Identification and Analysis of proteins which intreact with CRM1

CRM1是一种进化上保守的蛋白质，是依赖于亮氨酸的核输出信号(NES)依赖的蛋白质运输的受体，但在高等真核生物中，它在mRNA输出中的作用尚未确定。我们发现，用CRM1的特异性抑制剂软霉素B(LMB)处理哺乳动物细胞后，内源性mRNA的核积聚可能是由于抑制了内源性mRNA的输出。在裂解酵母中，在有限的温度下，用LMB处理的细胞或对温度敏感的CRM1突变体中也出现了mRNA的核积累。将合成的mRNA注入哺乳动物培养细胞的细胞核内，在5h内从细胞核中排出，这一排出可被小麦胚凝集素或40℃抑制。重要的是，这种信使核糖核酸的输出被LMB或过量的NES肽结合物抑制。这些结果表明，CRM1参与了真核细胞中的mRNA输出。我们先前证明了MAPK(MAPK)是MAPK的直接激活剂，是NES，并作为MAPK的细胞质锚。在这个项目中，我们揭示了MAPKK/MAPK复合体解离和/或结合的调节机制。酪氨酸(非洲爪哇MAPK中的Tyr190)被MAPKK磷酸化是解离复合体的必要条件和充分条件。

CRM1, an evolutionarily conserved protein, is shown to be a receptor for leucine-rich nuclear export signal (NES)-dependent protein transport, but its role in mRNA export has not been established in higher eukaryotes. We have found that treatment of mammalian cells with leptomycin B (LMB), a specific inhibitor of CRM1, induces nuclear accumulation of endogenous mRNA probably due to the inhibition of its export. In fission yeast, nuclear accumulation of mRNA also occurred in cells treated with LMB or in a temperature-sensitive crm1 mutant at a restrictive temperature. A synthetic mRNA that was injected into the nucleus of mammalian cultured cells was exported from the nucleus within 5 h. This export was inhibited by wheat germ agglutinin or at 40C. Importantly, this mRNA export was inhibited by LMB or by an excess amount of the NES peptideconjugates. These results suggest that CRM1 is involved in mRNA export in eukaryotic cells.We previously demonstrated that MAPKK(MAPK kinase),a direct activator for MAPK, as an NES and acts as a cytoplasmic anchor of MAPK. In this project, we revealed the regulatory mechanisms of the dissociation and/or the association of the MAPKK/MAPK complex. Tyrosine(Tyr190 in Xenopus MAPK)phosphorylation of MAPK by MAPKK is necessary and sufficient for the dissociation of the complex.