Detection of a variety of Ser/Thr protein kinases using a synthetic peptide with multiple phosphorylation sites.
Detection of a variety of Ser/Thr protein kinases using a synthetic peptide with multiple phosphorylation sites.
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使用具有多个磷酸化位点的合成肽检测多种 Ser/Thr 蛋白激酶。
DOI：
10.1093/oxfordjournals.jbchem.a022567
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发表时间：
1999
影响因子：
2.7
通讯作者：
H. Fujisawa
中科院分区：
文献类型：
--
作者：
I. Kameshita;S. Taketani;A. Ishida;H. Fujisawa
我们开发了一种具有多个磷酸化位点的新多肽，我们将其命名为MULDE，用于检测粗细胞提取物中的各种蛋白激酶。多肽KKRKSSLRRWSPLTPRQMSFDC被设计成在单一的多肽中包含各种Ser/Thr蛋白激酶的共同序列，包括cAMP依赖的蛋白激酶、蛋白激酶C、MAP激酶和Ca(2+)/钙调蛋白依赖的蛋白激酶。使用MULDE的凝胶内蛋白激酶分析被发现是非常有用的，以分析蛋白激酶的活性变化，以响应各种细胞外刺激。用5个具有不同磷酸化位点的多重类似物进一步确定了所检测到的蛋白激酶的底物特异性。
A novel peptide with multiple phosphorylation sites, which we designated as multide, was developed to detect a wide variety of protein kinases in crude cell extracts. Multide, KKRKSSLRRWSPLTPRQMSFDC, has been designed to contain consensus sequences for various Ser/Thr protein kinases including cAMP-dependent protein kinase, protein kinase C, MAP kinases, and Ca(2+)/calmodulin-dependent protein kinases in a single peptide. In-gel protein kinase assay using multide was found to be very useful for analyzing the activities of protein kinases that are altered in response to various extracellular stimuli. The substrate specificities of the protein kinases thus detected were further determined by using five multide analogs with different phosphorylation sites.