Sparks A B, Quilliam L A, Thorn J M, Der C J, Kay B K
Curriculum in Genetics and Molecular Biology, University of North Carolina, Chapel Hill 27599.
J Biol Chem. 1994 Sep 30;269(39):23853-6.
We have used the Src homology 3 (SH3) domain to screen two phage-displayed random peptide libraries, each containing 2 x 10(8) unique members, and have identified a series of high affinity peptide ligands. The peptides possess similar proline-rich regions, which yield a consensus Src SH3-binding motif of RPLPPLP. We have confirmed this motif by screening a phage-displayed peptide library biased for SH3 ligands and identifying the same consensus sequence. Binding studies using synthetic peptides suggest that the RPLPPLP motif is important for SH3 binding and confers specificity for the Src SH3 domain, and that residues which flank the motif may also contribute to binding. Peptides that contain the RPLPPLP motif compete Src, but not Abl or phospholipase C gamma, SH3 interactions with SH3-binding proteins from cell lysates (IC50 = 1-5 microM). Furthermore, RPLPPLP-related peptides are able to accelerate progesterone-induced maturation of Xenopus laevis oocytes. A similar acceleration has been observed in oocytes treated with activated, but not normal, Xenopus Src, suggesting the possibility that the peptides are able to antagonize the negative regulation of Src activity by Src SH3 in vivo.
我们利用Src同源结构域3（SH3）筛选了两个噬菌体展示的随机肽库，每个库包含2×10⁸个独特成员，并鉴定出一系列高亲和力肽配体。这些肽具有相似的富含脯氨酸区域，产生了RPLPPLP的共有Src SH3结合基序。我们通过筛选偏向于SH3配体的噬菌体展示肽库并鉴定相同的共有序列，证实了这一基序。使用合成肽的结合研究表明，RPLPPLP基序对SH3结合很重要，并赋予对Src SH3结构域的特异性，且该基序两侧的残基可能也有助于结合。含有RPLPPLP基序的肽可竞争Src，但不竞争Abl或磷脂酶Cγ，其SH3与细胞裂解物中的SH3结合蛋白相互作用（IC50 = 1 - 5 microM）。此外，与RPLPPLP相关的肽能够加速非洲爪蟾卵母细胞孕酮诱导的成熟。在用活化的而非正常的非洲爪蟾Src处理的卵母细胞中也观察到了类似的加速现象，这表明这些肽有可能在体内拮抗Src SH3对Src活性的负调控。
