Barradeau S, Imaizumi-Scherrer T, Weiss M C, Faust D M
Unité de Génétique de la Différenciation, Département de Biologie Moléculaire, Institut Pasteur, Unité de Recherche Associée 1773 du Centre National de la Recherche Scientifique, 25, Rue du Dr Roux, 75724 Paris Cedex 15, France.
Proc Natl Acad Sci U S A. 2001 Apr 24;98(9):5037-42. doi: 10.1073/pnas.081393598. Epub 2001 Apr 10.
In skeletal muscle, transcription of the gene encoding the mouse type Ialpha (RIalpha) subunit of the cAMP-dependent protein kinase is initiated from the alternative noncoding first exons 1a and 1b. Here, we report that activity of the promoter upstream of exon 1a (Pa) depends on two adjacent E boxes (E1 and E2) in NIH 3T3-transfected fibroblasts as well as in intact muscle. Both basal activity and MyoD transactivation of the Pa promoter require binding of the upstream stimulating factors (USF) to E1. E2 binds either an unknown protein in a USF/E1 complex-dependent manner or MyoD. Both E2-bound proteins seem to function as repressors, but with different strengths, of the USF transactivation potential. Previous work has shown localization of the RIalpha protein at the neuromuscular junction. Using DNA injection into muscle of plasmids encoding segments of RIalpha or RIIalpha fused to green fluorescent protein, we demonstrate that anchoring at the neuromuscular junction is specific to RIalpha subunits and requires the amino-terminal residues 1-81. Mutagenesis of Phe-54 to Ala in the full-length RIalpha-green fluorescent protein template abolishes localization, indicating that dimerization of RIalpha is essential for anchoring. Moreover, two other hydrophobic residues, Val-22 and Ile-27, are crucial for localization of RIalpha at the neuromuscular junction. These amino acids are involved in the interaction of the Caenorhabditis elegans type Ialpha homologue R(CE) with AKAP(CE) and for in vitro binding of RIalpha to dual A-kinase anchoring protein 1. We also show enrichment of dual A-kinase anchoring protein 1 at the neuromuscular junction, suggesting that it could be responsible for RIalpha tethering at this site.
在骨骼肌中，编码环磷酸腺苷（cAMP）依赖性蛋白激酶小鼠Iα（RIα）亚基的基因转录起始于可变的非编码第一外显子1a和1b。在此，我们报告外显子1a（Pa）上游启动子的活性在NIH 3T3转染的成纤维细胞以及完整肌肉中取决于两个相邻的E盒（E1和E2）。Pa启动子的基础活性和MyoD反式激活都需要上游刺激因子（USF）与E1结合。E2以USF/E1复合物依赖性方式结合一种未知蛋白或MyoD。两种与E2结合的蛋白似乎都作为USF反式激活潜能的抑制剂，但强度不同。先前的研究表明RIα蛋白定位于神经肌肉接头处。通过将编码与绿色荧光蛋白融合的RIα或RIIα片段的质粒DNA注射到肌肉中，我们证明在神经肌肉接头处的锚定是RIα亚基特有的，并且需要氨基末端残基1 - 81。在全长RIα - 绿色荧光蛋白模板中将苯丙氨酸 - 54突变为丙氨酸会消除定位，表明RIα的二聚化对于锚定至关重要。此外，另外两个疏水残基缬氨酸 - 22和异亮氨酸 - 27对于RIα在神经肌肉接头处的定位至关重要。这些氨基酸参与秀丽隐杆线虫Iα同源物R(CE)与AKAP(CE)的相互作用以及RIα与双A激酶锚定蛋白1的体外结合。我们还显示双A激酶锚定蛋白1在神经肌肉接头处富集，表明它可能负责RIα在此位点的拴系。
