Cellular functions of MNB/DYRK1A gene cloned from "Down syndrome critical region"

点击翻译按钮获取中文摘要

The human MNB/DYRK gene is a human homolog of Drosophila minibrain (mnb) and rat Dyrk (Dual-specificity tyrosine-phosphorylated and regulated kinase) genes. MNB/DYRK1A gene was first cloned from "Down syndrome critical region" on chromosome 21 and is a strong candidate for mental retardation associated with Down syndrome. We have previously reported that overexpression of MNB/DYRK1A induces multinucleation in HeLa cells through overproduction of the centrosome during interphase and production of aberrant spindles during mitosis. Thus, in this study, we determined phosphorylation state of MNB/DYRK1A during the cell cycle, because the activity of this kinase depends on the phosphorylation of tyrosine residues in the activation loop and possibly of Tyr219 in a tyrosine phosphorylation consensus motif. HeLa cells were synchronized at the G1/S boundary using thymidine block method. The cells were then released to progress cell cycle by removal of thymidine, and extracts were prepared from t … More hese cells at various time points after the removal. Western blot analysis of these extracts using anti-MNB/DYRK1A antibody revealed that all the extracts tested gave bands migrating at the same position. Thus, no modification of MNB/DYRK1A leading to band shift on SDS-PAGE occurred during the cell cycle. Next, HeLa cells were treated with nocodazole after the removal of thymidine and synchronized at M phase. The extract from the synchronized cells was analyzed for MNB/DYRK1A by western blot analysis. MNB/DYRK1A in the synchronized cells migrated slower than that in asynchronous cells. However, no band sift was observed if the extracts had been pretreated with λ-protein phosphatase, confirming that MNB/DYRK1A protein was phosphorylated in nocodazole-treated cells. When cells were treated with nocodazole but not arrested at M phase, such a band shift was not observed in these cells. Thus, this phosphorylation was induced in an M phase-specific manner. These results indicate that MNB/DYRK1A may be implicated in the spindle-attachment checkpoint mechanism which ensures that all chromosomes are properly attached to the microtubules of the spindle. Less