Proline-directed phosphorylation of the dopamine transporter N-terminal domain.
Proline-directed phosphorylation of the dopamine transporter N-terminal domain.
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DOI：
10.1021/bi801696n
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发表时间：
2009-02-10
期刊：
影响因子：
2.9
通讯作者：
Vaughan, Roxanne A.
中科院分区：
文献类型：
--
作者：
Gorentla, Balachandra K.;Moritz, Amy E.;Foster, James D.;Vaughan, Roxanne A.
多巴胺转运蛋白（DAT）在n端丝氨酸和未知苏氨酸上的磷酸化与PKC和底物诱导的转运蛋白活性、亚细胞分布和多巴胺外排的改变同时发生，但磷酸化的残基以及所涉及的蛋白激酶和磷酸酶的特性尚不清楚。作为研究这些问题的一种方法，我们重组表达了大鼠DAT （NDAT）的n端尾部，并在体外检测了其磷酸化和去磷酸化特性。我们发现NDAT可以被催化丝氨酸磷酸化的PKCα、PKA、PKG和CaMKII以及催化苏氨酸磷酸化的ERK1、JNK和p38显著磷酸化。我们鉴定了存在于膜近端脯氨酸定向激酶基元中的Thr53是在体外被ERK1、JNK和p38磷酸化的NDAT位点，并通过肽定位和诱变证实了Thr53在体内被磷酸化。去磷酸化研究表明，蛋白磷酸酶1在体外几乎完全催化了pkc α-磷酸化的NDAT的去磷酸化，与其在体内和体外对天然DAT的作用相似。这些发现证明了多种酶直接识别DAT n端结构域的能力，以及激酶在多个不同位点起作用的能力。NDAT和rDAT磷酸化特征之间的强烈对应表明，在体外对NDAT有活性的酶可能在体内作用于DAT，并表明NDAT对指导未来DAT磷酸化分析的有用性。
Phosphorylation of the dopamine transporter (DAT) on N-terminal serines and unidentified threonines occurs concomitantly with PKC- and substrate-induced alterations in transporter activity, subcellular distribution, and dopamine efflux, but the residues phosphorylated and identities of protein kinases and phosphatases involved are not known. As one approach to investigating these issues we recombinantly expressed the N-terminal tail of rat DAT (NDAT) and examined its phosphorylation and dephosphorylation properties in vitro. We found that NDAT could be phosphorylated to significant levels by PKCα, PKA, PKG, and CaMKII, which catalyzed serine phosphorylation, and ERK1, JNK, and p38, which catalyzed threonine phosphorylation. We identified Thr53, present in a membrane proximal proline-directed kinase motif as the NDAT site phosphorylated in vitro by ERK1, JNK and p38, and confirmed by peptide mapping and mutagenesis that Thr53 is phosphorylated in vivo. Dephosphorylation studies showed that protein phosphatase 1 catalyzed near-complete in vitro dephosphorylation of PKCα-phosphorylated NDAT, similar to its in vivo and in vitro effects on native DAT. These findings demonstrate the ability of multiple enzymes to directly recognize the DAT N-terminal domain and for kinases to act at multiple distinct sites. The strong correspondence between NDAT and rDAT phosphorylation characteristics suggests the potential for the enzymes that are active on NDAT in vitro to act on DAT in vivo and indicates the usefulness of NDAT for guiding future DAT phosphorylation analyses.
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影响因子：
4.8
作者：
Cervinski, MA;Foster, JD;Vaughan, RA
通讯作者：
Vaughan, RA
影响因子：
2.9
作者：
Indarte, Martin;Madura, Jeffry D.;Surratt, Christopher K.
通讯作者：
Surratt, Christopher K.
影响因子：
4.8
作者：
Foster, JD;Pananusorn, B;Vaughan, RA
通讯作者：
Vaughan, RA
影响因子：
4.8
作者：
Johnson, LA;Guptaroy, B;Gnegy, ME
通讯作者：
Gnegy, ME
影响因子：
15.9
作者：
KUHAR, MJ;RITZ, MC;BOJA, JW
通讯作者：
BOJA, JW