Stem Cell and Leukaemia Proteomics Laboratory, School of Cancer and Enabling Sciences, Manchester Academic Health Science Centre, University of Manchester, 27 Palatine Rd, Manchester, M20 4QL;
Mol Cell Proteomics. 2013 Nov;12(11):3319-29. doi: 10.1074/mcp.M112.024604. Epub 2013 Sep 1.
CXCL12 governs cellular motility, a process deregulated by hematopoietic stem cell oncogenes such as p210-BCR-ABL. A phosphoproteomics approach to the analysis of a hematopoietic progenitor cell line treated with CXCL12 and the Rac 1 and 2 inhibitor NSC23766 has been employed to objectively discover novel mechanisms for regulation of stem cells in normal and malignant hematopoiesis. The proteomic data sets identified new aspects of CXCL12-mediated signaling and novel features of stem cell regulation. We also identified a novel phosphorylation event in hematopoietic progenitor cells that correlated with motile response and governed by the chemotactic factor CXCL12. The novel phosphorylation site on PTPRC/CD45; a protein tyrosine phosphatase, was validated by raising an antibody to the site and also using a mass spectrometry absolute quantification strategy. Site directed mutagenesis and inhibitor studies demonstrated that this single phosphorylation site governs hematopoietic progenitor cell and lymphoid cell motility, lies downstream from Rac proteins and potentiates Src signaling. We have also demonstrated that PTPRC/CD45 is down-regulated in leukemogenic tyrosine kinase expressing cells. The use of discovery proteomics has enabled further understanding of the regulation of PTPRC/CD45 and its important role in cellular motility in progenitor cells.
CXCL12 调节细胞运动，这一过程被造血干细胞癌基因（如 p210-BCR-ABL）失调。采用磷酸化蛋白质组学方法分析经 CXCL12 和 Rac1 和 2 抑制剂 NSC23766 处理的造血祖细胞系，客观地发现了调节正常和恶性造血中干细胞的新机制。蛋白质组数据集确定了 CXCL12 介导的信号转导的新方面和干细胞调节的新特征。我们还在造血祖细胞中发现了一个与运动反应相关的新磷酸化事件，该事件受趋化因子 CXCL12 调节。在一种蛋白酪氨酸磷酸酶 PTPRC/CD45 上发现了一个新的磷酸化位点，该位点通过针对该位点的抗体和使用质谱绝对定量策略进行了验证。定点突变和抑制剂研究表明，这个单一的磷酸化位点调节造血祖细胞和淋巴样细胞的运动，位于 Rac 蛋白的下游，并增强了Src 信号。我们还证明，PTPRC/CD45 在表达致癌酪氨酸激酶的白血病细胞中下调。发现蛋白质组学的应用使我们进一步了解了 PTPRC/CD45 的调节及其在祖细胞中细胞运动中的重要作用。
