Tyrosine-specific dephosphorylation-phosphorylation with alkaline phosphatases and epidermal growth factor receptor kinase as evidenced by 31P NMR spectroscopy.
Tyrosine-specific dephosphorylation-phosphorylation with alkaline phosphatases and epidermal growth factor receptor kinase as evidenced by 31P NMR spectroscopy.
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酪氨酸特异性去磷酸化-用碱性磷酸酶和表皮生长因子受体激酶进行磷酸化，如 31P NMR 波谱所证实。
DOI：
10.1093/oxfordjournals.jbchem.a121975
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发表时间：
1987
影响因子：
2.7
通讯作者：
S. Taniguchi
中科院分区：
文献类型：
--
作者：
K. Takahashi;M. Shimidzu;H. Shindo;T. Kawamoto;M. Nishi;U. Matsumoto;S. Taniguchi
用~（31）P核磁共振波谱研究了小牛肠碱性磷酸酶（Al_2O_3）和大肠杆菌碱性磷酸酶（Al_2O_3）对磷酸化氨基酸的脱磷酸作用，以及人表皮样癌细胞A431表皮生长因子（EGF）受体激酶对牛血清白蛋白（BSA）的磷酸化作用。在单底物体系中，磷酸酪氨酸（P-Tyr）和磷酸丝氨酸（P-Ser）与丙氨酸脱磷酸的初始速率基本相同。然而，在双底物系统（P-Tyr + P-Ser）中，P-Tyr与P-Ser的初始速率的比率为2.4至4.5，这取决于所采用的缓冲液和pH条件。这首次证实了在游离氨基酸水平上AlPases对P-Tyr的特异性超过对P-Ser的特异性。在整个过程的稳定期，P-Ser的去磷酸化变得缓慢相比，在一个底物系统中的P-Tyr。P-Ser速率的降低在双底物系统中进一步明显。对于这种显着的效果，丝氨酸的再磷酸化是负责的，如在含有丝氨酸，Pi和丙氨酸的反应混合物中所证明的。EGF受体激酶磷酸化的BSA在中性pH下在0 ppm附近呈现尖锐的31 P共振，远离cAMP依赖性蛋白激酶磷酸化的组蛋白H1的峰位置（4.9 ppm）。这些NMR数据直接证明BSA仅在酪氨酰残基磷酸化，而组蛋白H1的磷酸化在丝氨酰残基。
The dephosphorylation of phospho-amino acids with alkaline phosphatase (AlPase) from calf intestine or Escherichia coli and the phosphorylation of bovine serum albumin (BSA) with epidermal growth factor (EGF) receptor kinase from human A431 epidermoid carcinoma cells were investigated by 31P NMR spectroscopy. The initial rates of the dephosphorylation of phospho-tyrosine (P-Tyr) and phosphoserine (P-Ser) with AlPase were essentially the same in the one-substrate system. In the two-substrate system (P-Tyr plus P-Ser), however, the ratio of the initial rate for P-Tyr vs. P-Ser was 2.4 to 4.5 depending on the buffer and pH conditions employed. This substantiates for the first time the specificity of AlPases to P-Tyr over P-Ser at the free amino acid level. In the stationary phase of the overall process, the dephosphorylation of P-Ser became slow compared to that of P-Tyr in the one-substrate system. The decrease in the rate for P-Ser was further pronounced in the two-substrate system. For this remarkable effect, the rephosphorylation of serine was responsible, as demonstrated in the reaction mixture containing serine, Pi, and AlPase. BSA phosphorylated by EGF receptor kinase exhibited sharp 31P resonances around 0 ppm at neutral pH, far distant from the peak positions (4.9 ppm) of histone H1 phosphorylated by cAMP-dependent protein kinase. These NMR data are directed evidence that BSA was phosphorylated exclusively at the tyrosyl residues, whereas the phosphorylation of histone H1 was at the seryl residues.