Mutations in PTEN-induced putative kinase 1 associated with recessive parkinsonism have differential effects on protein stability
Mutations in PTEN-induced putative kinase 1 associated with recessive parkinsonism have differential effects on protein stability
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DOI：
10.1073/pnas.0500617102
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发表时间：
2005-04-19
影响因子：
11.1
通讯作者：
Cookson, MR
中科院分区：
文献类型：
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作者：
Beilina, A;Van Der Brug, M;Cookson, MR
PTEN诱导的推定蛋白1(PINK1)基因的几个突变已被报道与隐性帕金森综合征有关。该编码蛋白被预测为针对线粒体的丝氨酸/苏氨酸蛋白激酶。在这项研究中，我们研究了突变对PINK1激酶活性的影响，以及对哺乳动物细胞表达水平和定位的影响。我们选择检测两个点突变：G309D和L347P。G309D最初被报道是稳定的，并在细胞中正确定位。L347P之所以引起人们的兴趣，是因为它在菲律宾存在于可察觉的载频。我们能够确认激酶的活性，并生产出稳定但缺乏活性的人工“激酶死亡”突变体。L347P突变严重破坏了PINK1的稳定性，并显著降低了激酶活性，而G309D在体外对这些参数的影响要小得多。这一发现与基于同源建模的预测一致。我们还通过使用在蛋白质两端标记了myc或GFP的构建物，检测了PINK1在转基因哺乳动物细胞中的定位。这些结果表明，PINK1在IN末端以与线粒体输入一致的方式被加工，但成熟蛋白也存在于细胞质中。这一观察结果的生理相关性尚不清楚，但它意味着PINK1的一部分可能在线粒体中加工后输出。
Several mutations in PTEN-induced putative kinase 1 (PINK1) gene have been reported to be associated with recessive parkinsonism. The encoded protein is predicted to be a Ser/Thr protein kinase targeted to mitochondria. In this study, we have investigated the effects of mutations on PINK1 kinase activity in vitro and on expression levels and localization in mammalian cells. We chose to examine two point mutations: G309D, which was originally reported to be stable and properly localized in cells and L347P, which is of interest because it is present at an appreciable carrier frequency in the Philippines. We were able to confirm kinase activity and produce artificial "kinase-dead" mutants that are stable but lack activity. The L347P mutation grossly destabilizes PINK1 and drastically reduces kinase activity, whereas G309D has much more modest effects on these parameters in vitro. This finding is in line with predictions based on homology modeling. We also examined the localization of PINK1 in transfected mammalian cells by using constructs that were tagged with myc or GFP at either end of the protein. These results show that PINK1 is processed at the IN terminus in a manner consistent with mitochondrial import, but the mature protein also exists in the cytosol. The physiological relevance of this observation is not yet clear, but it implies that a portion of PINK1 may be exported after processing in the mitochondria.