Crkl is constitutively tyrosine phosphorylated in platelets from chronic myelogenous leukemia patients and inducibly phosphorylated in normal platelets stimulated by thrombopoietin
Crkl is constitutively tyrosine phosphorylated in platelets from chronic myelogenous leukemia patients and inducibly phosphorylated in normal platelets stimulated by thrombopoietin
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DOI：
10.1182/blood.v88.11.4304.4304
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发表时间：
1996-12
期刊：
影响因子：
20.3
通讯作者：
A. Oda;Y. Miyakawa;B. Druker;A. Ishida;K. Ozaki;H. Ohashi;M. Wakui;M. Handa;Kiyoaki Watanabe;S. Okamoto;Y. Ikeda
中科院分区：
文献类型：
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作者：
A. Oda;Y. Miyakawa;B. Druker;A. Ishida;K. Ozaki;H. Ohashi;M. Wakui;M. Handa;Kiyoaki Watanabe;S. Okamoto;Y. Ikeda
慢性粒细胞白血病（CML）患者的血小板功能，如聚集和凝块收缩，经常异常。然而，这些功能改变的分子机制是未知的。由于p210 bcr-abl癌基因产物的表达，一种组成型活性酪氨酸激酶，已知在CML的发病机制中具有重要作用，并且酪氨酸磷酸化密切参与血小板活化的各个方面，我们通过单克隆抗磷酸酪氨酸抗体（4G 10）免疫印迹法检测了15例CML患者血小板中蛋白酪氨酸磷酸化的模式。凝血酶刺激前后，正常和CML血小板之间唯一一致的差异是存在一个相对分子量为39 kD的酪氨酸磷酸化蛋白。该蛋白被鉴定为crid，是一种含有SH 2、SH 3的衔接蛋白。因此，如先前对来自CML患者的中性粒细胞所证实的，p39 crkl的酪氨酸磷酸化在成熟血小板中持续存在。在正常血小板中，凝血酶刺激后未检测到crid的酪氨酸磷酸化。然而，crkl在凝血酶刺激后以依赖于血小板聚集的方式并入Triton X-100不溶性残留物中。此外，我们发现crkl是钙蛋白酶的内源性底物，钙蛋白酶是一种可能参与聚集后信号传导过程的蛋白酶。提示crkl可能参与正常血小板聚集过程中细胞骨架的重组，其酪氨酸磷酸化可能参与CML患者血小板功能异常。最后，我们发现血小板生成素诱导正常血小板和基因工程表达人c-Mpl的FDCP细胞中crk 1的酪氨酸磷酸化。这表明crk 1可以被p210 bcr-abl以外的激酶磷酸化，crk 1可能在血小板生成素的信号传导中起作用。
Platelet functions such as aggregation and clot retraction are often abnormal in chronic mylogenous leukemia (CML) patients. However, the molecular mechanisms of these altered functions are unknown. As expression of the p210bcr-abl oncogene product, a constitutively active tyrosine kinase, is known to have an essential role in the pathogenesis of CML and tyrosine phosphorylation is intimately involved in various aspects of platelet activation, we examined the pattern of protein tyrosine phosphorylation in platelets from 15 CML patients by immunoblotting with a monoclonal antiphosphotyrosine antibody (4G10). Before and after stimulation with thrombin, the only consistent difference between normal and CML platelets was the presence of a tyrosine phosphorylated protein with a relative molecular weight of 39 kD. This tyrosine phosphorylated protein was identified as crid, an SH2, SH3 containing adapter protein. Thus, as previously demonstrated for neutrophils from CML patients, tyrosine phosphorylation of p39crkl persists in mature platelets. No tyrosine phosphorylation of crid was detected following stimulation with thrombin in normal platelets. However, crkl became incorporated into the Triton X-100 insoluble residue following thrombin stimulation in a manner dependent on platelet aggregation. Further, we found that crkl is an endogenous substrate for calpain, a protease that may be involved in postaggregation signaling processes. This suggests that crkl may be involved in the reorganization of the cytoskeleton during normal platelet aggregation and its tyrosine phosphorylation in CML platelets may contribute to the abnormal platelet function in CML patients. Finally, we found that thrombopoietin induces tyrosine phosphorylation of crk1 in normal platelets and FDCP cells genetically engineered to express human c-Mpl. This suggests that crk1 can be phosphorylated by a kinase other than p210bcr-abl and that crk1 may have a role in signaling by thrombopoietin.