Disruption of the A-kinase anchoring domain in flagellar radial spoke protein 3 results in unregulated axonemal cAMP-dependent protein kinase activity and abnormal flagellar motility
Disruption of the A-kinase anchoring domain in flagellar radial spoke protein 3 results in unregulated axonemal cAMP-dependent protein kinase activity and abnormal flagellar motility
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DOI：
10.1091/mbc.e06-02-0095
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发表时间：
2006-06-01
影响因子：
3.3
通讯作者：
Sale, Winfield S.
中科院分区：
文献类型：
--
作者：
Gaillard, Anne R.;Fox, Laura A.;Sale, Winfield S.
对衣藻鞭毛轴丝的生化研究表明，放射状辐条蛋白 (RSP) 3 是一种 A 激酶锚定蛋白 (AKAP)。为了确定 PKA 锚定在轴丝中的生理作用，用在 PKA 结合域中含有突变的 RSP3 基因转化 RSP3 突变体 pf14。对几个独立转化体的分析表明，转化细胞表现出不寻常的表型：一部分细胞正常游泳；其余的细胞无力抽搐或瘫痪。异常/麻痹的运动并不是由于径向辐条组装的明显缺陷，并且表型与突变体RSP3共分离。我们推测瘫痪是由于轴丝 cAMP 依赖性蛋白激酶 (PKA) 的靶向和调节失败所致。为了测试这一点，在不存在或存在 PKA 抑制剂的情况下进行了脱膜细胞的再激活实验。重要的是，重新激活的细胞模型中的运动模拟了活细胞表型，运动细胞和瘫痪细胞的比例几乎相等。 PKA 抑制剂导致运动细胞数量增加两倍，从而挽救了瘫痪。这些结果证实鞭毛 RSP3 是 AKAP，并揭示 PKA 结合域的突变导致轴突 PKA 活性不受调节并抑制正常运动。
Biochemical studies of Chlamydomonas flagellar axonemes revealed that radial spoke protein (RSP) 3 is an A-kinase anchoring protein (AKAP). To determine the physiological role of PKA anchoring in the axoneme, an RSP3 mutant, pf14, was transformed with an RSP3 gene containing a mutation in the PKA-binding domain. Analysis of several independent transformants revealed that the transformed cells exhibit an unusual phenotype: a fraction of the cells swim normally; the remainder of the cells twitch feebly or are paralyzed. The abnormal/paralyzed motility is not due to an obvious deficiency of radial spoke assembly, and the phenotype cosegregates with the mutant RSP3. We postulated that paralysis was due to failure in targeting and regulation of axonemal cAMP-dependent protein kinase (PKA). To test this, reactivation experiments of demembranated cells were performed in the absence or presence of PKA inhibitors. Importantly, motility in reactivated cell models mimicked the live cell phenotype with nearly equal fractions of motile and paralyzed cells. PKA inhibitors resulted in a twofold increase in the number of motile cells, rescuing paralysis. These results confirm that flagellar RSP3 is an AKAP and reveal that a mutation in the PKA binding domain results in unregulated axonernal PKA activity and inhibition of normal motility.