Institute of Molecular Biology and Genetics, 150 Zabolotnogo Street, Kyiv 03680, Ukraine.
Biochem Biophys Res Commun. 2010 Aug 20;399(2):307-12. doi: 10.1016/j.bbrc.2010.07.080. Epub 2010 Jul 24.
SH3 domains function as protein-protein interaction modules in assembly of signalling and endocytic protein complexes. Here we report investigations of the mechanism of regulation of the binding properties of the SH3 domains of intersectin (ITSN1) and Src kinase by alternative splicing. Comparative sequence analysis of ITSN1 and Src genes revealed the conservation of alternatively spliced microexons affecting the structure of the SH3 domains in vertebrates. We show that neuron-specific ITSN1 transcripts containing the microexon 20 that encodes five amino acid residues within the SH3A domain are expressed in zebrafish from the earliest stages of the development of the nervous system. Models of alternative isoforms of the ITSN1 SH3A domain revealed that the insertion encoded by the microexon is located at the beginning of the n-Src loop of this domain causing a shift of negatively charged amino acids towards the interaction interface. Mutational analysis confirmed the importance of translocation of these negatively charged amino acids for interaction with dynamin 1. We also identified a residue within the microexon-encoded insert in the SH3 domain of brain-specific variant of Src that abolishes interaction of the domain with dynamin 1. Thus microexons provide a mechanism for the control of tissue-specific interactions of ITSN1 and Src with their partners.
SH3 结构域作为蛋白质-蛋白质相互作用模块，参与信号转导和内吞蛋白复合物的组装。在这里，我们报告了对衔接蛋白（ITSN1）和Src 激酶 SH3 结构域结合特性的调节机制的研究，该调节机制由选择性剪接引起。对 ITSN1 和 Src 基因的比较序列分析表明，在脊椎动物中，选择性剪接的微外显子影响 SH3 结构域的结构，从而保守存在。我们表明，在斑马鱼神经系统发育的最早阶段，就表达了含有编码 SH3A 结构域内五个氨基酸残基的微外显子 20 的神经元特异性 ITSN1 转录本。对 ITSN1 SH3A 结构域的选择性异构体模型的研究表明，微外显子编码的插入位于该结构域的 n-Src 环的起始处，导致带负电荷的氨基酸向相互作用界面转移。突变分析证实了这些带负电荷的氨基酸向相互作用界面转移对于与动力蛋白 1 相互作用的重要性。我们还在Src 的脑特异性变体的 SH3 结构域的微外显子编码插入中鉴定出一个残基，该残基消除了该结构域与动力蛋白 1 的相互作用。因此，微外显子为 ITSN1 和 Src 与其伴侣的组织特异性相互作用的控制提供了一种机制。
