Chou Chih-Ming, Chen Yi-Chung, Lee Ming-Ting, Chen Gen-Der, Lu I-Ching, Chen Shui-Tsung, Huang Chang-Jen
Department of Biochemistry, Taipei Medical University, Taipei, Taiwan.
Biochem Biophys Res Commun. 2006 Feb 17;340(3):767-75. doi: 10.1016/j.bbrc.2005.12.090. Epub 2005 Dec 27.
We have previously identified a novel protein kinase, pk146, in the brain of Tetraodon. In the present study, we cloned the homologous protein kinase gene encoding a protein of 385 amino acid residues from zebrafish. The overall amino acid sequence and the kinase domain of zebrafish BSK146 shows 48% and 69% identity to that of rat sbk, a SH3-containing serine/threonine protein kinase. By whole-mount in situ hybridization and RT-PCR, the expression of bsk146 mRNA was mainly in the brain. To explore the in vivo function of BSK146 during zebrafish development, we used morpholino knockdown approach and found that BSK146 morphants displayed enlarged hindbrain ventricle and smaller eyes. Whole-mount in situ hybridization was further performed to analyze the brain defects in BSK146-MO-injected embryos. The expression of brain-specific markers, such as otx2, pax2.1, and krox20, was found normal in morphant embryos at 24hpf, while expression of pax2.1 exerted changes in midbrain-hindbrain boundary and hindbrain in morphant embryos at 48hpf. These data suggest that BSK146 may play an important role in later ventricle expansion in zebrafish brain development. Although the recombinant BSK146 protein produced in insect cells was active and could phosphorylate both histone H1 and histone 2B, the endogenous substrate of BSK146 in the embryonic brain of zebrafish is not clear at the present time and needs further investigation.
我们之前在红鳍东方鲀的大脑中鉴定出一种新型蛋白激酶pk146。在本研究中，我们从斑马鱼中克隆了同源蛋白激酶基因，该基因编码一个由385个氨基酸残基组成的蛋白质。斑马鱼BSK146的整体氨基酸序列和激酶结构域与大鼠sbk（一种含SH3的丝氨酸/苏氨酸蛋白激酶）的相应序列分别具有48%和69%的同一性。通过整体原位杂交和RT-PCR分析，发现bsk146 mRNA主要在大脑中表达。为了探究BSK146在斑马鱼发育过程中的体内功能，我们采用了吗啉代敲低方法，发现BSK146敲低胚胎表现出后脑脑室扩大和眼睛变小。进一步进行整体原位杂交以分析注射了BSK146-MO的胚胎中的脑部缺陷。在24hpf时，发现脑特异性标记物如otx2、pax2.1和krox20在敲低胚胎中的表达正常，而在48hpf时，pax2.1在敲低胚胎的中脑-后脑边界和后脑的表达出现了变化。这些数据表明，BSK146可能在斑马鱼大脑发育后期的脑室扩张中发挥重要作用。尽管在昆虫细胞中产生的重组BSK146蛋白具有活性，并且能够磷酸化组蛋白H1和组蛋白2B，但目前尚不清楚斑马鱼胚胎大脑中BSK146的内源性底物，这需要进一步研究。
