The peptide microarray "ChloroPhos1.0" identifies new phosphorylation targets of plastid casein kinase II (pCKII) in Arabidopsis thaliana.
The peptide microarray "ChloroPhos1.0" identifies new phosphorylation targets of plastid casein kinase II (pCKII) in Arabidopsis thaliana.
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DOI：
10.1371/journal.pone.0108344
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Baginsky S
中科院分区：
文献类型：
--
作者：
Schönberg A;Bergner E;Helm S;Agne B;Dünschede B;Schünemann D;Schutkowski M;Baginsky S
我们报告了基于先前确定的叶绿体蛋白磷酸化位点的肽微阵列的发展。在玻片上，905个多肽在9个重复中被鉴定为15mers。我们使用微阵列进行体外磷酸化实验，并专门评估了叶绿体酪蛋白激酶II （pCKII）的肽底物光谱。为此，利用肝素- sepharose色谱法富集拟南芥和白芥叶绿体中的原生pCKII，并将其与重组拟南芥pCKII在芯片上的活性进行比较。这三种激酶制剂磷酸化了一组类似的肽，这些肽与对照实验中牛心脏蛋白激酶a （PKA）磷酸化的肽明显不同。大多数pCKII磷酸化靶点参与质体基因表达，支持pCKII早期命名为质体转录激酶（plastid transcription kinase， PTK）。此外，我们发现Alb3是pCKII底物，对于将光收集复合物亚基（LHC）整合到类囊体膜中至关重要。以重组野生型Alb3和磷酸化位点突变体为底物，在体外更详细地表征了质体CKII磷酸化活性，确定了S424为pCKII磷酸化位点。我们的数据表明，肽芯片ChloroPhos1.0是一种合适的工具，可以在体外鉴定新的激酶下游靶点，随后可以通过体内实验进行验证。
We report the development of a peptide microarray based on previously determined phosphorylation sites in chloroplast proteins. Altogether, 905 peptides were spotted as 15mers in nine replicates onto glass slides. We used the microarray for in vitro phosphorylation experiments and specifically assessed the peptide substrate spectrum of chloroplast casein kinase II (pCKII). To this end, native pCKII from Arabidopsis thaliana and Sinapis alba chloroplasts was enriched by Heparin-Sepharose chromatography and its activity on the microarray was compared to the activity of a recombinant Arabidopsis pCKII. All three kinase preparations phosphorylated a similar set of peptides that were clearly distinct from those phosphorylated by bovine heart protein kinase A (PKA) in control experiments. The majority of the pCKII phosphorylation targets are involved in plastid gene expression, supporting the earlier denomination of pCKII as plastid transcription kinase (PTK). In addition we identified Alb3 as pCKII substrate that is essential for the integration of light-harvesting complex subunits (LHC) into the thylakoid membrane. Plastid CKII phosphorylation activity was characterized in greater detail in vitro with recombinant wildtype Alb3 and phosphorylation site mutants as substrates, establishing S424 as the pCKII phosphorylation site. Our data show that the peptide microarray ChloroPhos1.0 is a suitable tool for the identification of new kinase downstream targets in vitro that can be validated subsequently by in vivo experiments.
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影响因子：
11.4
作者：
Hubbard, SR
通讯作者：
Hubbard, SR
影响因子：
10.5
作者：
Popescu, Sorina C.;Popescu, George V.;Dinesh-Kumar, Savithramma P.
通讯作者：
Dinesh-Kumar, Savithramma P.
影响因子：
5.1
作者：
Baginsky, S;Tiller, K;Link, G
通讯作者：
Link, G
影响因子：
5.1
作者：
Baginsky, S;Tiller, K;Link, G
通讯作者：
Link, G
影响因子：
3.5
作者：
KANEKATSU, M;MUNAKATA, H;OHTSUKI, K
通讯作者：
OHTSUKI, K