促红细胞生成素产生肝细胞受体A2(EphA2) SAM结构域对激酶结构域的脂质体结合能力与激酶活性的调控研究

促红细胞生成素产生肝细胞受体A2(EphA2) SAM结构域对激酶结构域的脂质体结合能力与激酶活性的调控研究

Erythropoietin-producing hepatocyte receptor (Eph receptor) is the largest subfamily in the receptor tyrosine kinase (RTK) family, and the bidirectional signal transduction it mediates has important regulatory effects on cell morphology, adhesion, movement, proliferation, survival and differentiation. EphA2 is an important subtype widely studied in the Eph receptor family and plays an important role in the pathogenesis of cataracts and breast cancer, etc. Previous studies have found that the kinase domain of the EphA2 receptor can bind to the cell membrane, and its kinase activity is regulated by the phospholipid membrane, but the influence of the adjacent SAM domain on the interaction between the kinase domain and the lipid membrane and the kinase activity is still unclear. In this study, through co-expression with the active fragment of phosphatase PTP1B1 - 301, the intracellular kinase - SAM tandem domain of the EphA2 receptor was expressed and purified. By comparing the liposome-binding ability of the intracellular kinase - SAM tandem domain with that of the single kinase domain and measuring the corresponding kinase activity, it was found that the SAM domain of the intracellular segment of the EphA2 receptor enhanced the binding ability of its kinase domain to liposomes (4 mg/mL) by about 6 times (P < 0.001); the ability of the phosphorylated intracellular kinase - SAM tandem domain of EphA2 to bind liposomes (4 mg/mL) was 2.5 times higher than that of the non-phosphorylated intracellular kinase - SAM tandem domain (P < 0.05); and after binding to liposomes, the kinase activity of the kinase domain was further increased, thus forming a positive feedback. In conclusion, the findings of this study suggest that the tyrosine kinase domain and the adjacent SAM domain in the intracellular segment of EphA2 can form a complete structural and functional unit, and its kinase activity and liposome-binding ability are significantly different from those of the single kinase domain. Our finding provides a reference and idea for further understanding the activity regulation of the kinase domains of other subtypes in the Eph receptor family.

促红细胞生成素产生肝细胞受体(Eph receptor)是受体酪氨酸激酶(RTK)家族中最大的亚家族,其介导的双向信号传导对细胞的形态、黏附、运动、增殖、生存及分化都有重要的调控作用。 EphA2是Eph受体家族中一个被广泛研究的重要亚型,在白内障和乳腺癌等病理发生过程中发挥了重要作用。既往研究发现:EphA2受体的激酶结构域可结合细胞膜,其激酶活性受磷脂膜的调控,但是相邻的SAM结构域对激酶结构域与脂膜的相互作用以及激酶活性的影响尚不清楚。在此项研究中,通过与磷酸酶PTP1B1-301活性片段共表达的方式,表达、纯化了EphA2受体的胞内段激酶-SAM串联结构域,通过比较胞内段激酶-SAM串联结构域与单独激酶结构域的脂质体结合能力,以及测定对应的激酶活性,发现:EphA2受体胞内段的SAM结构域使其激酶结构域与脂质体(4 mg/mL)的结合能力增强约6倍(P<0.001);磷酸化后的EphA2胞内段激酶-SAM串联结构域结合脂质体(4 mg/mL)的能力比非磷酸化的胞内段激酶-SAM串联结构域提高2.5倍(P<0.05);而结合脂质体后,激酶结构域的激酶活性也被进一步提高,从而形成正反馈。综上所述,本研究的发现提示:EphA2胞内段的酪氨酸激酶结构域与相邻的SAM结构域可形成一个完整的结构功能单位,其激酶活性和脂质体结合能力与单独的激酶结构域相比都形成了明显的差异,我们的这一发现对进一步理解Eph受体家族其他亚型的激酶结构域的活性调控提供了参考与思路。