The analysis of molecular mechanism for IRS-1 ubiquitination by nitric oxide

(1)一氧化氮对肿瘤细胞IGF-R/1RS-1 /PI3-K/Akt通路和MAPK通路的影响。一氧化氮（NO）供体下调MIAPaCa-2细胞（来源于胰腺癌）的胰岛素/IGF信号，包括IRS-1、Akt和GSK-3。另一方面，NO供体上调Erk 1/2的磷酸化。(2) NO供体蛋白酶抑制剂使IRS-1泛素化完全抑制了NO对MIAPaCa-2中IRS-1蛋白的降解。结果怀疑NO调控了MIAPaCa-2的泛素化和IRS-1蛋白的降解。我们制备了IRS-1延迟突变体的一些结构体，并使MIAPaCa-2表达它们。然后，我们找到了IRS-I蛋白被NO降解的重要位点。(3) NO对肿瘤增殖的影响NO可下调部分肿瘤细胞系的细胞生长。我们进行了稳定细胞系的克隆；MIAPaCa-2过表达IRS-1野生型蛋白，MIAPaCa-2过表达IRS-1显性阴性。过表达IRS-1野生型蛋白的细胞株对细胞生长具有较高的种子率，对NO具有较高的敏感性。另一方面，过表达IRS-1显性阴性的细胞系，细胞生长速度较慢，对NO的敏感性较小。这些结果表明NO通过抑制IRS-1蛋白下调IGF信号。这可能是NO下调肿瘤增殖的机制之一。

(1) The effects of nitric oxide on IGF-R/1RS-1 /PI3-K/Akt pathway and MAPK pathway in cancer cells.Nitric oxide (NO) donor down-regulated insulin/IGF signal including IRS-1, Akt, and GSK-3 in MIAPaCa-2 cells (derived from pancreatic cancer). On the other hand, NO donor up-regulated the phosphorylation of Erk 1/2.(2) IRS-1 ubiquitination by NO donorProtease inhibitor completely inhibited the degradation of IRS-1 protein by NO in MIAPaCa-2. The result suspected that NO regulated the ubiquitination and the degradation of IRS-1 protein in MIAPaCa-2. We produced the some constructs of IRS-1 delation mutants, and made the MIAPaCa-2 express them. Then, we found out the important site for IRS-I protein degradation by NO.(3) The effects of NO on cancer proliferationNO down-regulated the cell growth in some cancer cell lines.We performed the cloning of stable cell lines; MIAPaCa-2 over-expressing IRS-1 wild type protein, MIAPaCa-2 over-expressing IRS-1 dominant negative. The cell line, over-expressing IRS-1 wild type protein, show high seed for cell growth, and greater sensitivity for NO. On the other hand, cell line, over-expressing IRS-1 dominant negative, show low speed of cell growth, and smaller sensitivity for NO. These results indicates that NO down-regulates IGF signal by the depression of IRS-1 protein. That may be one of the mechanisms for the down-regulation of cancer proliferation by NO.