Swierczynski S L, Siddhanti S R, Tuttle J S, Blackshear P J
Howard Hughes Medical Institute, Durham, North Carolina 27710, USA.
Dev Biol. 1996 Oct 10;179(1):135-47. doi: 10.1006/dbio.1996.0246.
The myristoylated alanine-rich C kinase substrate, or MARCKS protein, is a widely expressed, prominent substrate for protein kinase C. Although the exact function of MARCKS has not been elucidated, targeted disruption of the MARCKS gene (Macs) in mice has shown that MARCKS plays a crucial role in the development of the central nervous system. Mice deficient in MARCKS exhibited universal perinatal death with defects in neurulation, fusion of the cerebral hemispheres, formation of the great forebrain commissures, and retinal and cortical lamination (Stumpo et al., Proc. Natl. Acad. Sci. USA 92, 944-948, 1995). In the present studies, a transgene consisting of approximately 3.4 kb of promoter from the human MARCKS gene (MACS), with an epitope tag sequence inserted at the carboxyl terminus of the MARCKS coding region, was able to complement completely MARCKS deficiency in mice. Thus, the human transgene contained all of the elements necessary for normal developmental expression of MARCKS. To test the importance of MARCKS myristoylation to its developmental role, an otherwise identical transgene was constructed in which the glycine at the amino terminus of MARCKS was mutated to an alanine. This mutation, which resulted in the expression of nonmyristoylated MARCKS, was successful in partially rescuing the Macs null phenotype. Specifically, about 25% of these mice survived the perinatal period; these survivors appeared to develop normally except for slightly decreased body size. In both the survivors and the nonsurvivors, all of the known anatomical defects associated with MARCKS deficiency were corrected by expression of the nonmyristoylated human protein. These results indicate that myristoylation of MARCKS is not required for the protein to correct many of the developmental abnormalities characteristic of its deficiency.
豆蔻酰化富含丙氨酸的蛋白激酶C底物，即MARCKS蛋白，是蛋白激酶C广泛表达的重要底物。尽管MARCKS的确切功能尚未阐明，但在小鼠中对MARCKS基因（Macs）进行靶向破坏显示，MARCKS在中枢神经系统发育中起关键作用。缺乏MARCKS的小鼠在围产期普遍死亡，伴有神经管形成缺陷、大脑半球融合、大脑前联合形成以及视网膜和皮质分层异常（Stumpo等人，《美国国家科学院院刊》92，944 - 948，1995）。在本研究中，一个由约3.4 kb人MARCKS基因（MACS）启动子组成的转基因，在MARCKS编码区羧基末端插入了一个表位标签序列，能够完全弥补小鼠中MARCKS的缺陷。因此，该人类转基因包含了MARCKS正常发育表达所需的所有元件。为了测试MARCKS豆蔻酰化对其发育作用的重要性，构建了一个除MARCKS氨基末端的甘氨酸突变为丙氨酸外其他完全相同的转基因。这种突变导致非豆蔻酰化MARCKS的表达，成功部分挽救了Macs基因敲除的表型。具体而言，约25%的这些小鼠在围产期存活下来；这些存活小鼠除体型略有减小外，似乎发育正常。在存活和未存活的小鼠中，所有与MARCKS缺乏相关的已知解剖学缺陷都通过非豆蔻酰化人类蛋白的表达得到了纠正。这些结果表明，MARCKS的豆蔻酰化对于该蛋白纠正其缺乏所特有的许多发育异常并非必需。
