Graziani Y, Erikson E, Erikson R L
J Biol Chem. 1983 May 25;258(10):6344-51.
This report describes quantitative results of in vitro phosphorylation of the Rous sarcoma virus transforming gene product, pp60src, using ATP or GTP as phosphate donors. The Km values for the phosphorylation of pp60src by ATP and GTP were similar (10-36 and 25-36 microM, respectively) and the Vmax values were different (5-7 and 1.5-1.7 nmol min-1 mg-1 of pp60src, respectively). The radiolabeling of pp60src by [gamma-32P] ATP was inhibited by ADP and dATP at 20-fold higher concentrations by 75 and 83%, respectively. Other nucleotides served as weaker inhibitors under the same conditions. The radiolabeling of pp60src by [gamma-32P]GTP had lower specificity for this nucleotide, since ATP, dATP, ADP, dGTP, GDP, and TTP had at least a 50% inhibitory effect. The phosphorylated products of approximate Mr = 60,000 that were produced with ATP or GTP were shown to be the same protein molecule since they both could be immunoprecipitated with antibody raised against p60src produced by bacterial recombinants. Structural analysis revealed that the use of GTP resulted in phosphorylation of a tyrosine residue on the COOH-terminal region of pp60src, apparently the same site which contains the tyrosine phosphorylated in infected cells. In contrast, the use of ATP resulted in phosphorylation of several additional tyrosine residues on the NH2-terminal region of the molecule. In thermolability studies, the t1/2 values for the phosphorylation of pp66src in preparations from wild type virus-infected chicken cells were 5.1 min for both ATP and GTP, whereas the t1/2 values for the phosphorylation of pp60src in preparations from temperature-sensitive transformation mutant-infected cells were 1.1 min for both phosphate donors. Similar observations were found with alpha-casein as substrate.
本报告描述了以ATP或GTP作为磷酸供体，对劳氏肉瘤病毒转化基因产物pp60src进行体外磷酸化的定量结果。ATP和GTP对pp60src磷酸化的Km值相似（分别为10 - 36和25 - 36微摩尔），而Vmax值不同（分别为5 - 7和1.5 - 1.7纳摩尔每分钟每毫克pp60src）。[γ-32P]ATP对pp60src的放射性标记分别被20倍高浓度的ADP和dATP抑制75%和83%。在相同条件下，其他核苷酸作为较弱的抑制剂。[γ-32P]GTP对pp60src的放射性标记对该核苷酸的特异性较低，因为ATP、dATP、ADP、dGTP、GDP和TTP至少有50%的抑制作用。用ATP或GTP产生的大约Mr = 60,000的磷酸化产物被证明是相同的蛋白质分子，因为它们都可以用针对细菌重组体产生的p60src的抗体进行免疫沉淀。结构分析表明，使用GTP导致pp60src羧基末端区域的一个酪氨酸残基磷酸化，显然是感染细胞中发生酪氨酸磷酸化的同一部位。相反，使用ATP导致该分子氨基末端区域的几个额外酪氨酸残基磷酸化。在热稳定性研究中，来自野生型病毒感染鸡细胞的制剂中pp66src磷酸化的t1/2值对于ATP和GTP均为5.1分钟，而来自温度敏感转化突变体感染细胞的制剂中pp60src磷酸化的t1/2值对于两种磷酸供体均为1.1分钟。以α-酪蛋白作为底物也发现了类似的观察结果。
