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.

项目摘要 双重特异性蛋白激酶在真核生物中发挥不可或缺的作用 信号转导途径和亚细胞隔室化的调节。在结构上，在 除了折叠的激酶结构域，它们由明显的长段组成 本质上无序。而这些激酶的折叠结构域是广泛研究的 （从生化和结构上），对无序的功能作用知之甚少 向极性和带电的氨基酸显示复合偏置的域。 在我们对所有人类双重特异性激酶的初步序列分析中，我们确定 CLK3具有最长的本质上无序区域。最近的进步表明CLK3已链接 多种癌症病理和腹腔疾病。例如，clk3的过表达为 与骨肉瘤，肝癌和胆汁癌有关。 CLK3基因的序列变化 与膀胱癌，克罗恩病和多种腹腔疾病有关。在功能上 CLK3被招募到核斑点，并在调节MRNA剪接的调节中起着至关重要的作用。 在此赠款应用中，我们建议表征序列确定的分子 CLK3核斑点募集的代码和功能作用，利用体外结合 和体内实验。基于我们的初步分析，我们肯定（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斑点定位/动力学的改变与疾病病理有关。我们 将利用整合生物物理，生化和细胞生物学检验这些假设 方法。为此，我们将利用剪接变体，疾病连接的突变体，并合理地 干扰的LCD变体系统地破解了LCD在CLK3功能/功能障碍中的作用。 成功完成拟议的研究不仅会阐明这种无序的作用 CLK3（Patho）生物学中的领域，但也为相互作用提供了重要的见解 双重特异性激酶功能和功能障碍中的LCD和激酶结构域。