Phosphopeptide mapping of proteins ectopically expressed in tissue culture cell lines.
Phosphopeptide mapping of proteins ectopically expressed in tissue culture cell lines.
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DOI：
10.1251/bpo69
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发表时间：
2004-01-01
影响因子：
6.4
通讯作者：
Firulli, Anthony B.
中科院分区：
文献类型：
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作者：
Firulli, Beth A.;Virshup, David M.;Firulli, Anthony B.
磷酸化等翻译后修饰在蛋白质功能调控中起着至关重要的作用。在我们对碱性螺旋-环-螺旋（bHLH）转录因子HAND1的研究中，我们怀疑HAND1在滋养层巨细胞分化过程中被磷酸化，并且组成活性激酶与HAND1的共表达导致了蛋白质二聚化谱的变化。为了准确记录HAND1磷酸化并确定被修饰的位点，我们使用代谢细胞（32）P标记组织培养细胞，这些细胞共同表达标记HAND1的flag表位以及许多活性激酶和磷酸酶亚基。我们使用下面描述的方法生成磷酸化HAND1的磷酸肽图，并将这些修饰与HAND1生物学功能的变化联系起来。
Post-translational modifications such as phosphorylation play a vital role in the regulation of protein function. In our study of the basic Helix-loop-Helix (bHLH) transcription factor HAND1, it was suspected that HAND1 was being phosphorylated during trophoblast giant cell differentiation and that coexpression of a constitutively active kinase with HAND1 resulted in changes in the proteins dimerization profile. In order to accurately document HAND1 phosphorylation and identify the resides being modified, we employed metabolic cell labeling with (32)P of tissue culture cells coexpressing a Flag-epitope tagged HAND1 along with a number of active kinases and phosphatase subunits. We generated phosphopeptide maps of the phosphorylated HAND1 using the methods described below and linked these modifications to changes in HAND1 biological function.