Hamada-Kawaguchi Noriko, Nishida Yasuyoshi, Yamamoto Daisuke
Department of Developmental Biology and Neurosciences, Tohoku University, Graduate School of Life Sciences, Sendai, Japan.
Department of Biological Science, Graduate School of Science, Nagoya University, Nagoya, Japan.
PLoS One. 2015 Mar 24;10(3):e0121484. doi: 10.1371/journal.pone.0121484. eCollection 2015.
Drosophila Btk29A is the ortholog of mammalian Btk, a Tec family nonreceptor tyrosine kinase whose deficit causes X-linked agammaglobulinemia in humans. The Btk29AficP mutation induces multiple abnormalities in oogenesis, including the growth arrest of ring canals, large intercellular bridges that allow the flow of cytoplasm carrying maternal products essential for embryonic development from the nurse cells to the oocyte during oogenesis. In this study, inactivation of Parcas, a negative regulator of Btk29A, was found to promote Btk29A accumulation on ring canals with a concomitant increase in the ring canal diameter, counteracting the Btk29AficP mutation. This mutation markedly reduced the accumulation of phosphotyrosine on ring canals and in the regions of cell-cell contact, where adhesion-supporting proteins such as DE-cadherin and β-catenin ortholog Armadillo (Arm) are located. Our previous in vitro and in vivo analyses revealed that Btk29A directly phosphorylates Arm, leading to its release from DE-cadherin. In the present experiments, immunohistological analysis revealed that phosphorylation at tyrosine 150 (Y150) and Y667 of Arm was diminished in Btk29AficP mutant ring canals. Overexpression of an Arm mutant with unphosphorylatable Y150 inhibited ring canal growth. Thus Btk29A-induced Y150 phosphorylation is necessary for the normal growth of ring canals. We suggest that the dissociation of tyrosine-phosphorylated Arm from DE-cadherin allows dynamic actin to reorganize, leading to ring canal expansion and cell shape changes during the course of oogenesis.
果蝇Btk29A是哺乳动物Btk的直系同源物，Btk是一种Tec家族非受体酪氨酸激酶，其缺陷会导致人类X连锁无丙种球蛋白血症。Btk29AficP突变在卵子发生过程中诱导多种异常，包括环管生长停滞，环管是一种大型细胞间桥，在卵子发生期间允许携带胚胎发育所需母体产物的细胞质从滋养细胞流向卵母细胞。在本研究中，发现Btk29A的负调节因子Parcas失活可促进Btk29A在环管上的积累，同时环管直径增加，抵消了Btk29AficP突变的影响。该突变显著降低了环管和细胞间接触区域中磷酸酪氨酸的积累，细胞间接触区域是诸如DE-钙黏蛋白和β-连环蛋白直系同源物犰狳（Arm）等黏附支持蛋白所在的位置。我们之前的体外和体内分析表明，Btk29A直接磷酸化Arm，导致其从DE-钙黏蛋白中释放。在本实验中，免疫组织学分析显示，Btk29AficP突变环管中Arm的酪氨酸150（Y150）和Y667位点的磷酸化减少。Y150不可磷酸化的Arm突变体的过表达抑制了环管生长。因此，Btk29A诱导的Y150磷酸化是环管正常生长所必需的。我们认为，酪氨酸磷酸化的Arm与DE-钙黏蛋白的解离使动态肌动蛋白得以重组，从而在卵子发生过程中导致环管扩张和细胞形状改变。
