Sburlati A R, De La Rosa A, Batey D W, Kurys G L, Manrow R E, Pannell L K, Martin B M, Sheeley D M, Berger S L
Section on Genes and Gene Products, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Biochemistry. 1993 May 4;32(17):4587-96. doi: 10.1021/bi00068a015.
Prothymosin alpha is post-translationally modified. When human myeloma cells were metabolically labeled with [32P]orthophosphoric acid, they synthesized [32P]prothymosin alpha. The incorporated radioactivity was resistant to DNase and RNases A, T1, and T2, but could be completely removed by alkaline phosphatase. No evidence was found for an RNA adduct as postulated by Vartapetian et al. [Vartapetian, A., Makarova, T., Koonin, E. V., Agol, V. I., & Bogdanov, A. (1988) FEBS Lett. 232, 35-38]. Thin-layer electrophoresis of partially hydrolyzed [32P]prothymosin alpha indicated that serine residues were phosphorylated. Analysis of peptides derived from bovine prothymosin alpha and human [32P]prothymosin alpha by treatment with endoproteinase Lys-C revealed that the amino-terminal 14-mer, with serine residues at positions 1, 8, and 9, was phosphorylated at a single position. Approximately 2% of the peptide in each case contained phosphate. Further digestion of the phosphopeptide with Asp-N followed by C18 reversed-phase column chromatography produced two peptides: a phosphate-free 9-mer containing amino acids 6-14 and a labeled peptide migrating slightly faster than the N-terminal 5-mer derived from the unmodified 14-mer. Positive identification of the phosphorylated amino acid was obtained by colliding the 14-residue phosphopeptide with helium in the mass spectrometer and finding phosphate only in a nested set of phosphorylated fragments composed of the first three, four, and five amino acids. The results prove that prothymosin alpha contains N-terminal acetylserine phosphate. In a synchronized population of human myeloma cells, phosphorylation occurred throughout the cell cycle. Furthermore, prothymosin alpha appeared to be stable, with a half-life slightly shorter than the generation time. Although prothymosin alpha is known to be essential for cell division, the constancy of both the amount of the protein and the degree of its phosphorylation suggests that prothymosin alpha does not directly govern mitosis.
前胸腺素α经过翻译后修饰。当用人骨髓瘤细胞进行[32P]正磷酸代谢标记时，它们合成了[32P]前胸腺素α。掺入的放射性对脱氧核糖核酸酶和核糖核酸酶A、T1及T2具有抗性，但可被碱性磷酸酶完全去除。未发现有Vartapetian等人[Vartapetian, A., Makarova, T., Koonin, E. V., Agol, V. I., & Bogdanov, A. (1988) FEBS Lett. 232, 35 - 38]所推测的RNA加合物的证据。对部分水解的[32P]前胸腺素α进行薄层层析表明丝氨酸残基被磷酸化。用内肽酶Lys - C处理牛前胸腺素α和人[32P]前胸腺素α得到的肽段分析显示，氨基末端的14肽（第1、8和9位为丝氨酸残基）在单个位置被磷酸化。每种情况下约2%的肽段含有磷酸基团。用天冬氨酸蛋白酶N进一步消化磷酸肽，随后进行C18反相柱层析，产生了两种肽段：一种不含磷酸的9肽，包含第6 - 14位氨基酸，以及一种标记肽，其迁移速度略快于从未修饰的14肽衍生的氨基末端5肽段。通过在质谱仪中使14个残基的磷酸肽与氦气碰撞，并仅在由前三个、四个和五个氨基酸组成的一组嵌套的磷酸化片段中发现磷酸基团，从而对磷酸化氨基酸进行了阳性鉴定。结果证明前胸腺素α含有氨基末端乙酰丝氨酸磷酸。在同步化的人骨髓瘤细胞群体中，磷酸化在整个细胞周期中都有发生。此外，前胸腺素α似乎是稳定的，其半衰期略短于细胞世代时间。尽管已知前胸腺素α对细胞分裂至关重要，但蛋白质数量及其磷酸化程度的恒定表明前胸腺素α并不直接控制有丝分裂。
