Site directed mutagenesis of Drosophila flightin disrupts phosphorylation and impairs flight muscle structure and mechanics
Site directed mutagenesis of Drosophila flightin disrupts phosphorylation and impairs flight muscle structure and mechanics
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DOI：
10.1007/s10974-007-9120-y
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发表时间：
2007-04-01
影响因子：
2.7
通讯作者：
Vigoreaux, Jim O.
中科院分区：
文献类型：
--
作者：
Barton, Byron;Ayer, Gretchen;Vigoreaux, Jim O.
Flightin是一种肌球蛋白结合蛋白，在果蝇中仅在异步间接飞行肌（IFM）中表达。飞行蛋白的过度磷酸化与肌原纤维组装的完成相一致，并且先于年轻成年人飞行能力的出现。为了研究flightin磷酸化在体内的作用，我们产生了三种flightin null（fln（0））果蝇品系，其表达具有两个（Thr 158，Ser 162）、三个（Ser 139，Ser 141，Ser 145）或全部五个潜在磷酸化位点突变为丙氨酸的突变flightin转基因。这些氨基酸取代导致比正常水平更低的flightin积累和不能拍打翅膀的转基因品系。在IFM蛋白的二维凝胶上，具有五个突变位点的转基因菌株（fln（5STA））缺乏所有磷酸化变体，具有两个突变位点的转基因菌株（fln（2TSA））仅表达九种磷酸化变体中酸性最小的两种，并且具有三个突变位点的转基因菌株（fln（3SA））表达所有九种磷酸化变体，与野生型菌株一样。这些结果表明，磷酸化的Thr158和/或Ser162是必要的后续磷酸化的其他网站。所有三种转基因株在新羽化的成虫中均显示正常但较长的IFM肌节。相比之下，完全成熟的fln（5STA）和fln（2TSA）成人的肌节显示出广泛的崩溃，而fln（3SA）中的肌节则不那么紊乱。表征fln（0）的纤维过度收缩表型在fln（5STA）和fln（2TSA）中完全明显，但在fln（3SA）中部分挽救。从新羽化苍蝇的皮肤纤维力学显示的变化，在粘滞模量的fln（5STA）supercript停止和fln（2TSA），导致在振荡功率输出显着减少。在野生型（fln（+）/fln（+））背景中，fln（5STA）和fln（2TSA）的表达，而非fln（3SA）的表达导致显性负效应，表现为飞行障碍和IFM纤维过度收缩。我们的研究表明，Thr158和/或Ser162是必不可少的flightin功能，并建议，磷酸化的一个或两个残基履行IFM的结构稳定性和力学的重要作用。
Flightin is a myosin rod binding protein that in Drosophila melanogaster is expressed exclusively in the asynchronous indirect flight muscles (IFM). Hyperphosphorylation of flightin coincides with the completion of myofibril assembly and precedes the emergence of flight competency in young adults. To investigate the role of flightin phosphorylation in vivo we generated three flightin null (fln(0)) Drosophila strains that express a mutant flightin transgene with two (Thr158, Ser 162), three (Ser139, Ser141, Ser145) or all five potential phosphorylation sites mutated to alanines. These amino acid substitutions result in lower than normal levels of flightin accumulation and transgenic strains that are unable to beat their wings. On two dimensional gels of IFM proteins, the transgenic strain with five mutant sites (fln(5STA)) is devoid of all phosphovariants, the transgenic strain with two mutant sites (fln(2TSA)) expresses only the two least acidic of the nine phosphovariants, and the transgenic strain with three mutant sites (fln(3SA)) expresses all nine phosphovariants, as the wild-type strain. These results suggest that phosphorylation of Thr158 and/or Ser162 is necessary for subsequent phosphorylation of other sites. All three transgenic strains show normal, albeit long, IFM sarcomeres in newly eclosed adults. In contrast, sarcomeres in fully mature fln(5STA) and fln(2TSA) adults show extensive breakdown while those in fln(3SA) are not as disordered. The fiber hypercontraction phenotype that characterizes fln(0) is fully evident in fln(5STA) and fln(2TSA) but partially rescued in fln(3SA). Mechanics on skinned fibers from newly eclosed flies show alterations in viscous modulus for fln(5STA) supercript stop and fln(2TSA) that result in a significant reduction in oscillatory power output. Expression of fln(5STA) and fln(2TSA), but not fln(3SA) , in a wild-type (fln(+)/fln(+)) background resulted in a dominant negative effect manifested as flight impairments and hypercontracted IFM fibers. Our studies indicate that Thr158 and/or Ser162 are (is) indispensable for flightin function and suggest that phosphorylation of one or both residues fulfills an essential role in IFM structural stability and mechanics.