Expression Studies of Other Unconventional Myosins

肌球蛋白VIIa是一种非传统的肌球蛋白，广泛表达于从阿米巴到哺乳动物的生物体中，已被证明在细胞粘附和吞噬中发挥重要作用。 我们研究了果蝇肌球蛋白VIIa在Sf 9细胞中的表达。 我们已经表明，这种肌球蛋白具有类似于进行性马达的高占空比动力学，但我们也表明，即使全长分子在Sf 9细胞中表达，它也不容易二聚化。 我们已经研究了全长肌球蛋白VIIa和各种C-末端截短片段的酶活性的调节。 全长肌球蛋白VIIa（FLM 7a）的Vmax约为每秒1次，但对肌动蛋白的表观亲和力较低，需要30-50 μ M acin才能达到最大激活的一半。 各种C-末端截短的片段具有相似的Vmax，但是对于半最大活化（0.5-1 μ M）需要少得多的肌动蛋白。 这意味着在5 μ M肌动蛋白时，片段的活性接近最大，而FLM 7a的活性仍然几乎没有被激活。 即使是最后99个氨基酸的去除也足以引起这种显著的活性变化。 我们探索了这种调节的结构基础，通过使用电子显微镜中的单粒子分析。 我们发现，在ATP的存在下，FLM 7a被紧密折叠成一个紧凑的结构，这样的肌球蛋白马达结构域不能被辨别，而在ATP的情况下，该分子是更广泛的，并显示出一个清晰可辨的马达结构域。 尾部的小片段的重叠也伴随着延伸的构象，这使我们认为紧凑的结构代表了分子的抑制状态。 我们现在正在寻找结合伴侣或翻译后修饰，将折叠的，无活性的FLM 7a转化为延伸的，活性的形式。 肌球蛋白XVIIa有一个大的预测卷曲螺旋形成序列，但肌球蛋白的结构还没有在单分子水平上检查。 我们表达了小鼠全长肌球蛋白XVIIIa，并显示它具有与肌球蛋白II相似的长尾。 我们正在研究这条尾巴是否能形成细丝。 关于III类非常规肌球蛋白的功能知之甚少，尽管它们具有N-末端激酶结构域，它们可能既是信号蛋白又是马达蛋白。鲎肌球蛋白III特别有趣，因为它是光感受器中丰富的磷蛋白，在夜间被蛋白激酶A磷酸化程度更高。这种增强的夜间磷酸化反应来自内源性生物钟的信号，并与感光器结构和功能的显著变化相关。我们试图了解鲎肌球蛋白III及其磷酸化在光感受器中的作用。在这里，我们确定的网站，成为磷酸化的鲎肌球蛋白III和研究其激酶，肌动蛋白结合，肌球蛋白ATP酶的活动。我们发现，鲎肌球蛋白III具有激酶活性，蛋白激酶A和自磷酸化的主要位点位于肌球蛋白结构域，一个重要的肌动蛋白结合区的环2内。我们还确定了一个额外的蛋白激酶A的磷酸化和自磷酸化位点附近的环2，和预测的磷酸化位点内的环2。我们发现，鲎肌球蛋白III的激酶结构域与蛋白激酶A共享一些药理学特性，并且它是一种潜在的视蛋白激酶。最后，我们证明，鲎肌球蛋白III结合肌动蛋白，但缺乏ATP酶活性。我们的结论是，鲎肌球蛋白III是一个肌动蛋白结合和信号蛋白，并推测肌动蛋白和鲎肌球蛋白III之间的相互作用调节的第二信使介导的磷酸化和自磷酸化的肌球蛋白结构域内和附近的环2。

Myosin VIIa is an unconventional myosin widely expressed in organisms ranging from amoebae to mammals that has been shown to play vital roles in cell adhesion and phagocytosis. We have studied Drosophila myosin VIIa that was expressed in Sf9 cells. We have shown that this myosin has high duty ratio kinetics similar to that of processive motors, but we also showed that it did not easily dimerize even if the full lenght molecule was expressed in Sf9 cells. We have examined the regulation of enzymatic activity of full length myosin VIIa and various C-terminally truncated fragments. Full length myosin VIIa (FLM7a) has a Vmax of about 1 per sec, but has a low apparent affinity for actin, requiring 30-50 uM acin for half-maximal activation. Various C-terminally truncated fragments have a similar Vmax, but much less actin is required for half maximal activation (0.5-1 uM). This means that at 5 uM actin the activity of the fragments is near maximal whereas that of the FLM7a is still barely activated. Removal of even the last 99 amino acids is sufficient to cause this remarkable change in activity. We explored the structural basis for this regulation by using single particle analysis in the electron microscope. We find that in the presence of ATP FLM7a is tightly folded into a compact structure such that the myosin motor domain cannot be discerned whereas in the absence of ATP the molecule is more extended and shows a clearly distinguishable motor domain. Removable of small bits of the tail also is accompanied by the extended conformation leading us to suggest that the compact structure represents an inhibited state of the molecule. We are now searching for binding partners or post-translation modifications that will convert the foled, inactive FLM7a into an extend, active form. Myosin XVIIa has a large predicted coiled-coil forming sequence, but the structure of the myosin has not been examined on a single molecule level. We expressed full length myosin XVIIIa from mouse and showed that it has a long tail similar to that of myosin II. We are examining whether this tail can form filaments. Little is known about the functions of class III unconventional myosins although, with an N-terminal kinase domain, they are potentially both signaling and motor proteins. Limulus myosin III is particularly interesting because it is a phosphoprotein abundant in photoreceptors that becomes more heavily phosphorylated at night by protein kinase A. This enhanced nighttime phosphorylation occurs in response to signals from an endogenous circadian clock and correlates with dramatic changes in photoreceptor structure and function. We seek to understand the role of Limulus myosin III and its phosphorylation in photoreceptors. Here we determined the sites that become phosphorylated in Limulus myosin III and investigated its kinase, actin binding, and myosin ATPase activities. We show that Limulus myosin III exhibits kinase activity and that a major site for both protein kinase A and autophosphorylation is located within loop 2 of the myosin domain, an important actin binding region. We also identify the phosphorylation of an additional protein kinase A and autophosphorylation site near loop 2, and a predicted phosphorylation site within loop 2. We show that the kinase domain of Limulus myosin III shares some pharmacological properties with protein kinase A, and that it is a potential opsin kinase. Finally, we demonstrate that Limulus myosin III binds actin but lacks ATPase activity. We conclude that Limulus myosin III is an actin-binding and signaling protein and speculate that interactions between actin and Limulus myosin III are regulated by both second messenger mediated phosphorylation and autophosphorylation of its myosin domain within and near loop 2.