Abstract:
SG2NA, a WD40 repeat protein of the Striatin subfamily, has four splicing and one messenger RNA edit variants. It is fast emerging as a scaffold for multimeric signaling complexes with roles in tissue development and disease. The green fluorescent protein (GFP)-tagged variants of SG2NA were ectopically expressed in NIH3T3 cells and their modulation by serum and GSK3β-ERK signaling were monitored. The 87, 78, and 35 kDa variants showed a biphasic modulation by serum till 24 h but the 52 kDa variant remained largely unresponsive. Inhibition of phosphatases by okadaic acid increased the levels of the endogenous 78 kDa and the ectopically expressed GFP-tagged 87 and 78 kDa SG2NAs. Contrastingly, okadaic acid treatment reduced the level of GFP-tagged 35 kDa SG2NA, suggesting differential modes of their stability through phosphorylation-dephosphorylation. The inhibition of GSK3β by LiCl showed a gradual decrease in the levels of 78 kDa. In the case of the other variants viz, GFP-tagged 35, 52, and 87 kDa, inhibition of GSK3β caused an initial increase followed by a decrease with a subtle difference in kinetics and intensities. Similar results were also seen upon inhibition of GSK3β by small interfering RNA. All the variants showed an increase followed by a decrease upon inhibition of extracellular-signal-regulated-kinase (ERK). These variants are localized in the plasma membrane, endoplasmic reticulum, mitochondria, and the nucleus with different propensities and no discernable subcellular distribution was seen upon stimulation by serum and the inhibition of phosphatases, GSK3β, and ERK. Taken together, the variants of SG2NA are modulated by the kinase-phosphatase network in a similar but characteristic manner.
摘要:
SG2NA,纹状体蛋白亚家族的WD40重复蛋白,具有四个剪接和一个信使RNA编辑变体。它作为在组织发育和疾病中起作用的多聚信号传导复合物的支架而迅速出现。绿色荧光蛋白(GFP)标记的SG2NA变体在NIH3T3细胞中异位表达,并监测其通过血清和GSK3β-ERK信号传导的调节。87、78和35kDa变体直到24小时都显示出血清的双相调节,但52kDa变体仍然基本上无反应。冈田酸对磷酸酶的抑制增加了内源性78kDa和异位表达的GFP标记的87和78kDaSG2NA的水平。相反,冈田酸处理降低了GFP标记的35kDaSG2NA的水平,通过磷酸化-去磷酸化提示其稳定性的不同模式。LiCl对GSK3β的抑制作用显示出78kDa的水平逐渐降低。在其他变体的情况下,即,GFP标记的35、52和87kDa,GSK3β的抑制导致最初的增加,然后是动力学和强度的细微差异。通过小干扰RNA抑制GSK3β也观察到类似的结果。所有变体在抑制细胞外信号调节激酶(ERK)后显示增加,随后减少。这些变体位于质膜中,内质网,线粒体,并且在血清刺激和磷酸酶抑制后,可以看到具有不同倾向且没有可辨别的亚细胞分布的细胞核,GSK3β,和ERK.一起来看,SG2NA的变体以相似但特征性的方式受到激酶-磷酸酶网络的调节。