LIM domain kinases: regulation and substrate recognition

LIM 结构域激酶：调节和底物识别

• 批准号: 10443356
• 负责人: Titus Jonathon Boggon
• 金额: $ 35.18万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10443356
• 关键词: Actins; Active Sites; Adaptor Signaling Protein; Address; Architecture; Area; Binding; Binding Proteins; Biochemical; Biological Models; Biophysics; CDC42 gene; Catalytic Domain; Cells; Cellular Morphology; Complement; Complex; Consensus Sequence; Coupled; Coupling; Cryoelectron Microscopy; Crystallization; Cues; Cytoplasmic Tail; Cytoskeleton; Data; Development; Distal; Enzymes; Eukaryotic Cell; Event; Family; Fibroblasts; Guanosine Triphosphate Phosphohydrolases; Homeostasis; Kinetics; LIM Domain; LIM Domain Kinase 1; LIMK1 gene; Laboratories; Libraries; MMP14 gene; Mediating; Microfilaments; Modeling; Molecular; Molecular Conformation; Monomeric GTP-Binding Proteins; Movement; Mutagenesis; Mutate; Myotonic Dystrophy; Peptides; Phosphorylation; Phosphorylation Site; Phosphotransferases; Process; Protein Kinase; Proteins; Regulation; Rho-associated kinase; Roentgen Rays; Role; Scanning; Signal Pathway; Signal Transduction; Specificity; Structure; Substrate Interaction; Surface; Variant; Work; X-Ray Crystallography; actin depolymerizing factor; base; biophysical techniques; cell growth regulation; cofilin; extracellular; insight; member; molecular dynamics; novel; p21 activated kinase; preference; recruit; rho GTP-Binding Proteins; upstream kinase

LIM Domain Kinases: Regulation and Substrate Recognition ABSTRACT Eukaryotic cells interpret extracellular and intrinsic cues to effect remodeling of the actin cytoskeleton, a process critical for controlling cell morphology, movement, and invasiveness. Tight control of signaling pathways impinging on the cytoskeleton is therefore essential to normal development and homeostasis. The RHO family GTPases RHO, RAC and CDC42 each directly activate kinases (RHO kinases, PAKs, and MRCKs) in a spatially restricted manner that in turn directly phosphorylate and activate the LIM domain kinases (LIMK1 and LIMK2). These kinase signaling cascades ultimately converge on phosphorylation of the cofilin/ADF (actin- depolymerizing factor) group of proteins, key molecules that mediate remodeling of actin filaments. Over the previous two periods we have leveraged the highly complementary expertise of our two laboratories to provide significant advances in two main areas: understanding the specificity and regulation of p21-activated kinases (PAKs) and revealing the basis for selective targeting of cofilin by LIMKs. We will now target our efforts toward answering outstanding questions that remain regarding regulation and function of LIMKs. Our preliminary data suggest that an intramolecular interaction between a LIM-PDZ module and the kinase domain, potentially involving evolutionarily conserved binding surfaces, is responsible for suppressing LIMK catalytic activity. Combining biophysical, biochemical, and cell-based approaches, we will address the hypothesis that disruption of this interaction results in activation of the LIM kinases, and we will reveal the structural basis for LIMK autoregulation. We will further investigate recent evidence that LIMKs can phosphorylate both Ser and Tyr residues by X-ray crystallography of LIMK-substrate complexes and molecular dynamics simulations. In this way LIMKs will serve as a general model for understanding substrate recognition by the various “dual specificity” kinase families. Finally, we will investigate the myotonic dystrophy related CDC42-binding protein kinases (MRCKs), a major group of LIMK activating kinases downstream of the GTPase CDC42, about which little is currently known. We will use structural, biophysical and biochemical approaches to define the basic architecture of MRCKβ studies and to probe how its activation is coupled to interactions with LIMKs through substrate adaptor proteins. Overall, our studies will provide a substantial advance in our molecular level understanding of signaling pathways downstream of the RHO family GTPases that impinge on regulation of the actin cytoskeleton.

LIM 结构域激酶：调节和底物识别 抽象的 真核细胞解释细胞外和内在线索来影响肌动蛋白细胞骨架的重塑，这是一个过程 对于控制细胞形态、运动和侵袭性至关重要。严格控制信号通路 因此，影响细胞骨架对于正常发育和体内平衡至关重要。 RHO家族 GTPases RHO、RAC 和 CDC42 各自在空间上直接激活激酶（RHO 激酶、PAK 和 MRCK） 进而直接磷酸化并激活 LIM 结构域激酶（LIMK1 和 LIMK2）。 这些激酶信号级联最终集中于肌动蛋白丝切蛋白/ADF（肌动蛋白- 解聚因子）一组蛋白质，介导肌动蛋白丝重塑的关键分子。超过 前两个时期，我们利用两个实验室高度互补的专业知识来提供 在两个主要领域取得重大进展：了解 p21 激活激酶的特异性和调节 （PAK）并揭示了 LIMK 选择性靶向丝切蛋白的基础。我们现在的努力目标是 回答有关 LIMK 的监管和功能的悬而未决的问题。我们的初步数据 表明 LIM-PDZ 模块和激酶结构域之间可能存在分子内相互作用 涉及进化保守的结合表面，负责抑制 LIMK 催化活性。 结合生物物理、生物化学和基于细胞的方法，我们将解决以下假设：破坏 这种相互作用导致 LIM 激酶激活，我们将揭示 LIMK 的结构基础 自动调节。我们将进一步研究 LIMK 可以磷酸化 Ser 和 Tyr 的最新证据 通过 LIMK-底物复合物的 X 射线晶体学和分子动力学模拟来分析残留物。这样 LIMK 将作为理解各种“双特异性”底物识别的通用模型 激酶家族。最后，我们将研究强直性肌营养不良相关的CDC42结合蛋白激酶 (MRCK)，GTPase CDC42 下游的一组主要 LIMK 激活激酶，但对此知之甚少。 目前已知。我们将使用结构、生物物理和生化方法来定义基本架构 MRCKβ 研究并探讨其激活如何通过底物适配器与 LIMK 相互作用耦合 蛋白质。总体而言，我们的研究将为我们对信号传导的分子水平理解提供实质性进展 RHO 家族 GTPases 下游的通路影响肌动蛋白细胞骨架的调节。