The loop connecting metal-binding domains 3 and 4 of ATP7B is a target of a kinase-mediated phosphorylation.
The loop connecting metal-binding domains 3 and 4 of ATP7B is a target of a kinase-mediated phosphorylation.
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DOI：
10.1021/bi900325k
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发表时间：
2009-06-23
期刊：
影响因子：
2.9
通讯作者：
Lutsenko, Svetlana
中科院分区：
文献类型：
--
作者：
Bartee, Mee Y.;Ralle, Martina;Lutsenko, Svetlana
Cu-ATP酶ATP 7 B（威尔逊病蛋白）将铜转运到高尔基体网络中，用于生物合成掺入血浆铜蓝蛋白，并将过量的铜螯合到内吞囊泡中，用于进一步输出到细胞外。ATP 7 B的活性和细胞内位置受铜水平调节; ATP 7 B在细胞区室之间的运输与蛋白磷酸化水平的变化相关联。磷酸化ATP 7 B的激酶的性质和磷酸化位点的位置都是未知的。我们证明，膜结合的ATP 7 B磷酸化的ATP依赖性，GTP独立的激酶，可以是可溶性或膜相关的。Mg ~（2+）或Mn ~（2+）是激酶活性所必需的。我们进一步表明，重组N-末端结构域的ATP 7 B（N-ATP 7 B）是一个特定的目标激酶介导的磷酸化在体外和细胞。虽然外源性添加铜不是激酶活性所必需的，但铜与N-ATP 7 B的结合显著改变了连接金属结合亚结构域（MBD）的环对蛋白水解的暴露，并促进磷酸化25- 30%。MBD 1 -2和MBD 4 -5接头受到保护，而MBD 2 -3和MBD 3 -4区域保持暴露。对于缺乏29 kDa N-末端片段的ATP 7 B变体（很可能由MBD 1 -3组成），也观察到磷酸化水平显著增加5倍。通过二维凝胶电泳和质谱分析磷酸化肽，指出连接MBD 3和MBD 4的环是磷酸化区域。总之，结果表明，激酶介导的ATP 7 B磷酸化的机制是由N-ATP 7 B的构象状态控制。
Cu-ATPase ATP7B (Wilson’s disease protein) transports copper into the trans-Golgi network for biosynthetic incorporation into ceruloplasmin and sequesters excess copper to endocytic vesicles for further export out of the cell. The activity and intracellular location of ATP7B are regulated by copper levels; the trafficking of ATP7B between cellular compartments is coupled to changes in the level of protein phosphorylation. Neither the nature of the kinase(s) phosphorylating ATP7B nor the location of phosphorylation sites is known. We demonstrate that the membrane-bound ATP7B is phosphorylated by an ATP-dependent, GTP-independent kinase that can be either soluble or membrane-associated. Mg2+ or Mn2+ is necessary for kinase activity. We further show that the recombinant N-terminal domain of ATP7B (N-ATP7B) is a specific target for a kinase-mediated phosphorylation in vitro and in cells. Although exogenous addition of copper is not required for kinase activity, copper binding to N-ATP7B markedly alters the exposure of loops connecting the metal-binding subdomains (MBDs) to proteolysis and facilitates phosphorylation by 25–30%. MBD1–2 and MBD4–5 linkers become protected, while MBD2–3 and MBD3–4 regions remain exposed. A significant, 5-fold increase in the level of phosphorylation is also observed for the ATP7B variant that lacks the 29 kDa N-terminal fragment (mostly likely comprised of MBD1–3). Analysis of phosphorylated peptides by two-dimensional gel electrophoresis and mass spectrometry points to the loop connecting MBD3 andMBD4 as a region of phosphorylation. Altogether, the results suggest a mechanism in which kinase-mediated phosphorylation of ATP7B is controlled by a conformational state of N-ATP7B.
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影响因子：
4.8
作者：
Tsivkovskii, R;MacArthurs, BC;Lutsenko, S
通讯作者：
Lutsenko, S
影响因子：
3.9
作者：
Vanderwerf, SM;Lutsenko, S
通讯作者：
Lutsenko, S
影响因子：
4.8
作者：
Tsivkovskii, R;Eisses, JF;Lutsenko, S
通讯作者：
Lutsenko, S
DOI：
10.1016/s0006-291x(03)00329-2
发表时间：
2003-03-28
影响因子：
3.1
作者：
Voskoboinik, I;Fernando, R;Camakaris, J
通讯作者：
Camakaris, J
影响因子：
2.9
作者：
DiDonato, M;Hsu, HF;Sarkar, B
通讯作者：
Sarkar, B