Department of Molecular and Cellular Biochemistry, College of Medicine, Ohio State University, Columbus, Ohio 43210, USA.
J Cell Biochem. 2010 Jul 1;110(4):846-56. doi: 10.1002/jcb.22593.
We have recently shown that the gene encoding the truncated form of protein tyrosine phosphatase receptor-type O (PTPROt) expressed predominantly in hematopoietic cells is epigenetically silenced in human primary chronic lymphocytic leukemia (B-CLL). To determine whether increased phosphorylation of the PTPROt substrates following PTPROt suppression alters signal transduction pathway(s) that impart a growth advantage to the leukemic lymphocytes, it is critical to discern the key substrates of PTPROt. Here, we used substrate-trapping assay to identify two novel substrates of PTPROt, the tyrosine kinases Lyn and ZAP70. Both Lyn and ZAP70 were dephosphorylated by wild-type PTPROt, but not by its catalytic site (CS) mutant. A critical phosphorylation site in Lyn, Y397, essential for its activity was dephosphorylated by PTPROt. Consequently, the activity of Lyn kinase was compromised when co-expressed with PTPROt-WT compared to vector control or upon co-expression with PTPROt-CS. Ectopic expression of PTPROt in Raji cells reduced phosphorylation of Lyn in the absence of any change in its protein levels. These results have revealed the physiological importance of PTPROt in regulating B-cell receptor signaling at Lyn kinase. Further, ectopic expression of PTPROt also sensitized the cells to the VEGF-R inhibitor Pazopanib.
我们最近发现，在造血细胞中表达的蛋白酪氨酸磷酸酶受体型 O（PTPROt）截断形式的基因在人原发性慢性淋巴细胞白血病（B-CLL）中被表观遗传沉默。为了确定 PTPROt 抑制后 PTPROt 底物的磷酸化增加是否改变赋予白血病淋巴细胞生长优势的信号转导途径，辨别 PTPROt 的关键底物至关重要。在这里，我们使用底物捕获测定法鉴定了 PTPROt 的两个新底物，即酪氨酸激酶 Lyn 和 ZAP70。野生型 PTPROt 可使 Lyn 和 ZAP70 去磷酸化，但不能使 PTPROt 的催化位点（CS）突变体去磷酸化。Lyn 的一个关键磷酸化位点 Y397 对于其活性是必需的，可被 PTPROt 去磷酸化。因此，与载体对照或与 PTPROt-CS 共表达相比，当与 PTPROt-WT 共表达时，Lyn 激酶的活性受到损害。在 Raji 细胞中异位表达 PTPROt 可降低 Lyn 的磷酸化，而其蛋白水平没有任何变化。这些结果揭示了 PTPROt 在调节 Lyn 激酶 B 细胞受体信号中的生理重要性。此外，PTPROt 的异位表达还使细胞对血管内皮生长因子受体抑制剂帕唑帕尼敏感。
