Characterization of a cAMP-stimulated cAMP phosphodiesterase in Dictyostelium discoideum
Characterization of a cAMP-stimulated cAMP phosphodiesterase in Dictyostelium discoideum
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DOI：
10.1074/jbc.m209648200
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发表时间：
2003-04-18
影响因子：
4.8
通讯作者：
Schaap, P
中科院分区：
文献类型：
--
作者：
Meima, ME;Weening, KE;Schaap, P
一种环核苷酸磷酸二酯酶（PdeE）在Dictyostelium中具有两个环核苷酸结合基序和一个双核Zn2+结合结构域。在其他真核生物中，Dictyostelium结构域与pre-mRNA切割和聚腺苷酸化特异性因子的73-kDa亚基具有最大的同源性。Dictyostelium PdeE基因在聚集期间表达最高水平，其破坏导致camp -磷酸二酯酶活性的丧失。pdeE无效突变体表现出正常的cAMP诱导的cGMP反应，cAMP诱导的cAMP接力增加1.5倍。pdee -黄色荧光蛋白（YFP）融合构建体的过表达会抑制聚集和cAMP中继反应的丧失，但细胞可以与野生型细胞协同聚集。采用免疫沉淀法部分纯化PdeE-YFP融合蛋白，并对其进行生化表征。PdeE和它的盘基骨柱同源物PdeD在pH 7.0时都具有最大的活性。这两种酶的活性都需要二价阳离子。常见的辅助因子Zn2+和Mg2+在10 mm处最大激活PdeE和PdeD，而Mn2+将酶的激活水平提高了4倍，一半最大激活在10到100 gm之间。PdeE是一种变结构酶，与cAMP的4倍活性相似，一半最大激活发生在10 muM左右，表观K-m接近1 mm。cGMP的降解速率比cAMP低6倍。cGMP和8-Br-cAMP都不是PdeE活性的有效激活剂。
A cyclic nucleotide phosphodiesterase, PdeE, that harbors two cyclic nucleotide binding motifs and a binuclear Zn2+-binding domain was characterized in Dictyostelium. In other eukaryotes, the Dictyostelium domain shows greatest homology to the 73-kDa subunit of the pre-mRNA cleavage and polyadenylation specificity factor. The Dictyostelium PdeE gene is expressed at its highest levels during aggregation, and its disruption causes the loss of a cAMP-phosphodiesterase activity. The pdeE null mutants show a normal cAMP-induced cGMP response and a 1.5-fold increase of cAMP-induced cAMP relay. Overexpression of a PdeE-yellow fluorescent protein (YFP) fusion construct causes inhibition of aggregation and loss of the cAMP relay response, but the cells can aggregate in synergy with wild-type cells. The PdeE-YFP fusion protein was partially purified by immunoprecipitation and biochemically characterized. PdeE and its Dictyostelium ortholog, PdeD, are both maximally active at pH 7.0. Both enzymes require bivalent cations for activity. The common cofactors Zn2+ and Mg2+ activated PdeE and PdeD maximally at 10 mm, whereas Mn2+ activated the enzymes to 4-fold higher levels, with half-maximal activation between 10 and 100 gm. PdeE is an allosteric enzyme, which is similar to4-fold activated by cAMP, with half-maximal activation occurring at about 10 muM and an apparent K-m of similar to1 mm. cGMP is degraded at a 6-fold lower rate than cAMP. Neither cGMP nor 8-Br-cAMP are efficient activators of PdeE activity.