Tornqvist H E, Pierce M W, Frackelton A R, Nemenoff R A, Avruch J
J Biol Chem. 1987 Jul 25;262(21):10212-9.
To identify the autophosphorylation sites on the human insulin receptor (IR), partially purified human IR was incubated in vitro in the presence of insulin and manganese [gamma-32P]ATP so as to achieve near-maximal activation of the histone 2b kinase activity. Approximately 70% of all beta subunit [32P]phosphotyrosine resides on two tryptic peptide segments identified by microsequencing as IR precursor (Ullrich, A., Bell, J. R., Chen, E.-Y., Herrera, R., Petruzelli, L. M., Dull, T. J., Gray, A., Coussens, L., Liao, Y.-C., Tsubokawa, M., Mason, A., Seeburg, P. H., Grunfeld, C., Rosen, O. M., and Ramachandran, J. (1985) Nature 313, 756-761) 1144-1152 (tyrosine at 1146, 1150, 1151, designated peptide 5) and 1315-1329 (tyrosine at 1316, 1322, designated peptide 8), which were recovered in approximately equal amounts. Half of the remaining unidentified [32P]phosphotyrosine residues reside on another tryptic peptide of Mr 4000-5000. Assignment of [32P]phosphotyrosine to specific residues required subdigestion and Edman degradation of 32P peptides covalently coupled to solid supports. Peptide 5 was recovered in triple and double phosphorylated forms in a molar ratio of about 2:1. Tyr-1146 contained 32P in both forms of peptide 5; in the double phosphorylated form, phenylthiohydantoin-[32P]phosphotyrosine was recovered at both Tyr-1150 and Tyr-1151, in a ratio of about 1:2. Thus, the double phosphorylated peptide 5 is presumably a mixture of Tyr-P-1146/1150 and Tyr-P-1146/1151, predominantly the latter. Peptide 8 was recovered only as the double phosphorylated form. We conclude that autophosphorylation of human IR in vitro leads to the phosphorylation of at least 6 of the 13 tyrosine residues on the beta subunit intracellular extension. Five of these tyrosines are clustered in two domains; one domain is in the structurally unique C-terminal tail and contains Tyr-1316 and -1322 which are both phosphorylated. The second domain is located in the segment of the tyrosine kinase region homologous to the major in vitro autophosphorylation site of pp60 v-src and contains Tyr-1146, which is fully phosphorylated, and Tyr-1150 and -1151; although the majority of IR beta subunits exhibit phosphorylation of both tyrosine 1150 and 1151, up to 20-25% of Tyr-1150 remains unphosphorylated at complete kinase activation.
为鉴定人胰岛素受体（IR）上的自身磷酸化位点，将部分纯化的人IR在胰岛素和锰[γ-32P]ATP存在的情况下于体外孵育，以实现组蛋白2b激酶活性的近乎最大程度激活。所有β亚基[32P]磷酸酪氨酸中约70%存在于通过微测序鉴定为IR前体（乌尔里希，A.，贝尔，J.R.，陈，E.-Y.，埃雷拉，R.，彼得鲁泽利，L.M.，杜尔，T.J.，格雷，A.，库森斯，L.，廖，Y.-C.，津川，M.，梅森，A.，西伯林，P.H.，格伦费尔德，C.，罗森，O.M.，和拉马钱德兰，J.（1985年）《自然》313, 756 - 761）的两个胰蛋白酶肽段上，即1144 - 1152（1146、1150、1151位的酪氨酸，称为肽段5）和1315 - 1329（1316、1322位的酪氨酸，称为肽段8），二者回收量大致相等。其余未鉴定的[32P]磷酸酪氨酸残基的一半存在于另一个分子量为4000 - 5000的胰蛋白酶肽段上。将[32P]磷酸酪氨酸分配到特定残基需要对共价偶联到固体支持物上的32P肽段进行亚消化和埃德曼降解。肽段5以三重磷酸化和双重磷酸化形式回收，摩尔比约为2:1。在两种形式的肽段5中，Tyr-1146均含有32P；在双重磷酸化形式中，在Tyr-1150和Tyr-1151处均回收了苯硫基乙内酰脲-[32P]磷酸酪氨酸，比例约为1:2。因此，双重磷酸化的肽段5大概是Tyr-P-1146/1150和Tyr-P-1146/1151的混合物，主要是后者。肽段8仅以双重磷酸化形式回收。我们得出结论，人IR在体外的自身磷酸化导致β亚基细胞内延伸段上13个酪氨酸残基中至少6个发生磷酸化。这些酪氨酸中有5个聚集在两个结构域中；一个结构域位于结构独特的C末端尾部，包含均被磷酸化的Tyr-1316和-1322。第二个结构域位于与pp60 v-src主要体外自身磷酸化位点同源的酪氨酸激酶区域片段中，包含完全磷酸化的Tyr-1146以及Tyr-1150和-1151；尽管大多数IRβ亚基在酪氨酸1150和1151处均发生磷酸化，但在激酶完全激活时，高达20 - 25%的Tyr-1150仍未磷酸化。
