Department of Cell Biology, Harvard Medical School, Boston, MA, USA.
Proteomics. 2012 Jul;12(13):2185-9. doi: 10.1002/pmic.201200003.
Developmental processes are governed by diverse regulatory mechanisms including a suite of signaling pathways employing reversible phosphorylation. With the advent of large-scale phosphoproteomics, it is now possible to identify thousands of phosphorylation sites from tissues at distinct developmental stages. We describe here the identification of over 6000 nonredundant phosphorylation sites from neonatal murine brain. When compared to nearly three times the number of phosphorylation sites identified from 3-week-old murine brain, remarkably one-third of the neonatal sites were unique. This fraction only dropped to one-quarter when allowing the site to stray plus or minus 15 residues. This provides evidence for considerable change in the profiles of developmentally regulated phosphoproteomes. Using quantitative MS we characterized a novel phosphorylation site (Ser265) identified uniquely in the neonatal brain on doublecortin (Dcx), a protein essential for proper mammalian brain development. While the relative levels of Dcx and phospho-Ser265 Dcx between embryonic and neonatal brain were similar, their levels fell precipitously by postnatal day 21, as did phospho-Ser297, a site required for proper neuronal migration. Both sites lie near the microtubule-binding domain and may provide functionally similar regulation via different kinases.
发育过程受多种调控机制控制，包括一系列采用可逆磷酸化的信号通路。随着大规模磷酸蛋白质组学的出现，现在可以从不同发育阶段的组织中鉴定出数千个磷酸化位点。我们在这里描述了从新生鼠脑中鉴定出超过 6000 个非冗余磷酸化位点。与从 3 周大的鼠脑中鉴定出的磷酸化位点数量相比几乎增加了两倍，令人惊讶的是，三分之一的新生位点是独特的。当允许该位点偏离 15 个残基左右时，这一比例仅下降到四分之一。这为发育调控磷酸蛋白质组的显著变化提供了证据。使用定量 MS，我们对在双皮质蛋白 (Dcx) 上唯一鉴定出的新生脑中的一个新磷酸化位点 (Ser265) 进行了表征，Dcx 是哺乳动物大脑正常发育所必需的蛋白质。虽然胚胎期和新生期大脑中 Dcx 和磷酸化 Ser265 Dcx 的相对水平相似，但它们的水平在出生后第 21 天急剧下降，磷酸化 Ser297 也是如此，后者是神经元迁移所必需的位点。这两个位点都位于微管结合域附近，可能通过不同的激酶提供类似的功能调节。
