The Role of X/Y Linker Region and N-terminal EF-hand Domain in Nuclear Translocation and Ca2+ Oscillation-inducing Activities of Phospholipase Cζ, a Mammalian Egg-activating Factor*
The Role of X/Y Linker Region and N-terminal EF-hand Domain in Nuclear Translocation and Ca2+ Oscillation-inducing Activities of Phospholipase Cζ, a Mammalian Egg-activating Factor*
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DOI：
10.1074/jbc.m603473200
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发表时间：
2006-09
影响因子：
4.8
通讯作者：
K. Kuroda;Masahiko Ito;T. Shikano;T. Awaji;A. Yoda;H. Takeuchi;K. Kinoshita;S. Miyazaki
中科院分区：
文献类型：
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作者：
K. Kuroda;Masahiko Ito;T. Shikano;T. Awaji;A. Yoda;H. Takeuchi;K. Kinoshita;S. Miyazaki
精子特异性磷脂酶C-Zeta(PLCζ)可引起细胞内钙离子振荡，从而激活卵子，并通过注射编码PLCζ的CRNA在小鼠卵子中表达，该基因由N端的四个EF-Hand结构域(Ef1-Ef4)、X和Y催化结构域以及C端C2结构域组成。在未受精卵或受精后的1-细胞胚胎中注射cRNA，表达标记有荧光蛋白Venus的PLCζ突变体，分析这些活性。核定位信号(NLS)存在于X/Y连接区的374-381处。用谷氨酸取代Arg376、Lys377、Arg378、Lys379或Lys381可使细胞核失去易位，而钙振荡则保持不变。X结构域C端的Lys299和/或Lys301或EF1的N端的Trp13、Phe14或Val18的点突变也没有核靶向性。前者使细胞内钙离子振荡诱导活性丧失，后者则显著抑制其活性。与金星融合的短序列374-383显示出活跃的易位到COS-7细胞的核中，而296-309或1-19则没有。尽管存在这些特殊区域，但由于EF1和EF2-4或C2结构域的缺失，这两种活性都被剥夺了。因此，PLCζ主要通过NLS和可能的调控位点进入细胞核，但似乎不仅是酶活性所必需的，而且核转位能力也需要协调的三维结构，可能是由X/Y连接子的折叠和紧密的EF/C2接触形成的，就像PLCδ1一样。
Sperm-specific phospholipase C-zeta (PLCζ) causes intracellular Ca2+ oscillations and thereby egg activation and is accumulated into the formed pronucleus (PN) when expressed in mouse eggs by injection of cRNA encoding PLCζ, which consists of four EF-hand domains (EF1-EF4) in the N terminus, X and Y catalytic domains, and C-terminal C2 domain. Those activities were analyzed by expressing PLCζ mutants tagged with fluorescent protein Venus by injection of cRNA into unfertilized eggs or 1-cell embryos after fertilization. Nuclear localization signal (NLS) existed at 374–381 in the X/Y linker region. Nuclear translocation was lost by replacement of Arg376, Lys377, Arg378, Lys379, or Lys381 with glutamate, whereas Ca2+ oscillations were conserved. Nuclear targeting was also absent for point mutation of Lys299 and/or Lys301 in the C terminus of X domain, or Trp13, Phe14, or Val18 in the N terminus of EF1. Ca2+ oscillation-inducing activity was lost by the former mutation and was remarkably inhibited by the latter. A short sequence 374–383 fused with Venus showed active translocation into the nucleus of COS-7 cells, but 296–309 or 1–19 did not. Despite the presence of these special regions, both activities were deprived by deletion of not only EF1 but also EF2–4 or C2 domain. Thus, PLCζ is driven into the nucleus primarily by the aid of NLS and putative regulatory sites, but coordinated three-dimensional structure, possibly formed by a folding in the X/Y linker and close EF/C2 contact as in PLCδ1, seems to be required not only for enzymatic activity but also for nuclear translocation ability.