Yamaguchi Akihiko, Katsu Yoshinao, Matsuyama Michiya, Yoshikuni Michiyasu, Nagahama Yoshitaka
Laboratory of Marine Biology, Department of Animal and Marine Bioresource Sciences, Faculty of Agriculture, Kyushu University, Hakozaki 6-10-1, Higashi-Ku, Fukuoka 812-8581, Japan.
Eur J Cell Biol. 2006 Jun;85(6):501-17. doi: 10.1016/j.ejcb.2006.02.002. Epub 2006 Apr 5.
The nuclear membranes surrounding fish and frog oocyte germinal vesicles (GVs) are supported by the lamina, an internal, mesh-like structure that consists of the protein lamin B3. The mechanisms by which lamin B3 is transported into GVs and is assembled to form the nuclear lamina are not well understood. In this study, we developed a heterogeneous microinjection system in which wild-type or mutated goldfish GV lamin B3 (GFLB3) was expressed in Escherichia coli, biotinylated, and microinjected into Xenopus oocytes. The localization of the biotinylated GFLB3 was visualized by fluorescence confocal microscopy. The results of these experiments indicated that the N-terminal domain plays important roles in both nuclear transport and assembly of lamin B3 to form the nuclear lamina. The N-terminal domain includes a major consensus phosphoacceptor site for the p34(cdc2) kinase at amino acid residue Ser-28. To investigate nuclear lamin phosphorylation, we generated a monoclonal antibody (C7B8D) against Ser-28-phosphorylated GFLB3. Two-dimensional (2-D) electrophoresis of GV protein revealed two major spots of lamin B3 with different isoelectric points (5.9 and 6.1). The C7B8D antibody recognized the pI-5.9 spot but not the pI-6.1 spot. The former spot disappeared when the native lamina was incubated with lambda phage protein phosphatase (lambda-PP), indicating that a portion of the lamin protein was already phosphorylated in the goldfish GV-stage oocytes. GFLB3 that had been microinjected into Xenopus oocytes was also phosphorylated in Xenopus GV lamina, as judged by Western blotting with C7B8D. Thus, lamin phosphorylation appears to occur prior to oocyte maturation in vivo in both these species. Taken together, our results suggest that the balance between phosphorylation by interphase lamin kinases and dephosphorylation by phosphatases regulates the conformational changes in the lamin B3 N-terminal head domain that in turn regulates the continual in vivo rearrangement and remodeling of the oocyte lamina.
围绕鱼类和蛙类卵母细胞生发泡（GVs）的核膜由核纤层支撑，核纤层是一种内部的网状结构，由蛋白质核纤层蛋白B3组成。核纤层蛋白B3被转运到GVs中并组装形成核纤层的机制尚未完全了解。在本研究中，我们开发了一种异源显微注射系统，其中野生型或突变型金鱼GV核纤层蛋白B3（GFLB3）在大肠杆菌中表达、生物素化，然后显微注射到非洲爪蟾卵母细胞中。通过荧光共聚焦显微镜观察生物素化GFLB3的定位。这些实验结果表明，N端结构域在核纤层蛋白B3的核转运和组装形成核纤层过程中都起着重要作用。N端结构域包括一个位于氨基酸残基Ser-28的p34(cdc2)激酶的主要共有磷酸化位点。为了研究核纤层蛋白的磷酸化，我们制备了一种针对Ser-28磷酸化GFLB3的单克隆抗体（C7B8D）。GV蛋白的二维（2-D）电泳显示核纤层蛋白B3有两个主要的等电点不同的斑点（5.9和6.1）。C7B8D抗体识别pI-5.9的斑点，但不识别pI-6.1的斑点。当天然核纤层与λ噬菌体蛋白磷酸酶（λ-PP）一起孵育时，前一个斑点消失，这表明在金鱼GV期卵母细胞中一部分核纤层蛋白已经被磷酸化。通过用C7B8D进行蛋白质免疫印迹分析判断，显微注射到非洲爪蟾卵母细胞中的GFLB3在非洲爪蟾GV核纤层中也被磷酸化。因此，在这两个物种中，核纤层蛋白的磷酸化似乎在体内卵母细胞成熟之前就发生了。综上所述，我们的结果表明，间期核纤层蛋白激酶的磷酸化和磷酸酶的去磷酸化之间的平衡调节了核纤层蛋白B3 N端头部结构域的构象变化，进而调节了卵母细胞核纤层在体内的持续重排和重塑。
