Identification and characterization of a mammalian 14-kDa phosphohistidine phosphatase
Identification and characterization of a mammalian 14-kDa phosphohistidine phosphatase
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DOI：
10.1046/j.1432-1033.2002.03206.x
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发表时间：
2002-10-01
期刊：
影响因子：
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通讯作者：
Zetterqvist, Ö
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文献类型：
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作者：
Ek, P;Pettersson, G;Zetterqvist, Ö
与蛋白质丝氨酸/苏氨酸和酪氨酸磷酸化相比，真核生物中蛋白质组氨酸磷酸化的研究较少。为了通过用含磷酸组氨酸的肽段琥珀酰 - 丙氨酸 - 组氨酸（P） - 脯氨酸 - 苯丙氨酸 - 对硝基苯胺（磷酸肽I）探测猪肝胞质溶胶来纠正这种情况，我们观察到一种对冈田酸和EDTA不敏感的磷酸酶活性。这表明存在一种不同于蛋白磷酸酶1、2A和2C的磷酸组氨酸磷酸酶。经过1000倍纯化达到表观同质性后，得到一种14 kDa的磷酸酶，在pH 7.5时，以7 μM磷酸肽I为底物，其比活性为3 μmol·min⁻¹·mg⁻¹。通过质谱对纯化的猪源酶进行部分氨基酸序列测定，发现其与一种代表人类9号染色体上迄今功能未知基因的人类序列具有相似性。从人胚胎肾细胞cDNA文库进行分子克隆，然后进行表达和纯化，得到一种分子量为13700 Da的蛋白质，对磷酸肽I具有9 μmol·min⁻¹·mg⁻¹的对EDTA不敏感的磷酸组氨酸磷酸酶活性。对一组磷酸丝氨酸、磷酸苏氨酸和磷酸酪氨酸肽段未检测到活性。Northern杂交分析表明，人磷酸组氨酸磷酸酶mRNA优先存在于心脏和骨骼肌中。这些结果为研究真核生物组氨酸磷酸化/去磷酸化提供了一种新工具。
Protein histidine phosphorylation in eukaryotes has been sparsely studied compared to protein serine/threonine and tyrosine phosphorylation. In an attempt to rectify this by probing porcine liver cytosol with the phosphohistidine-containing peptide succinyl-Ala-His(P)-Pro-Phe-p -nitroanilide (phosphopeptide I), we observed a phosphatase activity that was insensitive towards okadaic acid and EDTA. This suggested the existence of a phosphohistidine phosphatase different from protein phosphatase 1, 2A and 2C. A 1000-fold purification to apparent homogeneity gave a 14-kDa phosphatase with a specific activity of 3 mumol.min(-1) .mg(-1) at pH 7.5 with 7 mum phosphopeptide I as substrate. Partial amino-acid sequence determination of the purified porcine enzyme by MS revealed similarity with a human sequence representing a human chromosome 9 gene of hitherto unknown function. Molecular cloning from a human embryonic kidney cell cDNA-library followed by expression and purification, yielded a protein with a molecular mass of 13 700 Da, and an EDTA-insensitive phosphohistidine phosphatase activity of 9 mumol.min(-1) .mg(-1) towards phosphopeptide I. No detectable activity was obtained towards a set of phosphoserine-, phosphothreonine-, and phosphotyrosine peptides. Northern blot analysis indicated that the human phosphohistidine phosphatase mRNA was present preferentially in heart and skeletal muscle. These results provide a new tool for studying eukaryotic histidine phosphorylation/dephosphorylation.