NOVEL CYTOKINE RESPONSIVE NUCLEAR PROTEIN KINASE

我们的主要目标是鉴定对精氨酸敏感的核蛋白激酶 在淋巴细胞中，探索它们被激活的机制， 定义它们在淋巴细胞核事件中的作用。 我们最近 鉴定了一种新的85kD核激酶， 细胞因子和其他生长因子。 85kD激酶是 通过磷酸化激活，表明85kD激酶与 一个由精氨酸诱导的磷酸化级联反应 的目的 一个建议是克隆这种蛋白激酶，然后验证一个假设 这个85kD的激酶将细胞质到细胞核的磷酸化 调节基因表达的级联反应。 首先，我们将纯化和克隆该85kD核蛋白激酶。 （一） 小规模制备核提取物将用于开发一种 纯化85kD蛋白的方案。 (ii)扩大的程序将 以获得足够量的蛋白质用于蛋白水解 肽片段的消化和测序。 (iii)的氨基酸 蛋白水解片段的序列将用于设计合成的 用于筛选cDNA文库的简并寡核苷酸， 85kD核激酶基因的克隆。 第二，我们将探索85kD酶是否与一种 精氨酸诱导的磷酸化级联反应及85kD激酶的研究 效应器 (i)我们将确定是否在体内状态的 85kD蛋白的磷酸化受细胞因子调节。 (ii)我们 将测试已知的激酶和细胞质和核提取物， 经精氨酸处理的细胞的磷酸化和活化能力 85kD核激酶。 (iii)细胞转化为阴性或 将使用ras和其它关键转换器的激活显性突变体 为了评估85kD激酶是否与已知的生长有关， 因子触发的信号转导通路。 第三，我们将确定85kD激酶在精氨酸诱导的细胞凋亡中的作用。 核事件。 (i)85kD激酶的活性将与 在野生型和突变细胞系中具有确定的细胞应答。 （二） 我们将确定野生型和非野生型的过度表达的影响。 突变的85kD激酶。 (iii)突变细胞将转染 组成型活性的85kD激酶，试图补充其 信号转导缺陷 (iv)我们将测试有针对性的效果 在细胞培养物和"敲除"中破坏85kD激酶基因 老鼠. " 许多参与信号转导的蛋白激酶是 分布在整个亚细胞区室中并且是多功能的。 相比之下，我们已经鉴定的85kD激酶仅是 核武器 证实85kD激酶参与细胞因子- 诱导淋巴细胞基因表达、增殖或凋亡 开辟了利用85kD激酶作为模型系统设计 消除细胞核内信号转导的策略。 在精氨酸介导的疾病中，治疗性消除在 细胞核水平将使细胞膜、细胞质和其他重要的非细胞核水平得以保留。 核过程。

Our broad goal is to identify cytokine-responsive nuclear protein kinases in lymphocytes, explore mechanisms by which they are activated and define their role in lymphocyte nuclear events. We have recently identified a novel 85kD nuclear kinase that is activated by treatment of cells with cytokines and other growth factors. The 85kD kinase is activated by phosphorylation suggesting that the 85kD kinase is linked to a cytokine-induced phosphorylation cascade. The objective of this proposal is to clone this protein kinase, and then to test a hypothesis that this 85kD kinase links a cytoplasm-to-nucleus phosphorylation cascade that regulates gene expression. First, we will purify and clone this 85kD nuclear protein kinase. (i) Small scale preparation of nuclear extracts will be used to develop a scheme to purify the 85kD protein. (ii) Scaled up procedures will then be used to obtain sufficient amounts of the protein for proteolytic digestion and sequencing of peptide fragments. (iii) The amino acid sequence of the proteolytic fragments will be used to design synthetic degenerate oligonucleotides for the screening of cDNA libraries and cloning of the 85kD nuclear kinase gene. Second, we will explore whether or not the 85kD enzyme is linked to a cytokine-induced phosphorylation cascade and search for the 85kD kinase effector. (i) We will determine whether the in vivo state of phosphorylation of the 85kD protein is modulated by cytokines. (ii) We will test known kinases and cytoplasmic and nuclear extracts from cytokine-treated cells for their ability to phosphorylate and activate the 85kD nuclear kinase. (iii) Cells transformed by negative or activated dominant mutants of ras and other key transducers will be used to assess whether or not the 85kD kinase is linked to a known growth factor-triggered signal transduction pathway. Third, we will define the role of the 85kD kinase in cytokine-induced nuclear events. (i) The activity of the 85kD kinase will be correlated with defined cellular responses in wild-type and mutant cell lines. (ii) We will determine the effects of overexpression of the wild-type and mutated 85kD kinase. (iii) Mutant cells will be transfected with constitutively active 85kD kinase in an attempt to complement their signal transduction defect. (iv) We will test the effect of targeted disruption of the 85kD kinase gene in cell cultures and in "knockout mouse." Many of the protein kinases participating in signal transduction are distributed throughout subcellular compartments and are multifunctional. In contrast, the 85kD kinase that we have identified is exclusively nuclear. Demonstration of involvement of the 85kD kinase in cytokine- induced lymphocytes gene expression, proliferation or apoptosis would open up the way to use the 85kD kinase as a model system to devise strategies to abrogate signal transduction exclusively in the nucleus. In cytokine-mediated diseases therapeutic abrogation of signals at the nuclear level would spare membrane, cytoplasmic and other vital non- nuclear processes.