Profiling Protein Kinases and Other ATP Binding Proteins in Arabidopsis Using Acyl-ATP Probes*
Profiling Protein Kinases and Other ATP Binding Proteins in Arabidopsis Using Acyl-ATP Probes*
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DOI：
10.1074/mcp.m112.026278
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发表时间：
2013-05
影响因子：
7
通讯作者：
Joji Grace Villamor;F. Kaschani;T. Colby;Julian Oeljeklaus;David Zhao;M. Kaiser;M. Patricelli;R. V. D. van der Hoorn
中科院分区：
文献类型：
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作者：
Joji Grace Villamor;F. Kaschani;T. Colby;Julian Oeljeklaus;David Zhao;M. Kaiser;M. Patricelli;R. V. D. van der Hoorn
许多蛋白质的活性是由ATP结合和水解驱动的。在这里，我们探索ATP结合蛋白质组的模式植物拟南芥使用酰基ATP（AcATP）1探针。这些探针靶向ATP结合位点并共价标记ATP结合口袋中的赖氨酸残基。使用生物素化AcATP的基于凝胶的分析表明，标记依赖于pH和二价离子，并且可以被核苷酸竞争。这些AcATP标记的蛋白质中的绝大多数是已知的ATP结合蛋白。我们在凝胶内消化后对标记肽的搜索导致发现标记肽的生物素部分被氧化。凝胶内分析显示两种受体样激酶（RLK）的激酶结构域的分子量低于预期，表明这些RLK失去了细胞外结构域，可能是受体脱落的结果。使用无凝胶平台的修饰肽分析确定了拟南芥蛋白质组中AcATP的242个不同标记位点。每个单独的标记位点的检查揭示了在ATP结合口袋中标记的广泛多样性的ATP结合蛋白的偏好。其中，24个标记的肽来自不同范围的蛋白激酶，包括RLK，促分裂原活化蛋白激酶和钙依赖性激酶。标记位点的显著部分不能被分配到已知的核苷酸结合位点。然而，标记可以与ATP竞争的事实表明，这些标记位点可能代表以前未表征的核苷酸结合位点。光谱计数对表达水平的图说明AcATP探针对蛋白激酶和已知ATP结合蛋白的高特异性。这项工作介绍了在植物蛋白质组中的一个大的多样性的蛋白质的ATP结合活性的剖析。这些数据已存入ProteomeXchange，标识符为PXD 000188。
Many protein activities are driven by ATP binding and hydrolysis. Here, we explore the ATP binding proteome of the model plant Arabidopsis thaliana using acyl-ATP (AcATP)1 probes. These probes target ATP binding sites and covalently label lysine residues in the ATP binding pocket. Gel-based profiling using biotinylated AcATP showed that labeling is dependent on pH and divalent ions and can be competed by nucleotides. The vast majority of these AcATP-labeled proteins are known ATP binding proteins. Our search for labeled peptides upon in-gel digest led to the discovery that the biotin moiety of the labeled peptides is oxidized. The in-gel analysis displayed kinase domains of two receptor-like kinases (RLKs) at a lower than expected molecular weight, indicating that these RLKs lost the extracellular domain, possibly as a result of receptor shedding. Analysis of modified peptides using a gel-free platform identified 242 different labeling sites for AcATP in the Arabidopsis proteome. Examination of each individual labeling site revealed a preference of labeling in ATP binding pockets for a broad diversity of ATP binding proteins. Of these, 24 labeled peptides were from a diverse range of protein kinases, including RLKs, mitogen-activated protein kinases, and calcium-dependent kinases. A significant portion of the labeling sites could not be assigned to known nucleotide binding sites. However, the fact that labeling could be competed with ATP indicates that these labeling sites might represent previously uncharacterized nucleotide binding sites. A plot of spectral counts against expression levels illustrates the high specificity of AcATP probes for protein kinases and known ATP binding proteins. This work introduces profiling of ATP binding activities of a large diversity of proteins in plant proteomes. The data have been deposited in ProteomeXchange with the identifier PXD000188.