IDENTIFICATION OF PUTATIVE UBIQUINATION SITES ON CALPACTIN

钙肽酶上假定的泛化位点的鉴定

• 批准号: 7355121
• 负责人: KATHERINE AMBERSON HAJJAR
• 金额: $ 0.25万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-01 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7355121
• 关键词: IDENTIFICATION; PUTATIVE; UBIQUINATION; SITES; CALPACTIN

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 reticulum¿Golgi 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.

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，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）的纤溶酶原共受体，可刺激主要纤溶酶（纤溶酶）的活化60倍。内皮细胞膜联蛋白II是一种缺乏典型信号肽的蛋白，在体外和体内均可响应短暂的温度胁迫，在没有细胞死亡或细胞裂解的情况下从细胞质转移到胞质外膜。这种受调节的反应不依赖于新蛋白或mRNA的合成，也不需要经典的内质网高尔基通路。温度胁迫诱导的膜联蛋白II易位既依赖于蛋白p11 （S100A10）的表达，也依赖于膜联蛋白II的酪氨酸磷酸化，因为在p11缺失、酪氨酸激酶失活或酪氨酸23突变的情况下，膜联蛋白II的释放被完全消除。膜联蛋白II易位到细胞表面显著增加组织纤溶酶原激活剂依赖的纤溶酶原激活电位，可能代表一种新的应激诱导蛋白分泌途径。由两个膜联蛋白II和两个p11分子组成的膜联蛋白II异源四聚体的形成进一步诱导了纤溶蛋白的激活。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著降低，从而导致纤溶酶形成。我们已经通过质谱技术对p11上假定的泛素化位点进行了广泛的定位，目前已经覆盖了94%的蛋白质序列。剩余的序列区域位于p11的羧基端，包含三个赖基残基。这三个残基中哪一个实际上是被泛素化修饰的还有待确定。