Biomolecular Mass Spectrometry and Proteomics, Utrecht University, 3584 CH Utrecht, The Netherlands.
Bijvoet Center for Biomolecular Research, Utrecht University, 3584 CH Utrecht, The Netherlands.
Proc Natl Acad Sci U S A. 2017 Aug 29;114(35):E7255-E7261. doi: 10.1073/pnas.1620529114. Epub 2017 Aug 14.
Proteins can be modified by multiple posttranslational modifications (PTMs), creating a PTM code that controls the function of proteins in space and time. Unraveling this complex PTM code is one of the great challenges in molecular biology. Here, using mass spectrometry-based assays, we focus on the most common PTMs-phosphorylation and O-GlcNAcylation-and investigate how they affect each other. We demonstrate two generic crosstalk mechanisms. First, we define a frequently occurring, very specific and stringent phosphorylation/O-GlcNAcylation interplay motif, (pSp/T)P(V/A/T)(gS/gT), whereby phosphorylation strongly inhibits O-GlcNAcylation. Strikingly, this stringent motif is substantially enriched in the human (phospho)proteome, allowing us to predict hundreds of putative O-GlcNAc transferase (OGT) substrates. A set of these we investigate further and show them to be decent substrates of OGT, exhibiting a negative feedback loop when phosphorylated at the P-3 site. Second, we demonstrate that reciprocal crosstalk does not occur at PX(S/T)P sites, i.e., at sites phosphorylated by proline-directed kinases, which represent 40% of all sites in the vertebrate phosphoproteomes.
蛋白质可以通过多种翻译后修饰（PTMs）进行修饰，从而产生一种 PTM 密码，控制蛋白质在空间和时间上的功能。揭示这个复杂的 PTM 密码是分子生物学的重大挑战之一。在这里，我们使用基于质谱的测定方法，重点研究最常见的 PTMs——磷酸化和 O-GlcNAc 化，并研究它们如何相互影响。我们展示了两种通用的串扰机制。首先，我们定义了一个经常出现的、非常具体和严格的磷酸化/O-GlcNAc 相互作用基序（pSp/T）P（V/A/T）（gS/gT），其中磷酸化强烈抑制 O-GlcNAc 化。引人注目的是，这个严格的基序在人类（磷酸化）蛋白质组中大量富集，使我们能够预测数百种潜在的 O-GlcNAc 转移酶（OGT）底物。我们进一步研究了其中的一组，并证明它们是 OGT 的良好底物，在 P-3 位点磷酸化时表现出负反馈环。其次，我们证明在 PX（S/T）P 位点不会发生相互串扰，即不会发生在由脯氨酸定向激酶磷酸化的位点上，这些位点占脊椎动物磷酸蛋白质组中所有位点的 40%。
