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：调控和底物识别 摘要 真核细胞解释细胞外和内在的线索，以影响肌动蛋白细胞骨架的重塑， 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，对细胞骨架的冲击对于正常发育和体内平衡是必不可少的。RHO家族 GTP酶RHO、RAC和CDC 42各自在空间上直接激活激酶（RHO激酶、PAK和MRCK）。 限制性的方式，进而直接磷酸化和激活LIM结构域激酶（LIMK 1和LIMK 2）。 这些激酶信号级联最终汇聚在cofilin/ADF（actin-1）的磷酸化上。 解聚因子（解聚因子）是一组蛋白质，是介导肌动蛋白丝重塑的关键分子。来 在前两个阶段，我们利用两个实验室高度互补的专业知识， 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 （PAKs）的研究，并揭示LIMKs选择性靶向cofilin的基础。我们现在的目标是 回答关于LIMK的调节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的分子内相互作用， 涉及进化上保守的结合表面，负责抑制LIMK催化活性。 结合生物物理，生物化学和细胞为基础的方法，我们将解决的假设，破坏 这种相互作用的结果在激活的LIM激酶，我们将揭示LIMK的结构基础 自动调节我们将进一步研究最近的证据，LIMKs可以磷酸化丝氨酸和酪氨酸 通过LIMK-底物复合物的X射线晶体学和分子动力学模拟，以这种方式 LIMKs将作为理解底物识别的各种“双重特异性”的一般模型。 激酶家族最后，我们将研究强直性肌营养不良相关的CDC 42结合蛋白激酶 （MRCK），一组主要的LIMK激活激酶下游的GT3 CDC 42，关于这一点很少被提及。 目前已知。我们将使用结构，生物物理和生物化学的方法来定义基本架构 的MRCKβ研究，并探讨其激活如何耦合到与LIMKs的相互作用，通过底物衔接 proteins.总的来说，我们的研究将为我们在分子水平上对信号传导的理解提供实质性的进展 RHO家族GTP酶的下游通路，其影响肌动蛋白细胞骨架的调节。