IDENTIFICATION OF PUTATIVE UBIQUINATION SITES ON CALPACTIN

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。膜联蛋白II是纤溶酶原(Kd=114 nM)和组织型纤溶酶原激活剂(Kd=30 nM)的纤溶酶原结合受体，可刺激主要纤溶酶--纤溶酶的激活，激活倍数为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，在体外和体内都是在没有细胞死亡或细胞溶解的情况下，对短暂的温度应激做出反应，从细胞质转移到胞质外质膜上。这种受调控的反应不依赖于新的蛋白质或mRNA的合成，也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的易位依赖于蛋白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突变时被完全消除。膜联蛋白II转位到细胞表面显著增加了组织纤溶酶原激活剂依赖的纤溶酶原激活潜力，可能代表了一种新的应激诱导的蛋白质分泌途径。由两个Annexin II和两个p11分子组成的Annexin II异四聚体的形成，进一步诱导了纤溶酶的激活。我们已经证明，通过泛素化途径隔离p11降解导致纤溶酶原激活显著减少，从而形成纤溶酶。我们已经通过质谱学技术对p11上可能的泛素化位点进行了广泛的绘制，目前已经覆盖了94%的蛋白质序列。剩余的序列位于p11的羧基末端，含有三个赖氨酰残基。仍然需要确定这三个残基中的哪一个实际上被泛素化修饰了。

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.