Wu R, Durick K, Songyang Z, Cantley L C, Taylor S S, Gill G N
Department of Biology, University of California San Diego, La Jolla, California 92093-0650, USA.
J Biol Chem. 1996 Jul 5;271(27):15934-41. doi: 10.1074/jbc.271.27.15934.
LIM domains, Cys-rich motifs containing approximately 50 amino acids found in a variety of proteins, are proposed to direct protein*protein interactions. To identify structural targets recognized by LIM domains, we have utilized random peptide library selection, the yeast two-hybrid system, and glutathione S-transferase fusions. Enigma contains three LIM domains within its carboxyl terminus and LIM3 of Enigma specifically recognizes active but not mutant endocytic codes of the insulin receptor (InsR) (Wu, R. Y., and Gill, G. N. (1994) J. Biol. Chem. 269, 25085-25090). Interaction of two random peptide libraries with glutathione S-transferase-LIM3 of Enigma indicated specific binding to Gly-Pro-Hyd-Gly-Pro-Hyd-Tyr-Ala corresponding to the major endocytic code of InsR. Peptide competition demonstrated that both Pro and Tyr residues were required for specific interaction of InsR with Enigma. In contrast to LIM3 of Enigma binding to InsR, LIM2 of Enigma associated specifically with the receptor tyrosine kinase, Ret. Ret was specific for LIM2 of Enigma and did not bind other LIM domains tested. Mutational analysis indicated that the residues responsible for binding to Enigma were localized to the carboxyl-terminal 61 amino acids of Ret. A peptide corresponding to the carboxyl-terminal 20 amino acids of Ret dissociated Enigma and Ret complexes, while a mutant that changed Asn-Lys-Leu-Tyr in the peptide to Ala-Lys-Leu-Ala or a peptide corresponding to exon16 of InsR failed to disrupt the complexes, indicating the Asn-Lys-Leu-Tyr sequence of Ret is essential to the recognition motif for LIM2 of Enigma. We conclude that LIM domains of Enigma recognize tyrosine-containing motifs with specificity residing in both the LIM domains and in the target structures.
LIM结构域是在多种蛋白质中发现的富含半胱氨酸的基序，约含50个氨基酸，被认为可指导蛋白质与蛋白质的相互作用。为了鉴定LIM结构域识别的结构靶点，我们利用了随机肽库筛选、酵母双杂交系统和谷胱甘肽S-转移酶融合技术。Enigma在其羧基末端含有三个LIM结构域，Enigma的LIM3特异性识别胰岛素受体（InsR）的活性内吞密码而非突变型内吞密码（Wu，R.Y.，和Gill，G.N.（1994年）《生物化学杂志》269，25085 - 25090）。两个随机肽库与Enigma的谷胱甘肽S-转移酶-LIM3相互作用表明，其与对应于InsR主要内吞密码的Gly-Pro-Hyd-Gly-Pro-Hyd-Tyr-Ala有特异性结合。肽竞争实验表明，脯氨酸和酪氨酸残基对于InsR与Enigma的特异性相互作用都是必需的。与Enigma的LIM3结合InsR不同，Enigma的LIM2与受体酪氨酸激酶Ret特异性结合。Ret对Enigma的LIM2具有特异性，不与所测试的其他LIM结构域结合。突变分析表明，与Enigma结合的残基定位于Ret的羧基末端61个氨基酸。对应于Ret羧基末端20个氨基酸的肽可解离Enigma和Ret复合物，而将该肽中的Asn-Lys-Leu-Tyr突变为Ala-Lys-Leu-Ala的突变体或对应于InsR外显子16的肽未能破坏复合物，这表明Ret的Asn-Lys-Leu-Tyr序列对于Enigma的LIM2识别基序至关重要。我们得出结论，Enigma的LIM结构域识别含酪氨酸的基序，特异性存在于LIM结构域和靶结构中。
