IDENTIFICATION OF PUTATIVE UBIQUINATION SITES ON CALPACTIN

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 膜联蛋白II是纤溶酶原（Kd = 114 nM）和组织纤溶酶原激活剂（Kd = 30 nM）的促纤溶共受体，其刺激主要纤溶酶纤溶酶激活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，在体外和体内不存在细胞死亡或细胞溶解的情况下，响应于短暂的温度应激，从细胞质易位到细胞质外质膜。这种受调节的反应不依赖于新蛋白质或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易位依赖于蛋白质p11（S100 A10）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易位到细胞表面显着增加组织纤溶酶原激活剂依赖的纤溶酶原激活的潜力，并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活化。我们已经表明，通过泛素化途径降解p11的螯合导致纤溶酶原激活，从而纤溶酶形成显着减少。我们已经通过质谱技术对p11上推定的泛素化位点进行了广泛的作图，目前已覆盖了94%的蛋白质序列。剩余的序列区域位于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。

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.