Pomiès P, Louis H A, Beckerle M C
Department of Biology, University of Utah, Salt Lake City 84112-0840, USA.
J Cell Biol. 1997 Oct 6;139(1):157-68. doi: 10.1083/jcb.139.1.157.
Members of the cysteine-rich protein (CRP) family are LIM domain proteins that have been implicated in muscle differentiation. One strategy for defining the mechanism by which CRPs potentiate myogenesis is to characterize the repertoire of CRP binding partners. In order to identify proteins that interact with CRP1, a prominent protein in fibroblasts and smooth muscle cells, we subjected an avian smooth muscle extract to affinity chromatography on a CRP1 column. A 100-kD protein bound to the CRP1 column and could be eluted with a high salt buffer; Western immunoblot analysis confirmed that the 100-kD protein is alpha-actinin. We have shown that the CRP1-alpha-actinin interaction is direct, specific, and saturable in both solution and solid-phase binding assays. The Kd for the CRP1-alpha-actinin interaction is 1.8 +/- 0.3 microM. The results of the in vitro protein binding studies are supported by double-label indirect immunofluorescence experiments that demonstrate a colocalization of CRP1 and alpha-actinin along the actin stress fibers of CEF and smooth muscle cells. Moreover, we have shown that alpha-actinin coimmunoprecipitates with CRP1 from a detergent extract of smooth muscle cells. By in vitro domain mapping studies, we have determined that CRP1 associates with the 27-kD actin-binding domain of alpha-actinin. In reciprocal mapping studies, we showed that alpha-actinin interacts with CRP1-LIM1, a deletion fragment that contains the NH2-terminal 107 amino acids (aa) of CRP1. To determine whether the alpha-actinin binding domain of CRP1 would localize to the actin cytoskeleton in living cells, expression constructs encoding epitope-tagged full-length CRP1, CRP1-LIM1(aa 1-107), or CRP1-LIM2 (aa 108-192) were microinjected into cells. By indirect immunofluorescence, we have determined that full-length CRP1 and CRP1-LIM1 localize along the actin stress fibers whereas CRP1-LIM2 fails to associate with the cytoskeleton. Collectively these data demonstrate that the NH2-terminal part of CRP1 that contains the alpha-actinin-binding site is sufficient to localize CRP1 to the actin cytoskeleton. The association of CRP1 with alpha-actinin may be critical for its role in muscle differentiation.
富含半胱氨酸蛋白（CRP）家族的成员是含有LIM结构域的蛋白质，与肌肉分化有关。确定CRP增强肌生成机制的一种策略是表征CRP结合伴侣的种类。为了鉴定与CRP1相互作用的蛋白质，CRP1是成纤维细胞和平滑肌细胞中的一种重要蛋白质，我们将禽平滑肌提取物在CRP1柱上进行亲和层析。一种100-kD的蛋白质与CRP1柱结合，可用高盐缓冲液洗脱；Western免疫印迹分析证实该100-kD蛋白质是α-辅肌动蛋白。我们已经表明，在溶液和固相结合试验中，CRP1-α-辅肌动蛋白相互作用是直接、特异且可饱和的。CRP1-α-辅肌动蛋白相互作用的解离常数（Kd）为1.8±0.3μM。体外蛋白质结合研究的结果得到双标记间接免疫荧光实验的支持，该实验表明CRP1和α-辅肌动蛋白在鸡胚成纤维细胞（CEF）和平滑肌细胞的肌动蛋白应力纤维上共定位。此外，我们已经表明α-辅肌动蛋白与来自平滑肌细胞去污剂提取物中的CRP1共免疫沉淀。通过体外结构域定位研究，我们确定CRP1与α-辅肌动蛋白的27-kD肌动蛋白结合结构域相关联。在反向定位研究中，我们表明α-辅肌动蛋白与CRP1-LIM1相互作用，CRP1-LIM1是一个缺失片段，包含CRP1的NH2末端107个氨基酸（aa）。为了确定CRP1的α-辅肌动蛋白结合结构域是否会定位于活细胞中的肌动蛋白细胞骨架，将编码表位标记的全长CRP1、CRP1-LIM1（aa 1-107）或CRP1-LIM2（aa 108-192）的表达构建体显微注射到细胞中。通过间接免疫荧光，我们确定全长CRP1和CRP1-LIM1定位于肌动蛋白应力纤维，而CRP1-LIM2未能与细胞骨架相关联。这些数据共同表明，CRP1含有α-辅肌动蛋白结合位点的NH2末端部分足以将CRP1定位于肌动蛋白细胞骨架。CRP1与α-辅肌动蛋白的关联可能对其在肌肉分化中的作用至关重要。
