A regulatory light chain of ciliary outer arm dynein in Tetrahymena thermophila
A regulatory light chain of ciliary outer arm dynein in Tetrahymena thermophila
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DOI：
10.1074/jbc.m008412200
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发表时间：
2001-06-08
影响因子：
4.8
通讯作者：
Hamasaki, T
中科院分区：
文献类型：
--
作者：
Christensen, ST;Guerra, C;Hamasaki, T
纤毛搏动频率主要由外臂动力蛋白（22 S动力蛋白）调节，Chilcote和约翰逊（Chilcote，T. J.，和约翰逊，K. A.（1990）J. BiolChem，256，17257-17266）先前研究了分离的四膜虫22 S动力蛋白，鉴定了蛋白质p34，其显示cAMP依赖性磷酸化。在此，我们进一步表征了p34的分子生物化学，证明它是草履虫p34中22 S动力蛋白调节轻链p29的功能性直系同源物，在cAMP存在下，在分离的轴丝中硫代磷酸化，与22 S动力蛋白共纯化，而不与内臂动力蛋白（14 S动力蛋白）共纯化，含有磷酸化的p34的分离的22 S动力蛋白显示与其未磷酸化的对应物相比，体外微管易位速度增加约70%。提取的p34反弹到分离的22 S动力蛋白从四膜虫或草履虫，但不14 S动力蛋白从任何纤毛虫。放射性标记的p34与22 S动力蛋白的结合与p29竞争，磷酸化的p34不存在于从缺乏外臂的突变体分离的轴丝中。双向凝胶电泳和荧光成像显示至少有5个磷酸化的p34相关的斑点，与p34的多个磷酸化位点或p34的多个亚型相一致。这些新的特征表明，包括p34在内的一类外臂动力蛋白轻链通过磷酸化调节微管滑动速度，从而调节纤毛搏动频率。
Ciliary beat frequency is primarily regulated by outer arm dyneins (22 S dynein), Chilcote and Johnson (Chilcote, T. J., and Johnson, K. A. (1990) J. Biol Chem, 256, 17257-17266) previously studied isolated Tetrahymena 22 S dynein, identifying a protein p34, which showed cAMP-dependent phosphorylation, Here, we characterize the molecular biochemistry of p34 further, demonstrating that it is the functional ortholog of the 22 S dynein regulatory light chain, p29, in Paramecium, p34, thiophosphorylated in isolated axonemes in the presence of cAMP, co-purified with 22 S dynein and not with inner arm dynein (14 S dynein), Isolated 22 S dynein containing phosphorylated p34 showed similar to 70% increase in in vitro microtubule translocation velocity compared with its unphosphorylated counterpart. Extracted p34 rebound to isolated 22 S dynein from either Tetrahymena or Paramecium but not to 14 S dynein from either ciliate. Binding of radiolabeled p34 to 22 S dynein was competitive with p29, Phosphorylated p34 was not present in axonemes isolated from a mutant lacking outer arms. Two-dimensional gel electrophoresis followed by phosphorimaging revealed at least five phosphorylated p34-related spots, consistent with multiple phosphorylation sites in p34 or perhaps multiple isoforms of p34, These new features suggest that a class of outer arm dynein light chains including p34 regulates microtubule sliding velocity and consequently ciliary beat frequency through phosphorylation.