Clk Kinases and Splicing Regulation

描述(申请人提供)：尽管剪接位点的正确选择对基因组多样性、适应性生长和发育至关重要，但剪接错误会对功能产生巨大的有害影响，现在被认为是许多人类疾病的根本原因。事实上，剪接错误与肌肉营养不良、阿尔茨海默病、帕金森氏症、精神障碍、共济失调和癌症有关，这使得对控制剪接位点选择的因素的研究对人类疾病至关重要。剪接发生在剪接体，这是一个由几个RNA和大量蛋白质组成的大分子复合体。对于正常的基因剪接来说，关键是正确选择5‘和3’剪接位点，这些事件 发生在剪接体发育的早期，其特异性受称为SR蛋白的重要剪接因子家族的指导。SR蛋白的磷酸化状态直接影响其亚细胞定位和剪接活性，但我们对这些不同形式如何获得的理解充其量也是不完整的。SR蛋白的核进入和剪接功能是由SRPK家族蛋白激酶催化的基础水平的磷酸化(低磷酸化)驱动的。然而，随着第二个蛋白激酶家族作为关键的SR蛋白调节因子出现，这种一种激酶-一种底物的简单范例现在正受到挑战。与SRPKs相比，CLK家族能在更大程度上增加SR蛋白的磷酸化含量，从而产生结构和功能水平上基本未知的超磷酸化形式。虽然它们在许多方面与SRPKs有很大的不同，但最重要的是，CLK激酶拥有一个额外的非催化结构域，这是我们最近发现的，它是导致其独特的超磷酸化活性的原因。尽管它在控制SR蛋白功能和剪接方面具有重要意义，但人们对它知之甚少 CLK酶。使用新的磷酸定位和结构技术，结合基于细胞的分析，我们将研究CLK激酶如何过度磷酸化SR蛋白并调节剪接。 公共卫生相关性：尽管包括神经退行性疾病和癌症在内的许多人类疾病与基因剪接错误有关，但控制细胞内剪接位点选择的因素仍然知之甚少。为了了解疾病和基因处理之间的联系，我们正在研究一组重要的剪接因子(SR蛋白质)中独特的磷酸化状态是如何由CLK家族酶催化并调节正确剪接位点的选择的。

DESCRIPTION (provided by applicant): While the proper selection of splice sites is essential for genomic diversity, adaptive growth and development, errors in splicing can have enormous detrimental effects on function and are now recognized as the underlying cause for many human diseases. Indeed, splicing errors are associated with muscular dystrophy, Alzheimer's disease, parkinsonism, psychiatric disorders, ataxias and cancers making the study of factors that control splice-site selection vitally important for human disease. Splicing occurs at the spliceosome, a macromolecular complex composed of several RNAs and numerous proteins. Critical to normal gene splicing is the proper selection of the 5' and 3' splice sites, events that occur early in the development of the spliceosome and whose specificity is guided by an essential family of splicing factors known as SR proteins. The phosphorylation states of SR proteins directly impact their subcellular localization and splicing activities but our understandig of how these different forms are attained is, at best, incomplete. SR protein nuclear entry and splicing function are driven by a basal level of phosphorylation (hypo-phosphorylation) catalyzed by the SRPK family of protein kinases. However, this simple paradigm of one kinase-one substrate is now being challenged as a second protein kinase family has emerged as critical SR protein regulators. The Clk family of kinases can increase SR protein phosphoryl content to a greater extent than the SRPKs, generating hyper-phosphorylated forms that are largely uncharacterized at both structural and functional levels. While they differ substantially from the SRPKs in several ways, most importantly, Clk kinases possess an additional noncatalytic domain that we showed recently is responsible for its unique hyper-phosphorylating activity. Despite its significance in controlling SR protein function and splicing, little is known about the Clk enzymes. Using novel phosphate mapping and structural techniques combined with cell-based assays, we will investigate how the Clk kinases hyper-phosphorylate SR proteins and modulate splicing. PUBLIC HEALTH RELEVANCE: Although many human diseases including neurodegenerative disorders and cancers are linked to mistakes in gene splicing, what controls the selection of splice sites in the cell is still poorly understood. To understand the link between disease and gene processing, we are studying how unique phosphorylation states in an essential group of splicing factors (SR proteins) are catalyzed by the Clk family of enzymes and regulate the selection of correct splice sites.