Kim H S, Swierczynski S L, Tuttle J S, Lai W S, Blackshear P J
Office of Clinical Research and Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, 27709, USA.
Dev Biol. 1998 Aug 15;200(2):146-57. doi: 10.1006/dbio.1998.8952.
MARCKS is a widely expressed protein kinase C substrate that is essential for normal prenatal development of the central nervous system in mice. MARCKS-deficient mice exhibit universal perinatal mortality and numerous developmental abnormalities of the brain and retina. To determine which domains of the protein were important in complementing these neurodevelopmental anomalies, we have interbred MARCKS knockout mice with transgenic mice expressing an epitope-tagged human MARCKS transgene that can completely correct the MARCKS-deficient phenotype. Previous structure-function studies showed that a nonmyristoylatable form of MARCKS could correct all of the neuroanatomical abnormalities, and resulted in approximately 25% viable pups that grew to adulthood and were fertile. The present experiment attempted a similar complementation strategy in which a nonmyristoylatable, "pseudo-phosphorylated" form of the protein was used, which has been shown to be almost completely cytosolic in cell expression studies. Surprisingly, this transgene was able to complement almost all of the cerebral anatomical abnormalities characteristic of the knockout mice. However, these mice also exhibited a universal, novel phenotype: profound retinal ectopia, in which retinal tissue was often found in the vitreous humor as well as extraocularly. Retrospective evaluation of the original MARCKS knockout phenotype revealed that this anomaly was present in about 43% of the knockout mice, and was clearly detectable as early as embryonic day 12.5, before retinal cell differentiation begins. These data suggest that a nonmyristoylatable, pseudo-phosphorylated form of MARCKS can complement most if not all cerebral aspects of the MARCKS-deficient phenotype, but that it appears to worsen a retinal phenotype, perhaps by exerting a dominant-negative effect on a coexpressed MARCKS homologue.
MARCKS是一种广泛表达的蛋白激酶C底物，对小鼠中枢神经系统的正常产前发育至关重要。缺乏MARCKS的小鼠表现出普遍的围产期死亡率以及大脑和视网膜的众多发育异常。为了确定该蛋白的哪些结构域在补充这些神经发育异常方面很重要，我们将MARCKS基因敲除小鼠与表达可完全纠正MARCKS缺陷表型的表位标记人MARCKS转基因的转基因小鼠进行了杂交。先前的结构功能研究表明，一种不可肉豆蔻酰化形式的MARCKS可以纠正所有神经解剖学异常，并产生约25%存活至成年且可育的幼崽。本实验尝试了一种类似的互补策略，使用了该蛋白的一种不可肉豆蔻酰化的“假磷酸化”形式，在细胞表达研究中已证明其几乎完全位于细胞质中。令人惊讶的是，这种转基因能够补充基因敲除小鼠几乎所有典型的脑解剖学异常。然而，这些小鼠还表现出一种普遍的新表型：严重的视网膜异位，其中视网膜组织经常出现在玻璃体液以及眼外。对原始MARCKS基因敲除表型的回顾性评估显示，这种异常在约43%的基因敲除小鼠中存在，并且早在胚胎第12.5天视网膜细胞分化开始之前就可清晰检测到。这些数据表明，一种不可肉豆蔻酰化的假磷酸化形式的MARCKS可以补充MARCKS缺陷表型的大部分（如果不是全部）脑方面，但它似乎会使视网膜表型恶化，可能是通过对共表达的MARCKS同源物发挥显性负效应。
