Phosphorylation of DGCR8 increases its intracellular stability and induces a progrowth miRNA profile.
Phosphorylation of DGCR8 increases its intracellular stability and induces a progrowth miRNA profile.
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DOI：
10.1016/j.celrep.2013.10.017
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发表时间：
2013-11-27
期刊：
影响因子：
8.8
通讯作者：
Steitz JA
中科院分区：
文献类型：
--
作者：
Herbert KM;Pimienta G;DeGregorio SJ;Alexandrov A;Steitz JA
在miRNA生物发生过程中，微处理器复合物（MC），其最低限度地由Drosha（一种RNase III酶）和DGCR 8（一种双链RNA结合蛋白）组成，切割初级miRNA（pri-miRNA），以释放pre-miRNA茎环结构。使用磷酸化蛋白质组学，我们绘制了23个磷酸化位点的全长人类DGCR 8在昆虫或哺乳动物细胞中表达。DGCR 8可以被促有丝分裂ERK/MAPK磷酸化，表明DGCR 8磷酸化可能响应并整合细胞外信号。磷酸化模拟DGCR 8的表达或磷酸酶的抑制增加了DGCR 8和Drosha蛋白的细胞水平。磷酸化模拟DGCR 8水平的增加不是由于更高的mRNA水平、改变的DGCR 8定位或DGCR 8的自缔合能力，而是由于蛋白质稳定性的增加。结合磷酸突变体或磷酸模拟DGCR 8的MC在特定加工活性方面没有改变。然而，相对于表达磷酸化突变体DGCR 8的细胞，表达磷酸化模拟DGCR 8的HeLa细胞表现出促生长miRNA表达谱和增加的增殖和划痕闭合率。
During miRNA biogenesis, the microprocessor complex (MC), which is composed minimally of Drosha, an RNase III enzyme, and DGCR8, a double-stranded RNA-binding protein, cleaves the primary miRNA (pri-miRNA) in order to release the pre-miRNA stem-loop structure. Using phosphoproteomics, we mapped 23 phosphorylation sites on full-length human DGCR8 expressed in insect or mammalian cells. DGCR8 can be phosphorylated by mitogenic ERK/MAPK, indicating that DGCR8 phosphorylation may respond to and integrate extracellular cues. The expression of phosphomimetic DGCR8 or inhibition of phosphatases increased the cellular levels of DGCR8 and Drosha proteins. Increased levels of phosphomimetic DGCR8 were not due to higher mRNA levels, altered DGCR8 localization, or DGCR8’s ability to self-associate, but rather to an increase in protein stability. MCs incorporating phosphomutant or phosphomimetic DGCR8 were not altered in specific processing activity. However, HeLa cells expressing phosphomimetic DGCR8 exhibited a progrowth miRNA expression profile and increased proliferation and scratch closure rates relative to cells expressing phosphomutant DGCR8.
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