Phosphoproteomics reveals extensive in vivo phosphorylation of Arabidopsis proteins involved in RNA metabolism.
Phosphoproteomics reveals extensive in vivo phosphorylation of Arabidopsis proteins involved in RNA metabolism.
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DOI：
10.1093/nar/gkl429
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发表时间：
2006
影响因子：
14.9
通讯作者：
Hirt, Heribert
中科院分区：
文献类型：
--
作者：
de la Fuente van Bentem, Sergio;Anrather, Dorothea;Roitinger, Elisabeth;Djamei, Armin;Hufnagl, Thomas;Barta, Andrea;Csaszar, Edina;Dohnal, Ilse;Lecourieux, David;Hirt, Heribert
大多数调节途径由蛋白质的可逆磷酸化控制。基于质谱技术的最新发展允许大规模分析蛋白质磷酸化。在这里，我们展示了应用固定化金属亲和层析纯化拟南芥提取物中的磷酸肽。通过液相色谱-串联质谱法（LC-MS/MS/MS）鉴定磷酸肽序列。在22个磷酸化蛋白中共确定了79个独特的磷酸化位点，这些磷酸化蛋白在RNA代谢中具有假定的作用，包括mRNA的剪接。在这些磷蛋白中，鉴定了12个富含Ser/Arg（SR）的剪接因子。一个保守的磷酸化位点被发现在大多数的磷蛋白，包括SR蛋白，这表明这些蛋白质的目标是由相同的或高度相关的蛋白激酶。为了验证这一假设，拟南芥SR蛋白特异性激酶4（SRPK 4），最初被确定为SR蛋白的相互作用进行了测试，其磷酸化的SR蛋白RSp 31的能力。体外激酶试验表明，SRPK 4靶向RSp 31的所有体内磷酸化位点。这些数据表明，植物mRNA剪接机制是磷酸化的主要目标，并且参与RNA代谢的相当多的蛋白质可以被SRPKs靶向。
Most regulatory pathways are governed by the reversible phosphorylation of proteins. Recent developments in mass spectrometry-based technology allow the large-scale analysis of protein phosphorylation. Here, we show the application of immobilized metal affinity chromatography to purify phosphopeptides from Arabidopsis extracts. Phosphopeptide sequences were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS/MS). A total of 79 unique phosphorylation sites were determined in 22 phosphoproteins with a putative role in RNA metabolism, including splicing of mRNAs. Among these phosphoproteins, 12 Ser/Arg-rich (SR) splicing factors were identified. A conserved phosphorylation site was found in most of the phosphoproteins, including the SR proteins, suggesting that these proteins are targeted by the same or a highly related protein kinase. To test this hypothesis, Arabidopsis SR protein-specific kinase 4 (SRPK4) that was initially identified as an interactor of SR proteins was tested for its ability to phosphorylate the SR protein RSp31. In vitro kinase assays showed that all in vivo phosphorylation sites of RSp31 were targeted by SRPK4. These data suggest that the plant mRNA splicing machinery is a major target of phosphorylation and that a considerable number of proteins involved in RNA metabolism may be targeted by SRPKs.
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影响因子：
46.9
作者：
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通讯作者：
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