Regulation of Cellular Signaling Pathways by POSH

POSH 对细胞信号通路的调节

• 批准号: 7528685
• 负责人: CHARLES KENNETH KASSENBROCK
• 金额: $ 25.63万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7528685
• 关键词: Affect; Apoptosis; Apoptotic; Binding; Binding Sites; Biochemical; Biological Assay; Cell Death; Cell physiology; Cells; Cellular Stress; Class; Complex; Data; Degenerative Disorder; Future; GTP Binding; In Vitro; Injury; Lead; MAPK8 gene; Mammalian Cell; Mass Spectrum Analysis; Mediating; Minor; Modeling; Molecular; Nerve Growth Factors; Neurons; Numbers; PC12 Cells; Pathway interactions; Phosphorylation; Phosphorylation Site; Play; Point Mutation; Protein Kinase; Protein Overexpression; Proteins; Public Health; Regulation; Reporting; Rodent Model; Role; Scaffolding Protein; Serine; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Signaling Protein; Site; Stress; Stroke; Techniques; Testing; Ubiquitin-Protein Ligase Complexes; Ubiquitination; Withdrawal; Work; Yeasts; base; cell growth regulation; cell killing; cell type; clinically relevant; computerized data processing; improved; in vivo; mimetics; mutant; nervous system disorder; prevent; response; scaffold; stroke therapy; ubiquitin ligase; ubiquitin-protein ligase; yeast two hybrid system

DESCRIPTION (provided by applicant): The overall aim of these studies is to further understand the role of POSH (Plenty of SH3s) in apoptotic signaling in neurons and other cells. In response to a number of severe stresses, a signal transduction pathway is engaged leading from Rac to MLK to MKK to JNK and ending in apoptotic cell death. POSH is a recently discovered protein that acts as a molecular scaffold to bind Rac, MLKs and another group of scaffold proteins, JIPs, to facilitate apoptotic signaling through this pathway. This multi-protein signaling complex has been called the POSH-JIP Apoptotic Complex (PJAC). The role of POSH in this pathway appears to be essential; reducing POSH levels has been reported to reduce apoptosis in neurons and neuronally differentiated PC12 cells following nerve growth factor withdrawal, as well as in a rodent model of stroke. Understanding how POSH functions and is regulated is therefore critical to understanding stress-induced apoptosis, and ultimately may allow manipulation of the pathway in clinically useful ways. Over-expression of POSH induces apoptosis in neurons and other cell types examined, but can be prevented by the pro-survival protein kinase, Akt. We have shown that Akt directly phosphorylates POSH and blocks its ability to bind Rac. We have identified major and minor phosphorylation sites in POSH in vitro using mass spectrometry and we propose to use similar techniques to determine Akt phosphorylation sites in POSH in vivo, and to ask how phosphorylation at these sites affects POSH function. We also propose to investigate assembly of the PJAC complex, and ask what components of PJAC are constitutively bound to POSH in viable, unstressed cells, versus cells subjected to apoptotic stress. To further understand PJAC assembly, we will precisely identify the binding sites in POSH for MLK3 and JIP1, using yeast two-hybrid studies and biochemical studies with purified proteins. We also propose to identify critical regions in POSH by mutagenizing POSH and selecting for mutants that no longer induce apoptosis when over-expressed in mammalian cells. Finally, in addition to its function as a protein scaffold, POSH has ubiquitin ligase (E3) activity mediated by a RING domain. We will employ a variety of in vitro ubiquitination assays to test several models of how POSH's E3 activity might regulate apoptotic signaling within the cell. The proposed studies should lead to a greater understanding of how POSH promotes apoptotic signaling in neurons and other cells, and how this process is regulated. These studies are clinically relevant and may ultimately lead to improved therapies for stroke and other degenerative neurological diseases. PUBLIC HEALTH RELEVANCE: When nerve cells and other types of cells are subjected to severe stresses, it can trigger a special pathway that ends up killing the cells. This proposal seeks to better understand how that stress pathway works, so that we might be able to control it in the future. If we could control that pathway, nerve cells might be prevented from dying after some types of injuries or in some degenerative diseases.

描述（由申请人提供）：这些研究的总体目的是进一步了解POSH（大量SH3S）在神经元和其他细胞中凋亡信号传导中的作用。为了响应许多严重的应力，信号转导途径从RAC到MLK到MKK到JNK，并在凋亡细胞死亡中结束。 POSH是一种最近发现的蛋白质，可作为分子支架，与RAC，MLK和另一组脚手架蛋白JIPS结合，以通过该途径促进凋亡信号传导。这种多蛋白信号传导复合物称为POSH-JIP凋亡复合物（PJAC）。豪华在这一途径中的作用似乎是必不可少的。据报道，降低的posh水平可降低神经生长因子戒断后神经元和神经分化的PC12细胞的凋亡，以及中风模型。因此，了解豪华的功能和受调节方式对于理解压力引起的凋亡至关重要，最终可能允许以临床上有用的方式操纵该途径。 POSH的过度表达可诱导神经元和其他检查的细胞类型的凋亡，但可以通过促生物存在蛋白激酶（Akt）预防。我们已经表明，AKT直接磷酸化posh并阻止其结合RAC的能力。我们已经使用质谱法确定了POSH的主要磷酸化位点，我们建议使用类似的技术来确定体内POSH的Akt磷酸化位点，并询问这些位点的磷酸化如何影响POSH功能。我们还建议研究PJAC复合物的组装，并询问PJAC的哪些组成部分在可行的，无压力的细胞中与POSH结合，而受到凋亡应激的细胞。为了进一步了解PJAC组装，我们将使用酵母的两杂化研究和使用纯化蛋白质的酵母研究和生化研究，准确地确定MLK3和JIP1的POSH中的结合位点。我们还建议通过诱变POSH并选择在哺乳动物细胞中过表达时不再诱导凋亡的突变体来识别POSH中的关键区域。最后，除了其作为蛋白质支架的功能外，POSH还具有由环域介导的泛素连接酶（E3）活性。我们将采用各种体外泛素化测定法来测试一些模型，即POSH的E3活性如何调节细胞内的凋亡信号传导。拟议的研究应更深入地了解豪​​华的神经元和其他细胞中的凋亡信号传导以及如何调节该过程。这些研究在临床上是相关的，最终可能导致改善中风和其他退化性神经系统疾病的疗法。 公共卫生相关性：当神经细胞和其他类型的细胞受到严重的压力时，它可能会触发一条特殊的途径，最终杀死细胞。该提议试图更好地了解压力途径如何工作，以便我们将来能够控制它。如果我们能够控制该途径，则可能阻止神经细胞在某些类型的损伤或某些退化性疾病中死亡。