Tappia P S, Sharma R P, Sale G J
Department of Biochemistry, University of Southampton, U.K.
Biochem J. 1991 Aug 15;278 ( Pt 1)(Pt 1):69-74. doi: 10.1042/bj2780069.
The identity of protein-tyrosine-phosphatases (PTPases) active against autophosphorylated insulin receptor was probed by using an insulin-receptor-related peptide phosphorylated on tyrosine (peptide 1142-1153). Two major peaks of PTPase activity were resolved from the particulate (Triton X-100-soluble) fraction of human placenta by chromatography on DEAE-cellulose. The two peaks were purified 1300-2300-fold; other peaks of PTPase activity (greater than 15%) were not detected. Properties of the PTPases indicated that they corresponded to subtypes 1A and 1B. Both subtypes appeared capable of catalysing dephosphorylation of all autophosphorylation sites in three domains of the insulin receptor, with no appreciable difference in the pattern of dephosphorylation detected by two-dimensional tryptic-peptide mapping. The tyrosine-1150 domain of the insulin receptor in triply phosphorylated form was found to be highly sensitive to the action of both PTPases, and was dephosphorylated at least 4 times faster than the doubly and singly phosphorylated forms of the tyrosine-1150 domain or phosphorylation sites in other domains by either PTPase. This is significant, as the level of the triphosphotyrosine-1150 species has been shown to correlate well with the capacity of the insulin-receptor tyrosine kinase to phosphorylate other proteins. Both subtypes also dephosphorylated autophosphorylated epidermal-growth-factor (EGF) receptor by greater than 95%. Placental particulate (and cytosolic) PTPase activity against either receptor distributed approximately 2:1 between subtypes 1A and 1B as assayed in the presence of EDTA. In summary, PTPases within two major subtypes have been identified as phosphotyrosyl-insulin and -EGF-receptor phosphatases in vitro. The PTPases identified exhibit high affinities for substrates and high activities in cells, which is commensurate with the PTPases being important in vivo in controlling or reversing autophosphorylation-induced regulatory or signalling events.
通过使用在酪氨酸上磷酸化的胰岛素受体相关肽（肽1142 - 1153）来探究对自磷酸化胰岛素受体有活性的蛋白酪氨酸磷酸酶（PTPases）的特性。通过DEAE - 纤维素柱层析从人胎盘的颗粒（Triton X - 100可溶）部分分离出两个主要的PTPase活性峰。这两个峰被纯化了1300 - 2300倍；未检测到其他PTPase活性峰（大于15%）。这些PTPases的特性表明它们对应于1A和1B亚型。两种亚型似乎都能够催化胰岛素受体三个结构域中所有自磷酸化位点的去磷酸化，通过二维胰蛋白酶肽图谱检测到的去磷酸化模式没有明显差异。发现三重磷酸化形式的胰岛素受体酪氨酸 - 1150结构域对两种PTPases的作用高度敏感，其去磷酸化速度比酪氨酸 - 1150结构域的双重和单重磷酸化形式或其他结构域中的磷酸化位点被任何一种PTPase去磷酸化的速度至少快4倍。这很重要，因为已表明三磷酸酪氨酸 - 1150物种的水平与胰岛素受体酪氨酸激酶磷酸化其他蛋白质的能力密切相关。两种亚型还能使自磷酸化的表皮生长因子（EGF）受体去磷酸化超过95%。在EDTA存在的情况下进行测定时，胎盘颗粒（和胞质）对任一受体的PTPase活性在1A和1B亚型之间的分布约为2:1。总之，在体外已鉴定出两种主要亚型的PTPases为磷酸酪氨酸 - 胰岛素和 - EGF受体磷酸酶。所鉴定的PTPases对底物具有高亲和力且在细胞中具有高活性，这与它们在体内控制或逆转自磷酸化诱导的调节或信号事件中起重要作用是相符的。
