Kerk David, Conley Terry R, Rodriguez Flor A, Tran Hue T, Nimick Mhairi, Muench Douglas G, Moorhead Greg B G
Department of Biology, Point Loma Nazarene University, 3900 Lomaland Drive, San Diego, CA 92106, USA.
Plant J. 2006 May;46(3):400-13. doi: 10.1111/j.1365-313X.2006.02704.x.
Dual-specificity protein phosphatases (DSPs) are important regulators of a wide variety of protein kinase signaling cascades in animals, fungi and plants. We previously identified a cluster of putative DSPs in Arabidopsis (including At3g52180 and At3g01510) in which the phosphatase domain is related to that of laforin, the human protein mutated in Lafora epilepsy. In animal and fungal systems, the laforin DSP and the beta-regulatory subunits of AMP-regulated protein kinase (AMPK) and Snf-1 have all been demonstrated to bind to glycogen by a glycogen-binding domain (GBD). We present a bioinformatic analysis which shows that these DSPs from Arabidopsis, together with other related plant DSPs, share with the above animal and fungal proteins a widespread and ancient carbohydrate-binding domain. We demonstrate that DSP At3g52180 binds to purified starch through its predicted carbohydrate-binding region, and that mutation of key conserved residues reduces this binding. Consistent with its ability to bind exogenous starch, DSP At3g52180 was found associated with starch purified from Arabidopsis plants and suspension cells. Immunolocalization experiments revealed a co-localization with chlorophyll, placing DSP At3g52180 in the chloroplast. Gene-expression data from different stages of the light-dark cycle and across a wide variety of tissues show a strong correlation between the pattern displayed by transcripts of the At3g52180 locus and that of genes encoding key starch degradative enzymes. Taken together, these data suggest the hypothesis that plant DSPs could be part of a protein assemblage at the starch granule, where they would be ideally situated to regulate starch metabolism through reversible phosphorylation events.
双特异性蛋白磷酸酶（DSPs）是动物、真菌和植物中多种蛋白激酶信号级联反应的重要调节因子。我们之前在拟南芥中鉴定出一组假定的DSPs（包括At3g52180和At3g01510），其中磷酸酶结构域与拉福林（Laforin）的磷酸酶结构域相关，拉福林是在拉福拉癫痫（Lafora epilepsy）中发生突变的人类蛋白。在动物和真菌系统中，拉福林DSP以及AMP调节蛋白激酶（AMPK）和Snf-1的β调节亚基均已被证明通过糖原结合结构域（GBD）与糖原结合。我们进行了一项生物信息学分析，结果表明拟南芥中的这些DSPs与其他相关的植物DSPs与上述动物和真菌蛋白共享一个广泛且古老的碳水化合物结合结构域。我们证明DSP At3g52180通过其预测的碳水化合物结合区域与纯化的淀粉结合，并且关键保守残基的突变会降低这种结合。与其结合外源淀粉的能力一致，发现DSP At3g52180与从拟南芥植物和悬浮细胞中纯化的淀粉相关。免疫定位实验揭示了与叶绿素的共定位，将DSP At3g52180定位在叶绿体中。来自明暗周期不同阶段以及多种组织的基因表达数据显示，At3g52180基因座转录本所显示的模式与编码关键淀粉降解酶的基因模式之间存在强烈相关性。综上所述，这些数据提出了一个假说，即植物DSPs可能是淀粉颗粒处蛋白质组装体的一部分，在那里它们将处于理想位置，通过可逆磷酸化事件调节淀粉代谢。
