DEMONSTRATION OF THE PHOSPHORYLATION-DEPENDENT INTERACTION OF TRYPTOPHAN-HYDROXYLASE WITH THE 14-3-3-PROTEIN
DEMONSTRATION OF THE PHOSPHORYLATION-DEPENDENT INTERACTION OF TRYPTOPHAN-HYDROXYLASE WITH THE 14-3-3-PROTEIN
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DOI：
10.1006/bbrc.1993.1796
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发表时间：
1993-07-15
影响因子：
3.1
通讯作者：
ICHIMURA, T
中科院分区：
文献类型：
--
作者：
FURUKAWA, Y;IKUTA, N;ICHIMURA, T
色氨酸羟化酶的磷酸化依赖性激活的分子机制进行了研究，相对于14-3-3蛋白的作用。用纯化的TRH和14-3-3蛋白重建的系统的重新检查表明，TRH活性的水平与TRH中的Ca 2 +/钙调素或cAMP依赖性磷酸化的程度相关。该实验证实了活化需要14-3-3蛋白，但是加入到测定混合物中的14-3-3蛋白既不影响磷酸化的程度也不影响磷酸化的特异性。然而，在基于蝶啶的亲和柱上对测定混合物的分析表明TRH和14-3-3蛋白之间形成复合物，其中复合物的形成依赖于TRH的磷酸化。磷酸化的TRH和14-3-3蛋白之间的复合物也可以通过分析先前由内源性Ca 2 +/钙调蛋白依赖性蛋白激酶磷酸化的脑干粗提取物来检测。因此，14-3-3蛋白似乎是磷酸化依赖性TRH结合蛋白，其相互作用导致TRH的活化。
The molecular mechanism of the phosphorylation-dependent activation of tryptophan hydroxylase is studied with respect to the role of the 14-3-3 protein. Reexamination of the system reconstituted with the purified TRH and the 14-3-3 protein showed that the level of the TRH activity correlated with the extent of the Ca2+/calmodulin- or the cAMP-dependent phosphorylation in TRH. The experiment confirmed the requirement of the 14-3-3 protein for the activation, but the 14-3-3 protein added into the assay mixture did not affect either the extent nor the specificity of the phosphorylation. However, the analysis of the assay mixture on a pteridine-based affinity column indicated the formation of a complex between TRH and the 14-3-3 protein, where the complex formation depended on the phosphorylation of TRH. The complex between the phosphorylated TRH and the 14-3-3 protein could also be detected by the analysis of crude brainstem extract previously phosphorylated by endogeneous Ca2+/calmodulin-dependent protein kinase. The 14-3-3 protein, therefore, appears to be a phosphorylation-dependent TRH-binding protein whose interaction causes the activation of TRH.