Sawyer S T, Penta K
Department of Pharmacology and Toxicology, Medical College of Virginia Campus of Virginia Commonwealth University, Richmond, Virginia 23298-0613, USA.
J Biol Chem. 1996 Dec 13;271(50):32430-7. doi: 10.1074/jbc.271.50.32430.
Cytokine receptors act at least partially by associating with Janus tyrosine protein kinases at the conserved box one motif of the receptor. These receptor-associated kinases then activate STAT transcription factors through phosphorylation. We found that the 78-kDa erythropoietin receptor (EPOR), a highly modified form of the 66-kDa receptor which is abundant in HCD57 cells, was phosphorylated on serine residues without EPO stimulation. Coprecipitation experiments showed the 78-kDa EPOR but not the more abundant 66-kDa EPOR was associated with JAK2, a Janus protein kinase, in both the presence and absence of EPO. Solubilized 78-kDa EPOR bound to purified, genetically engineered JAK2 better than the 62-76-kDa receptor proteins, and additional phosphorylation of tyrosine residues further increased the binding of the 78-kDa EPOR to JAK2-agarose beads. STAT5 DNA binding was activated by 10-100-fold lower concentrations of EPO in HCD57 cells than in primary erythroid cells, and STAT5 associated with the EPOR in an EPO-dependent manner. These data suggest that phosphorylation of either serine or tyrosine residues of the EPOR can enhance the association of the receptor with JAK2, possibly increasing the sensitivity to EPO.
细胞因子受体至少部分通过与受体保守的盒状结构域1基序处的Janus酪氨酸蛋白激酶结合来发挥作用。这些与受体相关的激酶随后通过磷酸化激活STAT转录因子。我们发现，78 kDa的促红细胞生成素受体（EPOR），即66 kDa受体的高度修饰形式，在HCD57细胞中大量存在，在没有促红细胞生成素（EPO）刺激的情况下，其丝氨酸残基会发生磷酸化。共沉淀实验表明，无论有无EPO，78 kDa的EPOR而非更丰富的66 kDa的EPOR与Janus蛋白激酶JAK2相关联。溶解的78 kDa EPOR比62 - 76 kDa的受体蛋白与纯化的基因工程JAK2结合得更好，酪氨酸残基的额外磷酸化进一步增加了78 kDa EPOR与JAK2 - 琼脂糖珠的结合。与原代红细胞相比，HCD57细胞中10 - 100倍低浓度的EPO就能激活STAT5的DNA结合，并且STAT5以EPO依赖的方式与EPOR相关联。这些数据表明，EPOR的丝氨酸或酪氨酸残基的磷酸化可以增强受体与JAK2的结合，可能会增加对EPO的敏感性。
