Deciphering the role of low complexity domains in dual specificity kinase function

解读低复杂性结构域在双特异性激酶功能中的作用

• 批准号: 10217666
• 负责人: Priya R. Banerjee
• 金额: $ 15.83万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10217666
• 关键词: Address; Alternative Splicing; Amino Acid Sequence; Amino Acids; Applications Grants; Bile fluid; Biochemical; Bioinformatics; Biological Assay; Biology; Biophysics; C-terminal; Cell Culture Techniques; Cell physiology; Cells; Cellular biology; Charge; Code; Crohn' s disease; Disease; Dissection; Down Syndrome; Eukaryota; Fluorescence; Functional disorder; Genes; Genome; Goals; HIV; Human; In Vitro; Individual; Length; Light; Link; Malignant Neoplasms; Malignant neoplasm of liver; Malignant neoplasm of urinary bladder; Mediating; Mediator of activation protein; Membrane; Modeling; Molecular; Mutation; N-terminal; Nature; Nuclear; Nuclear Protein; Organelles; Pathologic; Pathology; Phase; Phosphotransferases; Physical condensation; Physiological; Physiology; Play; Process; Property; Protein Family; Protein Kinase; Proteins; RNA Binding; RNA Splicing; Regulation; Reporting; Role; Sequence Analysis; Signal Transduction; Signal Transduction Pathway; Specificity; Structure; Structure-Activity Relationship; Testing; Variant; base; biophysical properties; design; driving force; experimental study; in vivo; insight; mRNA Precursor; mutant; osteosarcoma; overexpression; prion-like; recruit; scaffold

Project Summary Dual specificity protein kinases perform indispensable roles in eukaryotes including the regulation of signal transduction pathways and subcellular compartmentalization. Structurally, in addition to their folded kinase domains, they are composed of significantly long segments that are intrinsically disordered. Whereas the folded domains of these kinases are studied extensively (both biochemically and structurally), little is known about the functional role of the disordered domains that display a compositional bias towards polar and charged amino acids. In our preliminary sequence analysis of all human dual specificity kinases, we identify that CLK3 has the longest intrinsically disordered region. Recent advances indicate that CLK3 is linked to multiple cancer pathologies and celiac disorders. For example, overexpression of CLK3 is associated with osteosarcoma, liver cancer and bile cancer. Sequence variations in CLK3 gene is associated with bladder cancer, Crohn’s disease and multiple celiac disorders. Functionally, CLK3 is recruited to nuclear speckles and plays vital roles in the regulation of pre-mRNA splicing. In this grant application, we propose to characterize the sequence determinants, molecular codes and functional roles of CLK3 nuclear speckle recruitment utilizing a combination of in vitro and in vivo experiments. Based on our preliminary analyses, we posit that (i) the low-complexity disordered domain of CLK3 (residue 1-285) drives the protein’s nuclear speckle localization, and (ii) alterations in CLK3 speckle localization/dynamics is associated with disease pathologies. We will test these hypotheses utilizing an integrative biophysical, biochemical and cell biology approach. To this end, we will utilize a splice variant, a disease-linked mutant, and rationally perturbed LCD variants to systematically decipher the LCD’s role in CLK3’s function/dysfunction. Successful completion of the proposed study will not only illuminate the role of this disordered domain in CLK3 (patho)biology, but also provide significant insights into the interplay between the LCDs and kinase domains in dual specificity kinase functions and dysfunctions.

项目概要 双特异性蛋白激酶在真核生物中发挥着不可或缺的作用，包括 从结构上讲，信号转导途径和亚细胞区室化的调节。 除了折叠的激酶结构域外，它们还由相当长的片段组成 本质上是无序的。 （生物化学和结构上），人们对无序的功能作用知之甚少。 显示出对极性和带电氨基酸的组成偏向的结构域。 在我们对所有人类双特异性激酶的初步序列分析中，我们发现 CLK3 具有最长的本质无序区域。最新进展表明 CLK3 是相连的。 例如，CLK3 的过度表达与多种癌症病理和乳糜泻有关。 与骨肉瘤、肝癌和回肠癌相关。 与膀胱癌、克罗恩病和多种功能性乳糜泻有关。 CLK3 被招募到核斑点中，并在前 mRNA 剪接的调节中发挥重要作用。 在本次拨款申请中，我们建议表征序列决定因素、分子 利用体外组合的 CLK3 核斑点募集的代码和功能作用 和体内实验基于我们的初步分析，我们假设（i）低复杂性。 CLK3（残基 1-285）的无序结构域驱动蛋白质的核斑点定位，并且 (ii) CLK3 散斑定位/动力学的改变与疾病病理相关。 将利用综合生物物理、生物化学和细胞生物学来测试这些假设 为此，我们将合理地利用剪接变体，即与疾病相关的突变体。 扰动 LCD 变体，系统地破译 LCD 在 CLK3 功能/功能障碍中的作用。 成功完成拟议的研究不仅将阐明这种无序的作用 CLK3（病理）生物学领域，而且还提供了对 CLK3（病理）生物学之间相互作用的重要见解 双特异性激酶功能和功能障碍中的 LCD 和激酶结构域。