FUNCTION AND REGULATION OF A SPLICING KINASE SRPK1

最近的研究表明，有一大类剪接因子 含有富含丝氨酸/精氨酸的结构域在这两种情况下都扮演着重要的角色 结构性和规律性剪接。家庭成员一般都是 称为SR蛋白。SR蛋白是一种磷化蛋白，并且 有证据表明它们是磷酸化调节的靶标。 前信使核糖核酸剪接。为了研究这一规定，我们最近发现 并克隆了一种细胞周期调节的激酶SRPK1，它似乎是 哺乳动物细胞中SR蛋白的特异性。我们已经证明了 SRPK1负责细胞周期依赖性的重组 核结构(核斑点)，其中SR蛋白和其他 剪接因素集中。因为相当数量的SRPK1 存在于细胞核中的间期细胞，我们将重点放在这个提议中 关于SRPK1的间期功能和调节的研究。 我们的具体目标1是通过一种 一系列体外实验。我们计划确定SRPK1在 剪接以及SRPK1对SR蛋白的磷酸化对剪接的影响 选址。此外，我们将确定SRPK1是否为 剪接体的组成部分，以及SRPK1如何参与剪接 它本身。一旦SRPK1在剪接中的作用被确立，我们将在AIM 2、解决SRPK1的调控问题。尽管SRPK1含有一种核 定位信号和一小部分激酶存在于 细胞核，SRPK1中的一个独特的序列似乎具有细胞质的功能 负责种群积累的保留信号 间期胞质内有SRPK1的表达。因此，SRPK1可能受到调控 通过核移位。我们计划确定最小序列 SRPK1通过缺失和连接子进行细胞质定位所必需的 扫描诱变，并鉴定一个潜在的细胞质锚定 与保留信号相互作用的蛋白质(S)。此外，我们还将 检视核移位的功能后果 在活体内拼接。在目标3中，我们将研究SRPK1的另一种机制 通过特定的抑制物进行调节。我们计划提纯抑制剂并 检查抑制的机制。我们还将调查 剪接调控中的抑制因子和剪接位点的选择。我们的 最终目标是探索信号可能被传递的可能性 通过SRPK1，调控细胞核内的Pre-mRNA剪接。

Recent studies have shown that a large family of splicing factors containing a serine/arginine-rich domain plays important roles in both constitutive and regulated splicing. The family members are generally referred to as SR proteins. SR proteins are phosphoproteins and increasing evidence suggests that they are targets for phosphorylation regulation in pre-mRNA splicing. To study this regulation, we have recently identified and cloned a cell cycle regulated kinase, SRPK1, that appears to be specific for SR proteins in mammalian cells. We have demonstrated that SRPK1 is responsible for the cell cycle dependent reorganization of a nuclear structure (the nuclear speckles) where SR proteins and other splicing factors are concentrated. Because a significant amount of SRPK1 is present in the nucleus in interphase cells, we focus in this proposal on investigating the interphase function and regulation of SRPK1. Our specific Aim 1 is to address the function of SRPK1 in splicing by a series of in vitro experiments. We plan to determine the role of SRPK1 in splicing, and the effect of SR protein phosphorylation by SRPK1 on splice site selection. In addition, we will determine whether SRPK1 is a component of the spliceosome, and how SRPK1 is involved in splicing itself. Once the role of SRPK1 in splicing is established, we will, in Aim 2, address the regulation of SRPK1. Although SRPK1 contains a nuclear localization signal and a fraction of the kinase is present in the nucleus, a unique sequence in SRPK1 appears to function as a cytoplasmic retention signal that is responsible for the accumulation of a population of SRPK1 in the cytoplasm in interphase. Therefore, SRPK1 may be regulated by nuclear translocation. We plan to determine the minimal sequence required for the cytoplasmic localization of SRPK1 by deletion and linker scanning mutagenesis, and to identify a potential cytoplasmic anchor protein(s) that interacts with the retention signal. Further, we will examine the functional consequence of the nuclear translocation on splicing in vivo. In Aim 3, we will study another mechanism of SRPK1 regulation by a specific inhibitor. We plan to purify the inhibitor and examine the mechanisms of inhibition. We will also investigate the role of the inhibitor in the regulation of splicing and splice site selection. Our ultimate goal is to explore the possibility that signals may be transduced through SRPK1,to regulate pre-mRNA splicing in the nucleus.