Mechanism of Lnk function in cytokine receptor signaling

说明(申请人提供)：促红细胞生成素(EPO)和血小板生成素(TPO)分别是调节红细胞生成、巨核细胞和血小板发育的主要细胞因子。接头蛋白LNK负性调节EPO受体(EPOR)和TPO受体(MPL)的信号转导，从而下调TPO介导的巨核细胞生成和EPO介导的红细胞生成。LNK抑制细胞因子受体整合伙伴JAK2酪氨酸激酶的活性。此外，LNK SH2结构域是其抑制功能所必需的，并且LNK与受体/JAK2复合体之间的功能相互作用依赖于磷酸化。这项资助的目的是解开LNK如何控制对细胞因子的定量反应以及其失调易导致白血病的机制。为了实现这一点，将使用生化、细胞和小鼠遗传学方法，重点研究LNK SH2结构域和酪氨酸残基。重组多肽文库筛选将建立LNK SH2结构域结合基序，质谱仪(MS)将在体内鉴定LNK SH2结构域相互作用蛋白。将开发新的原代造血细胞培养系统来研究LNK、细胞因子受体和JAK2之间的相互作用。此外，在体内被磷酸化并对其功能重要的LNK酪氨酸将被解剖。重要的是，LNK调节功能的机制将在生理环境中使用基因缺陷的原代造血细胞和产生基因去除的小鼠来测试。不受抑制和持续的细胞因子受体信号会导致致癌转化，因此了解LNK控制受体信号的幅度和持续时间的细胞机制将有助于从总体上理解细胞因子受体的调控。通过这项拟议的研究，申请者将接受结构和蛋白质组学生物学方面的培训，并将获得成为独立研究人员所需的技能和知识。

DESCRIPTION (provided by applicant): Erythropoietin (Epo) and thrombopoietin (Tpo) are the primary cytokines regulating red blood cell production, and megakaryocyte and platelet development, respectively. The adaptor protein Lnk negatively regulates signaling transduction of the Epo receptor (EpoR) and Tpo receptor (mpl), thereby downregulating Tpo-mediated megakaryocytopoiesis and Epo-mediated erythropoiesis. Lnk inhibits the activity of cytokine receptor integral partner, the JAK2 tyrosine kinase. Furthermore, the Lnk SH2 domain is essential for its inhibitory functions, and the functional interaction between Lnk and the receptor/JAK2 complex is phosphorylation-dependent. The purpose of this grant is to unravel the mechanism that encodes how Lnk controls the quantitative response to cytokines, and whose dysregulation predisposes to leukemia. To accomplish this, biochemical, cellular, and mouse genetic approaches will be used with the focus on the Lnk SH2 domain and tyrosine residues. Recombinant peptide library screens will establish Lnk SH2 domain binding motifs and mass spectrometry (MS) will identify Lnk SH2 domain interacting proteins in vivo. Novel primary hematopoietic cell culture systems will be developed to investigate the interactions among Lnk, cytokine receptors, and JAK2. Moreover, the Lnk tyrosines that are phosphorylated in vivo and important for its function will be dissected. Importantly, the mechanisms of Lnk regulatory function will be tested within physiological contexts using gene-deficient primary hematopoietic cells and generating gene-ablated mice. Unchecked and sustained cytokine receptor signaling leads to oncogenic transformation, therefore understanding the cellular mechanisms by which Lnk controls the amplitude and duration of receptor signaling will shed significant light on cytokine receptor regulation in general. Through this proposed research, the applicant will receive training in structural and proteomics biology and will gain the necessary skills and knowledge to become an independent investigator.