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 双特异性蛋白激酶在真核生物中起着不可或缺的作用，包括 调节信号转导途径和亚细胞区室化。在结构上，在 除了它们的折叠激酶结构域，它们还由相当长的片段组成， 本质上是无序的而这些激酶的折叠结构域被广泛研究 (both生物化学和结构），很少有人知道的功能作用的无序 这些结构域显示出对极性和带电氨基酸的组成偏好。 在我们对所有人类双特异性激酶的初步序列分析中，我们确定， CLK 3具有最长的固有无序区。最近的进展表明，CLK 3与 多种癌症病理和腹腔疾病。例如，CLK 3的过表达是 与骨肉瘤、肝癌和胆汁癌相关。CLK 3基因序列变异 与膀胱癌、克罗恩病和多种乳糜泻有关。在功能上， CLK 3被募集到核斑点中，并在前体mRNA剪接的调节中发挥重要作用。 在这项拨款申请中，我们建议描述序列决定簇，分子 利用体外细胞培养和细胞培养的组合， 和体内实验。基于我们的初步分析，我们认为（i）低复杂性 CLK 3的无序结构域（残基1-285）驱动蛋白质的核斑点定位， (ii)CLK 3斑点定位/动力学的改变与疾病病理学相关。我们 我将利用生物物理学、生物化学和细胞生物学的综合方法来检验这些假设 approach.为此，我们将利用一种剪接变异体，一种与疾病相关的突变体， 干扰LCD变体以系统地破译LCD在CLK 3的功能/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， 结构域在CLK 3（病理）生物学，而且还提供了重要的见解之间的相互作用， 双特异性激酶功能和功能障碍中的LCD和激酶结构域。