Msallam Muna, Sun Hao, Meledin Roman, Franz Pauline, Brik Ashraf
Schulich Faculty of Chemistry , Technion-Israel Institute of Technology , Technion City , Hailfa 32000 , Israel . Email:
Chem Sci. 2020 May 19;11(21):5526-5531. doi: 10.1039/c9sc06300e. eCollection 2020 Jun 7.
p19 plays an important role in the regulation of the cell cycle by inhibiting the function of cyclin-dependent kinases 4/6 that is responsible for the phosphorylation and deactivation of the retinoblastoma protein (pRb) tumour suppressor. Recently, it was reported that phosphorylation of p19 at Ser76 and Ser66 causes structural changes, which lead to its ubiquitination and degradation. Yet the exact contribution of each phosphorylation site remains unclear. To shed light on the role of these sites, we developed the chemical synthesis of unmodified, mono- and doubly phosphorylated p19 using state of the art methods for chemical protein synthesis. The synthesized proteins were characterized by circular dichroism and biochemical methods to examine the effect of phosphorylation on the thermal stability and ubiquitination, respectively. Our results provide clear determination of p19 stability upon phosphorylation at different sites and reveal that phosphorylation of both Ser residues might be necessary for promoting ubiquitination of p19.
p19通过抑制细胞周期蛋白依赖性激酶4/6的功能在细胞周期调控中发挥重要作用，该激酶负责视网膜母细胞瘤蛋白（pRb）肿瘤抑制因子的磷酸化和失活。最近，有报道称p19在Ser76和Ser66处的磷酸化会导致结构变化，进而导致其泛素化和降解。然而，每个磷酸化位点的确切作用仍不清楚。为了阐明这些位点的作用，我们采用先进的化学蛋白质合成方法，对未修饰的、单磷酸化和双磷酸化的p19进行了化学合成。通过圆二色性和生化方法对合成的蛋白质进行了表征，分别研究了磷酸化对热稳定性和泛素化的影响。我们的结果明确了不同位点磷酸化后p19的稳定性，并揭示两个丝氨酸残基的磷酸化可能是促进p19泛素化所必需的。
