Kempler Karen, Tóth Judit, Yamashita Roxanne, Mapel Gretchen, Robinson Kimberly, Cardasis Helene, Stevens Stanley, Sellers James R, Battelle Barbara-Anne
Whitney Laboratory for Marine Bioscience and the Department of Neuroscience, University of Florida, St. Augustine, Florida 32080, USA.
Biochemistry. 2007 Apr 10;46(14):4280-93. doi: 10.1021/bi062112u. Epub 2007 Mar 17.
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.
尽管III类非常规肌球蛋白具有N端激酶结构域，可能既是信号蛋白又是运动蛋白，但其功能却鲜为人知。鲎肌球蛋白III特别有趣，因为它是一种在光感受器中丰富的磷蛋白，在夜间会被蛋白激酶A磷酸化程度更高。这种增强的夜间磷酸化是对内源性生物钟信号的响应，并且与光感受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，我们确定了鲎肌球蛋白III中发生磷酸化的位点，并研究了其激酶、肌动蛋白结合和肌球蛋白ATP酶活性。我们表明，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的环2内，这是一个重要的肌动蛋白结合区域。我们还确定了环2附近另一个蛋白激酶A和自磷酸化位点以及环2内一个预测的磷酸化位点的磷酸化情况。我们表明，鲎肌球蛋白III的激酶结构域与蛋白激酶A具有一些药理学特性，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。我们得出结论，鲎肌球蛋白III是一种肌动蛋白结合和信号蛋白，并推测肌动蛋白与鲎肌球蛋白III之间的相互作用受环2内及其附近的第二信使介导的磷酸化和其肌球蛋白结构域的自磷酸化调节。
