In vivo phosphorylation of regulatory light chain of myosin II in sea urchin eggs and its role in controlling myosin localization and function during cytokinesis.
In vivo phosphorylation of regulatory light chain of myosin II in sea urchin eggs and its role in controlling myosin localization and function during cytokinesis.
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DOI：
10.1002/cm.20246
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发表时间：
2008-02
影响因子：
--
通讯作者：
Ryota Uehara;H. Hosoya;I. Mabuchi
中科院分区：
文献类型：
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作者：
Ryota Uehara;H. Hosoya;I. Mabuchi
肌球蛋白调节轻链 (RLC) Ser19 处的磷酸化（单磷酸化）可促进丝组装并增强非肌肉肌球蛋白的肌动蛋白激活的 ATP 酶活性，而 Ser19 和 Thr18 处的磷酸化（二磷酸化）可进一步增强 ATP 酶活性。然而，尚不清楚哪种类型的磷酸化对于胞质分裂过程中肌球蛋白的调节很重要。在这里，我们通过定量生化和时空细胞学方法研究了海胆卵中单磷酸化和二磷酸化 RLC 的亚细胞定位。在整个胞质分裂过程中，单磷酸化的 RLC 在赤道皮质中占主导地位。抑制肌球蛋白轻链激酶 (MLCK) 可减少皮质和卵裂沟中的单磷酸化 RLC，并阻止收缩环的形成和收缩。两种不同类型的 ROCK 抑制剂给出了不一致的结果：H1152 阻断裂沟中的 RLC 单磷酸化和收缩环的收缩，而 Y27632 既不影响单磷酸化，也不影响细胞分裂。这些结果表明，除了ROCK之外，H1152可能还有其他靶标，ROCK参与卵裂沟中的RLC磷酸化。此外，研究还表明，肌球蛋白重链在卵裂沟中的定位，而不是在皮质中，受到 RLC 单磷酸化抑制的干扰。这些结果表明，两种以上 RLC 激酶的 RLC 单磷酸化在海胆卵分裂中肌球蛋白的调节和定位中起主要作用。
Phosphorylation of myosin regulatory light chain (RLC) at Ser19 (mono-phosphorylation) promotes filament assembly and enhances actin-activated ATPase activity of non-muscle myosin, while phosphorylation at both Ser19 and Thr18 (di-phosphorylation) further enhances the ATPase activity. However, it has not well been addressed which type of phosphorylation is important in regulating myosin during cytokinesis. Here, we investigated subcellular localization in sea urchin eggs of mono-phosphorylated and di-phosphorylated RLC by both quantitative biochemical and spatiotemporal cytological approaches. Mono-phosphorylated RLC was dominant in the equatorial cortex throughout the whole process of cytokinesis. Inhibition of myosin light chain kinase (MLCK) decreased mono-phosphorylated RLC both in the cortex and in the cleavage furrow, and blocked both formation and contraction of the contractile ring. Two different types of ROCK inhibitor gave inconsistent results: H1152 blocked both RLC mono-phosphorylation in the cleavage furrow and contraction of the contractile ring, while Y27632 affected neither the mono-phosphorylation nor cell division. These results suggest that there may be other targets of H1152 than ROCK, which is involved in the RLC phosphorylation in the cleavage furrow. Furthermore, it was revealed that localization of myosin heavy chain in the cleavage furrow, but not in the cortex, was perturbed by inhibition of RLC mono-phosphorylation. These results suggested that RLC mono-phosphorylation by more than two RLC kinases play a main role in regulation and localization of myosin in the dividing sea urchin eggs.