Santini Valeria, Scappini Barbara, Grossi Alberto, Gozzini Antonella, Bonsi Laura, Pagliai Gabriella, Rossi Ferrini Pierluigi, Bagnara Gian Paolo
Dept. of Hematology, University of Florence, Policlinico di Careggi, viale Morgagni 85, Italy.
Haematologica. 2002 Dec;87(12):1242-7.
B1647 is a cell line derived from bone marrow cells of a patient with acute myeloid leukemia (M2) with a complete erythro-megakaryocytic phenotype and bears both k and p isoforms of c-mpl. Interestingly, spontaneous B1647 cell proliferation is significantly potentiated by thrombopoietin (TPO).
We aimed to evaluate the proliferative signal transduction events following the activation of c-mpl and we stimulated B1647 cells with TPO 40 ng/mL for 3, 7, 15 and 30 minutes; cells were then lysed and whole lysates were immunoprecipitated with anti-phosphotyrosine antibodies.
In our hands, TPO stimulation induced phosphorylation of several substrate proteins in B1647 cells. The increase in tyrosine phosphorylation from background spontaneous activation was transient, maximal after 10 minutes and declined to reach constitutive levels after 30 minutes. In particular, protein substrates between 50 and 140 kDa appeared to be selectively phosphorylated by TPO. We demonstrated that Jak2, Stat3 and Shc were activated in B1647 cells after TPO, as already shown for different cell lines by other authors. Moreover, Lyn kinase activation was detected. Grb2 co-immunoprecipitated with phosphorylated proteins. The phosphorylation of Syk kinase was not demonstrated, whereas Vav was activated by TPO.
The pattern of protein phosphorylation determined in B1647 cells by TPO testifies the role of this cytokine in sustaining cell growth and indicates Lyn tyrosine kinase as a possible target protein in transduction of the TPO proliferative signal.
B1647是一种源自急性髓系白血病（M2）患者骨髓细胞的细胞系，具有完整的红系-巨核系表型，同时表达c-mpl的k和p两种亚型。有趣的是，血小板生成素（TPO）能显著增强B1647细胞的自发增殖。
我们旨在评估c-mpl激活后的增殖信号转导事件，用40 ng/mL的TPO刺激B1647细胞3、7、15和30分钟；然后裂解细胞，用抗磷酸酪氨酸抗体对全细胞裂解物进行免疫沉淀。
在我们的实验中，TPO刺激诱导了B1647细胞中几种底物蛋白的磷酸化。酪氨酸磷酸化从背景自发激活状态的增加是短暂的，10分钟后达到最大值，30分钟后降至基础水平。特别是，50至140 kDa之间的蛋白质底物似乎被TPO选择性磷酸化。我们证明，TPO作用后B1647细胞中的Jak2、Stat3和Shc被激活，其他作者在不同细胞系中也有类似发现。此外，检测到Lyn激酶激活。Grb2与磷酸化蛋白共免疫沉淀。未证实Syk激酶的磷酸化，而Vav被TPO激活。
TPO在B1647细胞中所确定的蛋白质磷酸化模式证明了这种细胞因子在维持细胞生长中的作用，并表明Lyn酪氨酸激酶可能是TPO增殖信号转导中的靶蛋白。
