Genome Biology Unit, ‡Proteomics Core Facility and §Structural and Computational Biology Unit, European Molecular Biology Laboratory , Meyerhofstrasse 1, 69117 Heidelberg, Germany.
ACS Chem Biol. 2014 Jul 18;9(7):1451-9. doi: 10.1021/cb500240n. Epub 2014 May 9.
Vaccinia H1-related (VHR) phosphatase is a dual specificity phosphatase that is required for cell-cycle progression and plays a role in cell growth of certain cancers. Therefore, it represents a potential drug target. VHR is structurally and biochemically well characterized, yet its regulatory principles are still poorly understood. Understanding its regulation is important, not only to comprehend VHR's biological mechanisms and roles but also to determine its potential and druggability as a target in cancer. Here, we investigated the functional role of the unique "variable insert" region in VHR by selectively introducing the photo-cross-linkable amino acid para-benzoylphenylalanine (pBPA) using the amber suppression method. This approach led to the discovery of VHR dimerization, which was further confirmed using traditional chemical cross-linkers. Phe68 in VHR was discovered as a residue involved in the dimerization. We demonstrate that VHR can dimerize inside cells, and that VHR catalytic activity is reduced upon dimerization. Our results suggest that dimerization could occlude the active site of VHR, thereby blocking its accessibility to substrates. These findings indicate that the previously unknown transient self-association of VHR acts as a means for the negative regulation of its catalytic activity.
痘苗 H1 相关（VHR）磷酸酶是一种双特异性磷酸酶，它是细胞周期进程所必需的，并且在某些癌症的细胞生长中发挥作用。因此，它代表了一个潜在的药物靶点。VHR 在结构和生化上都得到了很好的表征，但它的调节原理仍知之甚少。了解其调控机制不仅对于理解 VHR 的生物学机制和作用很重要，而且对于确定其作为癌症靶点的潜力和可成药性也很重要。在这里，我们通过使用琥珀酸抑制方法选择性地引入光交联氨基酸对苯甲酰苯丙氨酸（pBPA），研究了 VHR 独特的“可变插入”区域的功能作用。这种方法发现了 VHR 的二聚化，并用传统的化学交联剂进一步证实了这一点。发现 VHR 中的苯丙氨酸 68 残基参与二聚化。我们证明 VHR 可以在细胞内二聚化，并且二聚化会降低 VHR 的催化活性。我们的结果表明，二聚化可能会封闭 VHR 的活性位点，从而阻止其与底物的结合。这些发现表明，以前未知的 VHR 瞬时自缔合是其催化活性负调控的一种手段。
