Department of Cell Biology & Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX 79430, United States.
Mol Cell Endocrinol. 2011 May 16;338(1-2):79-83. doi: 10.1016/j.mce.2011.03.009. Epub 2011 Mar 30.
Jak2/RUSH-mediated prolactin signaling culminates in RUSH-1α-DNA-binding. Heretofore, Jak2-specific phosphorylation residues in RUSH were unknown. Genpathway's discovery approaches correlated RUSH-DNA binding (-126/-121) in uteroglobin's proximal promoter with recruitment of the transcriptional machinery. NetPhos 2.0 server found a single tyrosine phosphorylation site in RUSH's minimal DNA-binding domain. Y195 had identical context and prediction scores (0.52) for rabbit and human (HLTF) orthologs. The mouse ortholog (Hltf) had a higher prediction score (0.897). Affinity purified RUSHY195ph antibodies recognized native tyrosine phosphorylated RUSH protein immunoprecipitated from nuclear extracts. When R5020-treated HRE-H9 cells±the Jak2 inhibitor, Tyrene CR4, were stimulated with prolactin, confocal immunofluorescence images provided conclusive evidence that Jak2 mediated the availability of phosphorylated RUSHY195 in nucleus and cytoplasm. Catalytically active Jak2 is ipso facto a RUSH site-specific tyrosine kinase. Immunoprecipitation/Western blotting revealed both phosphorylation at Y195 and the physical interaction between p-Jak2/RUSH/HLTF/Hltf are evolutionarily conserved across three mammalian (rabbit, human, mouse) orthologs.
Jak2/RUSH 介导的催乳素信号转导最终导致 RUSH-1α-DNA 结合。迄今为止，RUSH 中的 Jak2 特异性磷酸化残基尚不清楚。Genpathway 的发现方法将 RUSH-DNA 结合（-126/-121）与近端启动子中的尿促球蛋白与转录机制的募集相关联。NetPhos 2.0 服务器在 RUSH 的最小 DNA 结合结构域中发现了一个单一的酪氨酸磷酸化位点。Y195 在兔和人（HLTF）同源物中具有相同的上下文和预测评分（0.52）。小鼠同源物（Hltf）具有更高的预测评分（0.897）。亲和纯化的 RUSHY195ph 抗体识别从核提取物中免疫沉淀的天然酪氨酸磷酸化 RUSH 蛋白。当用 Jak2 抑制剂 Tyrene CR4 处理 R5020 处理的 HRE-H9 细胞并用催乳素刺激时，共聚焦免疫荧光图像提供了确凿的证据，表明 Jak2 介导了细胞核和细胞质中磷酸化 RUSHY195 的可用性。催化活性 Jak2 实际上是一种 RUSH 特异性酪氨酸激酶。免疫沉淀/Western 印迹揭示了 Y195 的磷酸化以及 p-Jak2/RUSH/HLTF/Hltf 之间的物理相互作用在三种哺乳动物（兔、人、鼠）同源物中都是保守的。
