Quantitative phosphoproteomics reveals that nestin is a downstream target of dual leucine zipper kinase during retinoic acid-induced neuronal differentiation of Neuro-2a cells.

IF 2.4 3区 生物学 Q4 CELL BIOLOGY
Guillaume St-Cyr, Daniel Garneau, Nicolas Gévry, Richard Blouin
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Abstract

Background: Dual leucine zipper kinase (DLK) is critical for neurite outgrowth in the developing nervous system and during nerve regeneration, but the underlying mechanisms remain largely unknown. To address this issue, we generated stable shRNA-mediated DLK-depleted Neuro-2a cell lines and analyzed their phosphoproteome after induction of neuronal differentiation by retinoic acid (RA).

Results: Here, we report the identification of 32 phosphopeptides that exhibited significant differences in relative abundance between control and DLK-depleted cells. Two of the most downregulated phosphopeptides identified after DLK depletion were derived from nestin, a type VI intermediate filament (IF) protein typically expressed in neural progenitor cells. The reduced abundance of these phosphopeptides in response to DLK knockdown was validated using parallel reaction monitoring (PRM)-based quantitative proteomics and paired with a concomitant reduction in nestin mRNA and protein expression, indicating that the decrease in nestin phosphorylation was due to a decrease in total nestin in DLK-depleted cells compared to control cells. This DLK-mediated regulation of nestin expression had no apparent effect on neurite formation because nestin knockdown alone was not sufficient to impair RA-induced neurite extension in parental Neuro-2a cells, and nestin overexpression failed to rescue the neurite outgrowth defect observed in DLK-depleted Neuro-2a cells.

Conclusions: Together, these results demonstrate that nestin is a novel downstream target of DLK signaling but not a mediator of its ability to promote neurite outgrowth during neuronal differentiation.

定量磷酸化蛋白质组学表明,巢蛋白是双亮氨酸拉链激酶在视黄酸诱导的神经细胞分化过程中的下游靶点。
背景:双亮氨酸拉链激酶(Dual leucine zipper kinase, DLK)在发育中的神经系统和神经再生过程中对神经突的生长至关重要,但其潜在机制仍不清楚。为了解决这个问题,我们生成了稳定的shrna介导的dlk缺失的neuro2a细胞系,并在维甲酸(RA)诱导神经元分化后分析了它们的磷酸化蛋白质组。结果:在这里,我们报告了32个磷酸肽的鉴定,在对照和dlk -缺失细胞之间表现出显著的相对丰度差异。DLK耗竭后发现的两个最下调的磷酸肽来源于巢蛋白,巢蛋白是一种通常在神经祖细胞中表达的VI型中间丝(IF)蛋白。基于平行反应监测(PRM)的定量蛋白质组学验证了这些磷酸化肽丰度的降低对DLK敲除的响应,并伴随着巢蛋白mRNA和蛋白表达的减少,表明巢蛋白磷酸化的减少是由于DLK缺失细胞中总巢蛋白的减少。这种dlk介导的巢蛋白表达调节对神经突的形成没有明显的影响,因为巢蛋白单独敲低并不足以损害亲代神经2a细胞中ra诱导的神经突延伸,巢蛋白过表达也不能修复在dlk缺失的神经2a细胞中观察到的神经突生长缺陷。总之,这些结果表明,巢蛋白是DLK信号的一个新的下游靶点,但不是其在神经元分化过程中促进神经突生长的能力的中介。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
BMC Molecular and Cell Biology
BMC Molecular and Cell Biology Biochemistry, Genetics and Molecular Biology-Cell Biology
CiteScore
5.50
自引率
0.00%
发文量
46
审稿时长
27 weeks
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