Identification of c-Src tyrosine kinase substrates using mass spectrometry and peptide microarrays.
Identification of c-Src tyrosine kinase substrates using mass spectrometry and peptide microarrays.
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DOI：
10.1021/pr800198w
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发表时间：
2008-09
影响因子：
4.4
通讯作者：
Pandey, Akhilesh
中科院分区：
文献类型：
--
作者：
Amanchy, Ramars;Zhong, Jun;Molina, Henrik;Chaerkady, Raghotharna;Iwahori, Akiko;Kalume, Dario Eluan;Gronborg, Mads;Joore, Jos;Cope, Leslie;Pandey, Akhilesh
c-Src酪氨酸激酶在生长因子受体、整合素和G蛋白偶联受体下游的信号转导中起关键作用。我们使用稳定同位素标记与氨基酸在细胞培养（SILAC）的方法来确定其他底物的c-Src酪氨酸激酶在人胚肾293 T细胞。我们已经确定了10个已知的基板和相互作用的c-Src和Src家族激酶沿着与26个新的基板。我们已经实验验证了4种新的蛋白质（NICE-4，RNA结合基序10，FUSE结合蛋白1和TRK融合基因）作为直接底物的c-Src使用体外激酶测定和共转染实验。值得注意的是，使用c-Src特异性抑制剂，我们还能够在PDGF信号传导中涉及3种新的底物（RNA结合基序10、EWS 1和Bcl-2相关转录因子）。最后，为了鉴定在新的c-Src底物上被c-Src磷酸化的确切酪氨酸残基，我们设计了定制的肽微阵列，其包含所有可能的含酪氨酸的肽（312种独特的肽）和它们的突变体对应物，所述突变体对应物包含来自14种鉴定的底物的Tyr → Phe取代。利用这个平台，我们鉴定了34个被c-Src磷酸化的肽。我们已经证明，SILAC为基础的定量蛋白质组学方法是适合于非受体酪氨酸激酶的底物的鉴定，并可以与肽微阵列的高通量鉴定底物磷酸肽。本研究描述了使用定量质谱和肽微阵列相结合的c-Src激酶底物的鉴定。一些已知的和新的底物的c-Src的确定使用稳定的同位素标记与氨基酸在细胞培养物（SILAC）耦合到串联质谱。定制肽微阵列被设计用于确定由SILAC实验鉴定的蛋白质底物上的c-Src磷酸化的确切位点。我们证明，SILAC为基础的定量蛋白质组学方法可以耦合肽微阵列高通量识别酪氨酸激酶底物和磷酸化位点。
c-Src tyrosine kinase plays a critical role in signal transduction downstream of growth factor receptors, integrins and G protein-coupled receptors. We used stable isotope labeling with amino acids in cell culture (SILAC) approach to identify additional substrates of c-Src tyrosine kinase in human embryonic kidney 293T cells. We have identified 10 known substrates and interactors of c-Src and Src family kinases along with 26 novel substrates. We have experimentally validated 4 of the novel proteins (NICE-4, RNA binding motif 10, FUSE-binding protein 1 and TRK-fused gene) as direct substrates of c-Src using in vitro kinase assays and cotransfection experiments. Significantly, using a c-Src specific inhibitor, we were also able to implicate 3 novel substrates (RNA binding motif 10, EWS1 and Bcl-2 associated transcription factor) in PDGF signaling. Finally, to identify the exact tyrosine residues that are phosphorylated by c-Src on the novel c-Src substrates, we designed custom peptide microarrays containing all possible tyrosine-containing peptides (312 unique peptides) and their mutant counterparts containing a Tyr → Phe substitution from 14 of the identified substrates. Using this platform, we identified 34 peptides that are phosphorylated by c-Src. We have demonstrated that SILAC-based quantitative proteomics approach is suitable for identification of substrates of nonreceptor tyrosine kinases and can be coupled with peptide microarrays for high-throughput identification of substrate phosphopeptides. This study describes identification of c-Src kinase substrates using a combination of quantitative mass spectrometry and peptide microarrays. A number of known and novel substrates of c-Src were identified using stable isotope labeling with amino acids in cell culture (SILAC) coupled to tandem mass spectrometry. Custom peptide microarrays were designed for determining the exact sites phosphorylated by c-Src on the protein substrates identified by SILAC experiments. We demonstrate that SILAC-based quantitative proteomics approaches can be coupled with peptide microarrays for high-throughput identification of tyrosine kinase substrates and sites of phosphorylation.
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影响因子：
7.4
作者：
Molina, H;Parmigiani, G;Pandey, A
通讯作者：
Pandey, A
影响因子：
64.8
作者：
BAGRODIA, S;CHACKALAPARAMPIL, I;SHALLOWAY, D
通讯作者：
SHALLOWAY, D
影响因子：
4.8
作者：
Huang, C;Liu, JL;Zhan, X
通讯作者：
Zhan, X
影响因子：
4.8
作者：
Nozawa, S;Ohno, T;Shimizu, K
通讯作者：
Shimizu, K
影响因子：
46.9
作者：
Ficarro, SB;McCleland, ML;White, FM
通讯作者：
White, FM