J Proteome Res. 2019 Apr 5;18(4):1679-1690. doi: 10.1021/acs.jproteome.8b00942. Epub 2019 Mar 21.
Ableson tyrosine kinase (ABL) plays essential roles in cell differentiation, division, adhesion, and stress response. However, fusion of the breakpoint cluster region (BCR) to ABL produces constitutive kinase activity that causes chronic myelogenous leukemia (CML). Small molecule tyrosine kinase inhibitors (TKIs) such as imatinib revolutionized the treatment of CML and other cancers, but acquired resistance to these inhibitors is rising. Thus, careful dissection of ABL signaling pathways is needed to find novel drug targets. Here we present a refined proteomic approach for elucidation of direct kinase substrates called kinase assay linked phosphoproteomics (KALIP). Our strategy integrates in vitro kinase assays at both the peptide and protein levels with quantitative tyrosine phosphoproteomics in response to treatment by multiple TKIs. Utilizing multiple TKIs permits elimination of off-target effects of these drugs, and overlapping the in vivo and in vitro data sets allows us to define a list of the most probable kinase substrates. Applying our approach produced a list of 60 ABL substrates, including novel and known proteins. We demonstrate that spleen tyrosine kinase (SYK) is a novel direct substrate of ABL, and we predict our proteomic strategy may facilitate identification of substrates in other cancers that have disrupted kinase signaling.
阿莱斯酮酪氨酸激酶（ABL）在细胞分化、分裂、黏附和应激反应中发挥着重要作用。然而，断裂点簇区（BCR）与 ABL 的融合产生了组成性激酶活性，导致慢性髓细胞白血病（CML）。小分子酪氨酸激酶抑制剂（TKI），如伊马替尼，彻底改变了 CML 和其他癌症的治疗方法，但这些抑制剂的获得性耐药性正在上升。因此，需要仔细剖析 ABL 信号通路，以寻找新的药物靶点。在这里，我们提出了一种改进的蛋白质组学方法，用于阐明称为激酶测定相关磷酸蛋白质组学（KALIP）的直接激酶底物。我们的策略将体外激酶测定整合到肽和蛋白质水平，并结合定量酪氨酸磷酸蛋白质组学，以响应多种 TKI 的治疗。利用多种 TKI 可以消除这些药物的脱靶效应，并且将体内和体外数据集重叠，使我们能够定义最可能的激酶底物列表。应用我们的方法产生了 60 个 ABL 底物的列表，包括新的和已知的蛋白质。我们证明了脾酪氨酸激酶（SYK）是 ABL 的一个新的直接底物，并且我们预测我们的蛋白质组学策略可能有助于鉴定其他具有激酶信号中断的癌症中的底物。
