Department of Biochemistry, Purdue University, West Lafayette, IN 47907, USA.
Proc Natl Acad Sci U S A. 2012 Apr 10;109(15):5615-20. doi: 10.1073/pnas.1119418109. Epub 2012 Mar 26.
Our understanding of the molecular control of many disease pathologies requires the identification of direct substrates targeted by specific protein kinases. Here we describe an integrated proteomic strategy, termed kinase assay linked with phosphoproteomics, which combines a sensitive kinase reaction with endogenous kinase-dependent phosphoproteomics to identify direct substrates of protein kinases. The unique in vitro kinase reaction is carried out in a highly efficient manner using a pool of peptides derived directly from cellular kinase substrates and then dephosphorylated as substrate candidates. The resulting newly phosphorylated peptides are then isolated and identified by mass spectrometry. A further comparison of these in vitro phosphorylated peptides with phosphopeptides derived from endogenous proteins isolated from cells in which the kinase is either active or inhibited reveals new candidate protein substrates. The kinase assay linked with phosphoproteomics strategy was applied to identify unique substrates of spleen tyrosine kinase (Syk), a protein-tyrosine kinase with duel properties of an oncogene and a tumor suppressor in distinctive cell types. We identified 64 and 23 direct substrates of Syk specific to B cells and breast cancer cells, respectively. Both known and unique substrates, including multiple centrosomal substrates for Syk, were identified, supporting a unique mechanism that Syk negatively affects cell division through its centrosomal kinase activity.
我们对许多疾病病理的分子控制的理解需要确定特定蛋白激酶的直接靶标。在这里，我们描述了一种综合蛋白质组学策略，称为激酶测定与磷酸化蛋白质组学相结合，该策略将敏感的激酶反应与内源性激酶依赖性磷酸化蛋白质组学相结合，以鉴定蛋白激酶的直接底物。独特的体外激酶反应以高效的方式进行，使用直接来自细胞激酶底物的肽池，并作为候选底物进行去磷酸化。然后通过质谱法分离和鉴定新磷酸化的肽。将这些体外磷酸化肽与源自激酶活性或抑制的细胞中分离的内源性蛋白质的磷酸肽进行进一步比较，揭示了新的候选蛋白底物。激酶测定与磷酸化蛋白质组学策略被应用于鉴定脾酪氨酸激酶（Syk）的独特底物，Syk 是一种具有癌基因和肿瘤抑制子双重特性的蛋白酪氨酸激酶，在不同的细胞类型中具有独特的特性。我们分别鉴定了 64 种和 23 种特定于 B 细胞和乳腺癌细胞的 Syk 直接底物。包括 Syk 多个中心体底物在内的已知和独特的底物都被鉴定出来，支持了一种独特的机制，即 Syk 通过其中心体激酶活性对细胞分裂产生负面影响。
