMAT2嵌合体和定点诱变的分析将磷酸化位点定位到VMAT2羧基末端的丝氨酸512和514。由于这些残基位于酸性区域，我们测试了嗜酸性激酶酪蛋白激酶I（CKI）和酪蛋白激酶II（CKII）对含有VMAT2羧基末端的细菌融合蛋白进行磷酸化的能力。纯化的CKI和CKII可磷酸化VMAT2的野生型羧基末端，但不能磷酸化丝氨酸512和514均被丙氨酸取代的双突变体。蛋白激酶抑制剂CKI-7和未标记的GTP均可阻断细胞匀浆的体外磷酸化，表明CKII以及可能的CKI在体内发挥作用。这两种激酶对VMAT2融合蛋白的磷酸化程度远高于含有VMAT1羧基末端的类似融合蛋白，这与在完整细胞中观察到的两种转运体的差异磷酸化情况一致。这些结果首次证明了囊泡神经递质转运体的磷酸化以及两种VMATs差异调节的潜在机制。
