IDENTIFICATION OF PUTATIVE UBIQUINATION SITES ON CALPACTIN

钙肽酶上假定的泛化位点的鉴定

• 批准号: 7722228
• 负责人: KATHERINE AMBERSON HAJJAR
• 金额: $ 0.11万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-03-01 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7722228
• 关键词: ANXA2 gene; Alteplase; Amino Acid Sequence; Calpactin; Cell Death; Cell membrane; Cell surface; Cells; Computer Retrieval of Information on Scientific Projects Database; Cytolysis; Cytoplasm; Endothelial Cells; Funding; Grant; In Vitro; Institution; Maps; Messenger RNA; Mutation; Pathway interactions; Peptide Sequence Determination; Peptide Signal Sequences; Plasmin; Plasminogen; Protein Secretion; Protein Tyrosine Kinase; Proteins; Research; Research Personnel; Resources; S100 family protein p11; Site; Source; Stress; Stress-Induced Protein; Techniques; Temperature; Tyrosine; Tyrosine Phosphorylation; Ubiquitination; United States National Institutes of Health; in vivo; novel; plasminogen receptor; protein expression; response

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Annexin II is a profibrinolytic co-receptor for plasminogen (Kd = 114 nM) and tissue plasminogen activator (Kd = 30nM), which stimulates activation of the major fibrinolysin, plasmin, 60-fold. Endothelial cell annexin II, a protein that lacks a typical signal peptide, is translocated from the cytoplasm to the extracytoplasmic plasma membrane in response to brief temperature stress both in vitro and in vivo in the absence of cell death or cell lysis. This regulated response is independent of new protein or mRNA synthesis and does not require the classical endoplasmic reticulumGolgi pathway. Translocation of annexin II induced by temperature stress is dependent on both expression of protein p11 (S100A10) and tyrosine phosphorylation of annexin II because the release of annexin II is completely eliminated on depletion of p11, inactivation of tyrosine kinase, or mutation of tyrosine 23. Translocation of annexin II to the cell surface dramatically increases tissue plasminogen activator-dependent plasminogen activation potential and may represent a novel stress-induced protein secretion pathway. The formation of the annexin II heterotetramer, composed of two annexin II and two p11 molecules, induces activation of plasmin even further. We have shown that sequestering of p11 for degradation via the ubiquitination pathway leads to a significant decrease in plasminogen activation, thus plasmin formation. We have undergone extensive mapping of the putative ubiquitination sites on p11 by mass spectrometric techniques and have currently covered 94% of the protein sequence. The remaining sequence region is located on the carboxyl terminal of p11 and contains three lysyl residues. It still remains to ascertain which one the three residues is actually being modified by ubiquitination.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 膜联蛋白 II 是纤溶酶原 (Kd = 114 nM) 和组织纤溶酶原激活剂 (Kd = 30nM) 的纤溶原共受体，可刺激主要纤溶酶纤溶酶的激活 60 倍。内皮细胞膜联蛋白 II 是一种缺乏典型信号肽的蛋白质，在没有细胞死亡或细胞裂解的情况下，响应体外和体内的短暂温度应激，从细胞质转移到胞质外质膜。这种调节反应独立于新蛋白质或 mRNA 合成，并且不需要经典的内质网高尔基体途径。温度应激诱导的膜联蛋白 II 易位取决于蛋白 p11 (S100A10) 的表达和膜联蛋白 II 的酪氨酸磷酸化，因为膜联蛋白 II 的释放在 p11 耗尽、酪氨酸激酶失活或酪氨酸 23 突变时完全消除。膜联蛋白 II 易位到细胞表面会显着增加组织纤溶酶原激活剂依赖性纤溶酶原 激活潜力，可能代表一种新的应激诱导的蛋白质分泌途径。由两个膜联蛋白 II 和两个 p11 分子组成的膜联蛋白 II 异四聚体的形成进一步诱导纤溶酶的激活。我们已经证明，通过泛素化途径隔离 p11 进行降解会导致纤溶酶原激活显着减少，从而导致纤溶酶形成。我们通过质谱技术对 p11 上假定的泛素化位点进行了广泛的定位，目前已覆盖了 94% 的蛋白质序列。其余序列区域位于 p11 的羧基末端，包含三个赖氨酰残基。仍有待确定这三个残基中的哪一个实际上被泛素化修饰。