Cheng H C, Bjorge J D, Aebersold R, Fujita D J, Wang J H
Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria, Australia.
Biochemistry. 1996 Sep 10;35(36):11874-87. doi: 10.1021/bi9603940.
The C-terminal src kinase (CSK) is a ubiquitously expressed, cytosolic enzyme capable of phosphorylating and inactivating several plasma membrane-bound src-family protein tyrosine kinases in vitro [Nada, S., Okada, M., MacAuley, A., Cooper, J.A., & Nakagawa, H. (1990) Nature 351, 69-72; Bergman, M., Mustelin, T., Oetken, C., Partanen, J., Flint, N.A., Amrein, K.E., Autero, M., Burn, P., & Alitalo, K. (1992) EMBO J. 11, 2919-2924]. We purified CSK to apparent homogeneity from bovine thymus cytosol to study in vitro how the purified enzyme recognizes the various src-family kinases as its substrates. A novel assay method was developed for assaying the ability of CSK to inactivate src-family tyrosine kinases. With this assay method, we demonstrated that CSK inactivated p56lyn with a significantly higher efficiency than pp60c-src. Phosphopeptide mapping of CSK-phosphorylated p56lyn and pp60c-src shows that the consensus tyrosine residue (also termed tail tyrosine) in the C-terminal regulatory domain of p56lyn was phosphorylated by CSK with an efficiency much higher than that of pp60c-src. Thus, the higher efficiency of inactivation of p56lyn by CSK is a result of the ability of p56lyn to serve as a better substrate of CSK. The synthetic peptides derived from the C-terminal portion of p56lyn and pp60c-src were much poorer substrates than the intact src-family kinases for CSK, indicating that the local structure around the tail tyrosine is not sufficient to direct efficient phosphorylation of p56lyn by CSK. Nevertheless, the slightly higher efficiency displayed by CSK in phosphorylating the peptide derived from the C-terminal portion of p56lyn than that from pp60c-src suggests that the structural differences between the C-terminal portions of p56lyn and pp60c-src contribute to the differential efficiencies displayed by CSK in phosphorylating the two kinases. Determination of the CSK-phosphorylation site in the src-C-terminal peptide by phosphopeptide mapping reveals that the whole C-terminal regulatory domain and an adjacent part of the protein kinase domain contain some of the structural determinants directing CSK to phosphorylate the consensus tail tyrosine of the src-family kinases.
C 端 src 激酶（CSK）是一种广泛表达的胞质酶，在体外能够磷酸化并使几种质膜结合的 src 家族蛋白酪氨酸激酶失活 [Nada, S., Okada, M., MacAuley, A., Cooper, J.A., & Nakagawa, H. (1990) 《自然》351, 69 - 72；Bergman, M., Mustelin, T., Oetken, C., Partanen, J., Flint, N.A., Amrein, K.E., Autero, M., Burn, P., & Alitalo, K. (1992) 《欧洲分子生物学组织杂志》11, 2919 - 2924]。我们从牛胸腺胞质溶胶中纯化 CSK 至表观均一性，以在体外研究纯化后的酶如何将各种 src 家族激酶识别为其底物。开发了一种新的检测方法来检测 CSK 使 src 家族酪氨酸激酶失活的能力。通过这种检测方法，我们证明 CSK 使 p56lyn 失活的效率显著高于 pp60c - src。对 CSK 磷酸化的 p56lyn 和 pp60c - src 进行磷酸肽图谱分析表明，p56lyn C 端调节结构域中的共有酪氨酸残基（也称为尾部酪氨酸）被 CSK 磷酸化的效率远高于 pp60c - src。因此，CSK 对 p56lyn 失活效率更高是因为 p56lyn 能够作为 CSK 更好的底物。从 p56lyn 和 pp60c - src 的 C 端部分衍生的合成肽作为 CSK 的底物比完整的 src 家族激酶差得多，这表明尾部酪氨酸周围的局部结构不足以指导 CSK 对 p56lyn 进行高效磷酸化。然而，CSK 磷酸化来自 p56lyn C 端部分的肽的效率略高于来自 pp60c - src 的肽，这表明 p56lyn 和 pp60c - src C 端部分之间的结构差异导致了 CSK 磷酸化这两种激酶时显示出不同的效率。通过磷酸肽图谱分析确定 src - C 端肽中的 CSK 磷酸化位点表明，整个 C 端调节结构域和蛋白激酶结构域的相邻部分包含一些指导 CSK 磷酸化 src 家族激酶共有尾部酪氨酸的结构决定因素。
