Hypophosphorylated neurofilament subunits undergo axonal transport more rapidly than more extensively phosphorylated subunits in situ.
Hypophosphorylated neurofilament subunits undergo axonal transport more rapidly than more extensively phosphorylated subunits in situ.
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DOI：
10.1002/1097-0169(200010)47:2
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发表时间：
2000-10
影响因子：
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通讯作者：
C. Jung;J. Yabe;Sangmook Lee;Thomas B. Shea
中科院分区：
文献类型：
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作者：
C. Jung;J. Yabe;Sangmook Lee;Thomas B. Shea
神经丝（NF）的轴突运输一直被认为是由磷酸化调节。我们目前的证据表明，在正常小鼠的视神经轴突，NF轴突运输率与NF磷酸化状态呈负相关。除了200 kDa NF-H和145 kDa NF-M外，CNS的轴突细胞骨架还含有一系列在160-200 kDa之间迁移的NF-H磷酸化变体和在97-145 kDa迁移的NF-M磷酸化变体。虽然NF-H的160 kDa磷酸化变体已经被很好地表征，但是我们确认了先前描述的97 kDa种类作为NF-M的低磷酸化变体的身份，因为（1）脉冲追踪代谢标记确认了97 kDa种类是新的合成产物，其随时间通过磷酸化转化为以145 kDa迁移的形式，（2）97 kDa蛋白与多种NF-M抗体反应，包括对低磷酸化NF-M特异的一种，和（3）去磷酸化将NF-M同种型转化为97 kDa。放射自显影分析后代谢放射性标记表明，hypophosphorylated NF-H和NF-M亚型经历了更快的运输原位比广泛磷酸化亚型，而NF-H亚基轴承发育延迟的C-末端磷酸化表位的速度慢于总200 kDa的NF-H。磷酸化变体的差异转运还突出了这些变体在NF之间不是均匀分布的，而是在一定程度上在不同的NF群体之间分离，尽管可能重叠，表明轴突NF在磷酸化状态方面是不均匀的。
Axonal transport of neurofilaments (NFs) has long been considered to be regulated by phosphorylation. We present evidence that in optic axons of normal mice, the rate of NF axonal transport is inversely correlated with the NF phosphorylation state. In addition to 200 kDa NF-H and 145 kDa NF-M, axonal cytoskeletons from CNS contained a range of phospho-variants of NF-H migrating between 160-200 kDa, and of NF-M migrating at 97-145 kDa. While 160 kDa phospho-variants of NF-H have been well characterized, we confirmed the identity of the previously-described 97 kDa species as a hypophospho-variant of NF-M since (1) pulse-chase metabolic labeling confirmed the 97 kDa species to be a new synthesis product that was converted by phosphorylation over time into a form migrating at 145 kDa, (2) the 97 kDa protein reacted with multiple NF-M antibodies, including one specific for hypophosphorylated NF-M, and (3) dephosphorylation converted NF-M isoforms to 97 kDa. Autoradiographic analyses following metabolic radiolabeling demonstrated that hypophosphorylated NF-H and NF-M isoforms underwent substantially more rapid transport in situ than did extensively phosphorylated isoforms, while NF-H subunits bearing a developmentally delayed C-terminal phospho-epitope transported at a rate slower than that of total 200 kDa NF-H. Differential transport of phospho-variants also highlights that these variants are not homogeneously distributed among NFs, but are segregated to some extent among distinct, although probably overlapping, NF populations, indicating that axonal NFs are not homogeneous with respect to phosphorylation state.