Nuclear Translocation of PKCdelta in Salivary Acinar Cells

唾液腺细胞中 PKCdelta 的核转位

• 批准号: 8010663
• 负责人: Tariq Sami Adwan
• 金额: $ 2.91万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-02-01 至 2011-08-17
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8010663
• 关键词: Adverse effects; Apoptosis; Apoptosis Regulator; Apoptotic; Binding; Binding Sites; Biological Assay; Catalytic Domain; Cell Nucleus; Cells; Cytoplasm; Cytosol; Disease; Effectiveness; Epithelial Cells; Event; Genotoxic Stress; Goals; Head and Neck Cancer; Head and neck structure; Importins; In Vitro; Induction of Apoptosis; Ligands; Medical; Molecular; Mus; National Institute of Dental and Craniofacial Research; Nuclear; Nuclear Import; Nuclear Localization Signal; Nuclear Translocation; Pathway interactions; Patients; Phosphorylation; Phosphotransferases; Protein Binding; Protein Tyrosine Kinase; Protein-Serine-Threonine Kinases; Quality of life; Radiation; Radiation induced damage; Regulation; Resistance; Role; SH3 Domains; Saliva; Salivary; Salivary Glands; Sequence Analysis; Signal Transduction; Site; Site-Directed Mutagenesis; Sjogren' s Syndrome; Stimulus; Therapeutic; Tyrosine; Tyrosine Phosphorylation; Xerostomia; cancer therapy; design; head and neck cancer patient; in vivo; insight; irradiation; mutant; prevent; protein kinase C-delta; research study; response; salivary acinar cell; therapeutic development

DESCRIPTION (provided by applicant): Xerostomia is the medical term for dry mouth due to a lack of saliva because of salivary glad hypofunction. Xerostomia is a side effect of irradiation treatment administered to head and neck cancers and greatly diminishes the effectiveness of anti-cancer therapies as well as the life quality of patients. The main cause of Xerostomia is salivary epithelial cells apoptosis. Our lab has identified PKC5 as a critical regulator of apoptosis in the salivary gland both in vitro and in vivo. We have also shown that nuclear accumulation of PKC5 is both required and sufficient for apoptosis, suggesting that tight regulation of the nuclear of this ubiquitous kinase is essential to avoid in-appropriate induction of apoptosis. Here we are exploring a regulatory interaction between the regulatory of PKC6, where we have shown that phosphryaltion of Y64 and Y155 is essential for nuclear localization of PKC5 and c-terminal Nuclear localization signal (NLS) that while required for nuclear accumulation of PKC5 is not sufficient. Interestingly, we have identified a PxxP motif overlapping the NLS that resembles a an SH3 binding site such that found in SH3 containing tyrosine kinases. Here, we are exploring the hypothesis that binding of tyrosine Kinase to the PxxP site, regulates phosphorylation of Y64 and Y155 which induces a conformational change allowing access of nuclear import machinery to the NLS. In aim 1 I will use site directed mutagenesis to determine the role of the PxxP site in regulating nuclear import of PKC5 and phosphoryaltion at Y64 and Y155, in addition I will use pull down assays to determine if nuclear import in ultimately regulated by binding of the NLS to nuclear import machinery. In Aim 2 I will use in and in vitro binding assays to determine which of the candidate tyrosine kinases that interact with PKC6 bind to it via the PxxP site, and assess the role of this interaction in the context of nuclear import of PKC5 using knockdown of the tyrosine kinase. We believe that our findings here will greatly contribute to public contribute to public as understanding the mechanism regulating nuclear import of PKC5 will give insight into mechanism by which PKC6 regulates apoptosis in the salivary epithelial cells which is crucial for the development of therapeutic strategies to protect against salivary gland apoptosis.

描述（由申请人提供）：由于唾液性良好的功能障碍，由于缺乏唾液，切除口径是干口的医学术语。静态症是针对头颈癌的辐射治疗的副作用，并大大降低了抗癌疗法的有效性以及患者的生活质量。静态的主要原因是唾液上皮细胞凋亡。我们的实验室已将PKC5确定为体外和体内唾液腺凋亡的关键调节剂。我们还表明，PKC5的核积累既需要凋亡，也足以使这种无处不在的激酶对核的严格调节对于避免不适当的凋亡诱导至关重要。在这里，我们正在探索PKC6调节性之间的调节相互作用，在该调节中，我们表明Y64和Y155的磷酸化对于PKC5和C端核定位信号（NLS）的核定位至关重要，尽管PKC5的核积累需要PKC5的核定位。有趣的是，我们已经确定了一个类似于sh3结合位点的NLS的PXXP基序，因此在含有酪氨酸激酶的SH3中发现。在这里，我们探讨了以下假设：酪氨酸激酶与PXXP位点结合，调节Y64和Y155的磷酸化，从而诱导构象变化，从而允许将核进口机械访问NLS。在AIM 1中，我将使用定向诱变来确定PXXP位点在调节Y64和Y155时调节PKC5和磷酸化的核进口中的作用，此外，我还将使用下拉测定来确定NLS与NLS结合到核进口机械的结合最终调节的核进口。在AIM 2中，我将在体外结合测定法中确定与PKC6相互作用的候选酪氨酸激酶通过PXXP位点结合，并评估这种相互作用在使用酪氨酸激酶敲低PKC5的核进口核导入的背景下的作用。我们认为，我们这里的发现将极大地为公众做出贡献，因为理解调节PKC5核进口的机制将洞悉PKC6在唾液上皮细胞中调节凋亡的机制，这对于唾液上皮细胞中的凋亡对于开发治疗策略至关重要，以防止唾液腺凋亡。