Molecular mechanisms of ERM regulation

ERM调节的分子机制

• 批准号: 7965921
• 负责人: James Shaw
• 金额: $ 25.68万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7965921
• 关键词: Actins; Acute; Binding; Biological Assay; Biology; CD44 gene; Cell membrane; Cells; Cellular biology; Complex; Cytoplasmic Tail; Cytoskeleton; Cytosol; Dependence; Event; Family; Hydrolysis; In Vitro; Journals; Lipids; Lymphocyte; Lymphocyte Activation; Mediating; Mediator of activation protein; Membrane; Modeling; Molecular; Mutation; N-terminal; Paper; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Phospholipase C; Phospholipids; Phosphoric Monoester Hydrolases; Phosphorylation; Process; Protein Dephosphorylation; Proteins; Publishing; Regulation; Role; Signal Transduction; Stromal Cell-Derived Factor 1; Transfection; chemokine; ezrin; inhibitor/antagonist; moesin; mutant; protein protein interaction; radixin protein

We first described several years ago the finding that chemokine (SDF-1) stimulation of lymphocytes induces very rapid (onset <=5 sec) ERM dephosphorylation and inactivation. Given the evidence that phosphorylation contributes to ERM activation, it was plausible that dephosphorylation mediated the inactivation. However, membrane phospho-lipid, especially PIP2, is an additional key mediator of ERM activation. We hypothesized that acute reduction of PIP2 could contribute to ERM inactivation. Specifically, we considered the possibility that chemokine: 1) activates phospholipase C (PLC); which 2) mediates reduction of phosphatidylinositol 4,5-bisphosphate (PIP2); which 3) releases ERM from its association with the plasma membrane; and 4) triggers ERM dephosphorylation. This proposed model is supported by multiple findings we have made, including the following. PLC inhibitor blocks these events. Translocation of GFP-tagged moesin into cytosol and ERM dephosphorylation can be induced either by: transfection with active PLC construct; or by acute targeting of phosphoinositide 5-phosphatase to the plasma membrane. PIP2 dependence cannot be replaced by phosphorylation (modeled by T558D mutation) in either of two assays: association with plasma membrane in cells or in vitro association with cytoplasmic tails of proteins like CD44. Moreover, induction of PIP2 hydrolysis is sufficient to induce ERM dephosphorylation, indicating that PLC activation can be sufficient to induce ERM dephosphorylation and that PLC activation may be the initiating event in lymphocytes rather than independent activation of an ERM phosphatase. Key findings made with moesin constructs have been confirmed with ezrin constructs, indicating that these are general principles pertinent to both ezrin and moesin. These results demonstrate a powerful new role of PLCs in rapid cytoskeletal remodeling and an additional key role of PIP2 in ERM biology, namely hydrolysis-mediated ERM inactivation. These studies are described in a paper published this year in Journal of Cell Biology. The complex N-terminal domain of ERM is called the FERM domain which binds to the C-terminus and thereby participates in autoinhibition. In our view, the release of ERM autoinhibition by acidic phospholipid binding is complex and incompletely understood. Therefore, we are conducting additional mutational analysis of FERM domain residues and analyzing the mutant proteins for protein-protein interaction and membrane localization to extend our functional understanding of these key processes.

几年前，我们首先描述了趋化因子（SDF-1）刺激淋巴细胞的刺激会诱导非常快速（发作<= 5 sec）ERM磷酸化和失活。鉴于证据表明磷酸化有助于ERM激活，因此去磷酸化介导了失活是合理的。然而，膜磷酸脂质，尤其是PIP2是ERM活化的另一个关键介体。我们假设PIP2的急性减少可能导致ERM失活。具体而言，我们考虑了趋化因子的可能性：1）激活磷脂酶C（PLC）； 2）介导磷脂酰肌醇4,5-双磷酸的还原（PIP2）； 3）释放ERM与质膜的关联； 4）触发ERM去磷酸化。我们提出的模型得到了我们已经做出的多个发现的支持，包括以下内容。 PLC抑制剂阻止这些事件。 GFP标记的Moesin转移到细胞质中，ERM去磷酸化可以通过以下方式诱导：用活性PLC构建体转染；或通过将磷酸肌醇5-磷酸酶急性靶向到质膜。 PIP2依赖性不能被两种测定中的任何一个中的任何一个磷酸化（由T558D突变建模）所取代：与细胞中的质膜相关，或与CD44（如CD44）的细胞质尾巴相关。此外，PIP2水解的诱导足以诱导ERM去磷酸化，表明PLC激活足以诱导ERM脱磷酸化，并且PLC激活可能是淋巴细胞中的起始事件，而不是独立的ERM磷酸酶激活。用Ezrin构建体证实了用Moesin构建体制成的主要发现，表明这些是与Ezrin和Moesin有关的一般原则。这些结果表明，PLC在快速细胞骨架重塑中起着强大的新作用，以及PIP2在ERM生物学中的其他关键作用，即水解介导的ERM失活。这些研究在今年发表在细胞生物学杂志上的论文中进行了描述。 ERM的复杂N末端结构域称为FERM结构域，该结构域与C末端结合，从而参与自身抑制。我们认为，通过酸性磷脂结合释放ERM自身抑制作用是复杂且未完全理解的。因此，我们正在对FERM结构域残基进行其他突变分析，并分析突变蛋白的蛋白质 - 蛋白质相互作用和膜定位，以扩展我们对这些关键过程的功能理解。