Kinome profiling for studying lipopolysaccharide signal transduction in human peripheral blood mononuclear cells
Kinome profiling for studying lipopolysaccharide signal transduction in human peripheral blood mononuclear cells
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DOI：
10.1074/jbc.m405028200
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发表时间：
2004-11-19
影响因子：
4.8
通讯作者：
Peppelenbosch, MP
中科院分区：
文献类型：
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作者：
Diks, SH;Kok, K;Peppelenbosch, MP
DNA阵列技术允许对基因组和转录组进行全面分析，但基于高通量阵列的细胞内信号转导评估仍然很麻烦。本研究的目的是测试一种新的肽阵列技术，用于研究全细胞裂解物中所有激酶的活性，即kinome。用脂多糖刺激前后的人外周血单核细胞裂解液，用玻璃上标记的由192个肽（激酶底物）组成的[γ - p -33] ATP阵列进行体外磷酸化。肽阵列对研究信号转导的有用性通过对脂多糖刺激诱导的磷酸化事件的时间动力学的第一次全面描述得到了证明。进一步分析获得的信号表明，脂多糖激活了p21Ras，并通过直接测量脂多糖刺激的人外周血单个核细胞中p21Ras GTP水平证实了这一点，这是第一次直接证明p21Ras通过刺激Toll受体家族成员而激活。脂多糖刺激细胞的Western blot分析进一步证实了肽阵列技术在研究信号转导方面的有效性，这证实了使用肽阵列获得的信号，也证实了激酶抑制剂对肽阵列磷酸化模式的影响与这些抑制剂的预期作用一致。我们得出的结论是，这种第一个代谢阵列是一种有用的方法来确定一大批激酶的酶活性，提供细胞代谢和信号转导的高通量分析。
The DNA array technique allows comprehensive analysis of the genome and transcriptome, but the high throughput array-based assessment of intracellular signal transduction remains troublesome. The goal of this study was to test a new peptide array technology for studying the activity of all kinases of whole cell lysates, the kinome. Cell lysates from human peripheral blood mononuclear cells before and after stimulation with lipopolysaccharide were used for in vitro phosphorylation with [gamma-P-33] ATP arrays consisting of 192 peptides ( substrates for kinases) spotted on glass. The usefulness of peptide arrays for studying signal transduction was demonstrated by the generation of the first comprehensive description of the temporal kinetics of phosphorylation events induced by lipopolysaccharide stimulation. Furthermore analysis of the signals obtained suggested activation of p21Ras by lipopolysaccharide, and this was confirmed by direct measurement of p21Ras GTP levels in lipopolysaccharide-stimulated human peripheral blood mononuclear cells, which represents the first direct demonstration of p21Ras activation by stimulation of a Toll receptor family member. Further confidence in the usefulness of peptide array technology for studying signal transduction came from Western blot analysis of lipopolysaccharide-stimulated cells, which corroborated the signals obtained using peptide arrays as well as from the demonstration that kinase inhibitors effected peptide array phosphorylation patterns consistent with the expected action of these inhibitors. We conclude that this first metabolic array is a useful method to determine the enzymatic activities of a large group of kinases, offering high throughput analysis of cellular metabolism and signal transduction.