Luo K, Zhou P, Lodish H F
Whitehead Institute for Biomedical Research, Nine Cambridge Center, MA 02142, USA.
Proc Natl Acad Sci U S A. 1995 Dec 5;92(25):11761-5. doi: 10.1073/pnas.92.25.11761.
Type I and II receptors for the transforming growth factor beta (TGF-beta) are transmembrane serine/threonine kinases that are essential for TGF-beta signaling. However, little is known about their in vivo substrates or signal transduction pathways. To determine the substrate specificity of these kinases, we developed combinatorial peptide libraries synthesized on a hydrophilic matrix that is easily accessible to proteins in aqueous solutions. When we subjected these libraries to phosphorylation by the cAMP-dependent protein kinase, we obtained the optimal peptide sequence RRXS (I/L/V), in perfect agreement with the substrate sequence deduced from mutagenesis and crystal structure analyses. By using the same libraries, we showed that the optimal substrate peptide for both the type I and II TGF-beta receptors was KKKKKK(S/T)XXX. Since the two kinases are thought to play different roles in intracellular signal transduction, it was a surprise to find that they have almost identical substrate specificity. Our method is direct, sensitive, and simple and provides information about the kinase specificity for all the amino acid residues at each position.
转化生长因子β（TGF-β）的I型和II型受体是跨膜丝氨酸/苏氨酸激酶，对TGF-β信号传导至关重要。然而，关于它们在体内的底物或信号转导途径知之甚少。为了确定这些激酶的底物特异性，我们开发了在亲水性基质上合成的组合肽库，该基质在水溶液中易于被蛋白质接近。当我们使这些文库接受环磷酸腺苷依赖性蛋白激酶的磷酸化作用时，我们获得了最佳肽序列RRXS（I/L/V），这与通过诱变和晶体结构分析推导的底物序列完全一致。通过使用相同的文库，我们表明I型和II型TGF-β受体的最佳底物肽都是KKKKKK(S/T)XXX。由于这两种激酶被认为在细胞内信号转导中发挥不同作用，所以发现它们具有几乎相同的底物特异性令人惊讶。我们的方法直接、灵敏且简单，并提供了关于每个位置所有氨基酸残基的激酶特异性的信息。
