Activation of the 43 kDa inositol polyphosphate 5-phosphatase by 14-3-3 zeta
Activation of the 43 kDa inositol polyphosphate 5-phosphatase by 14-3-3 zeta
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DOI：
10.1021/bi9708085
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发表时间：
1997-12-09
期刊：
影响因子：
2.9
通讯作者：
Mitchell, CA
中科院分区：
文献类型：
--
作者：
Campbell, JK;Gurung, R;Mitchell, CA
43 kDa的肌醇多磷酸5-磷酸酶（5-磷酸酶）在信号终止反应中水解并使第二信使分子肌醇1,4,5-三磷酸[Ins(1,4,5)P-3]和肌醇1,3,4,5-四磷酸失活。最近的研究表明，血小板蛋白pleckstrin与43 kDa 5-磷酸酶形成复合物，并激活2倍的Ins(1,4,5)P-3水解[Auethavekiat， V., Abrams， C. S., & Majerus， P. W. (1997) J. Biol。[j].中国科学，1999,17(2):1 - 7。我们现在发现，另一种血小板蛋白14-3-3 zeta与43 kDa的5-磷酸酶形成复合物，从而激活Ins(1,4,5)P-3的水解。pleckstrin和14-3-3 zeta都含有一个或多个pleckstrin同源结构域，它们都存在于血小板细胞质中，并且都与其他信号蛋白二聚并形成复合物。纯化后的血小板pleckstrin和14-3-3 zeta分别使43 kDa 5-磷酸酶对Ins(1,4,5)P-3的水解率提高了1.9倍和3.8倍，但对75 kDa 5-磷酸酶没有激活作用。我们已经证明了14-3-3 zeta活化5-磷酸酶的机制是由43 kDa 5-磷酸酶和14-3-3 zeta之间形成特异性复合物引起的。重组43 kDa 5-磷酸酶结合重组谷胱甘肽s -转移酶(GST)/14-3-3 zeta融合蛋白，但不是单独的GST，固定在谷胱甘肽- sepharose上。一个潜在的14-3-3结合基序位于43 kDa，而不是75 kDa的5-磷酸酶。motif“(RSESEE)-R-363”存在于43 kDa 5-磷酸酶的催化结构域附近。与推测的14-3-3结合基序相对应的合成肽与纯化的I-125-14-3-3具有特异性，饱和结合，K-d为92 nM。此外，血小板胞质5-磷酸酶结合重组14-3-3 zeta固定在谷胱甘肽- sepharose。因此，14-3-3 zeta可在人血小板中激活43 kDa 5-磷酸酶，从而可在未受刺激的血小板中防止Ins(1,4,5) p -3介导的钙释放的产生。
The 43 kDa inositol polyphosphate 5-phosphatase (5-phosphatase) hydrolyzes and thereby inactivates the second messenger molecules inositol 1,4,5-trisphosphate [Ins(1,4,5)P-3] and inositol 1,3,4,5-tetrakisphosphate in a signal terminating reaction. Recent studies have shown that the platelet protein pleckstrin forms a complex with the 43 kDa 5-phosphatase and activates Ins(1,4,5)P-3 hydrolysis 2-fold [Auethavekiat, V., Abrams, C. S., & Majerus, P. W. (1997) J. Biol. Chern. 272, 1786-1790]. We now show that another platelet protein, 14-3-3 zeta, forms a complex with the 43 kDa 5-phosphatase and thereby activates the hydrolysis of Ins(1,4,5)P-3. Both pleckstrin and 14-3-3 zeta contain one or more pleckstrin-homology domains, both are present in platelet cytosol, and both dimerize and form complexes with other signalling proteins. Purified platelet pleckstrin and 14-3-3 zeta enhanced the rate of the hydrolysis of Ins(1,4,5)P-3 by the 43 kDa 5-phosphatase 1.9- and 3.8-fold, respectively, but did not activate the 75 kDa 5-phosphatase. We have demonstrated that the mechanism of 5-phosphatase activation by 14-3-3 zeta results from specific complex formation between the 43 kDa 5-phosphatase and 14-3-3 zeta. Recombinant 43 kDa 5-phosphatase bound to recombinant glutathione S-transferase (GST)/14-3-3 zeta fusion protein, but not GST alone, immobilized on glutathione-Sepharose. A potential 14-3-3 binding motif was located in the 43 kDa, but not the 75 kDa, 5-phosphatase. The motif ''(RSESEE)-R-363'' is present in close proximity to the proposed catalytic domain of the 43 kDa 5-phosphatase. A synthetic peptide corresponding to the putative 14-3-3 binding motif demonstrated specific, saturable binding to purified I-125-14-3-3, with a K-d of 92 nM. In addition, platelet cytosolic 5-phosphatase bound to recombinant 14-3-3 zeta immobilized on glutathione-Sepharose. Thus, 14-3-3 zeta serves in human platelets to activate the 43 kDa 5-phosphatase and may thereby function to prevent generation of Ins(1,4,5)P-3-mediated calcium release in unstimulated platelets.