Tiganis T, Bennett A M, Ravichandran K S, Tonks N K
Cold Spring Harbor Laboratory, New York 11724, USA.
Mol Cell Biol. 1998 Mar;18(3):1622-34. doi: 10.1128/MCB.18.3.1622.
T-cell protein tyrosine phosphatase (TCPTP) exists as two forms generated by alternative splicing: a 48-kDa endoplasmic reticulum (ER)-associated form (TC48) and a 45-kDa nuclear form (TC45). To identify TCPTP substrates, we have generated substrate-trapping mutants, in which the invariant catalytic acid of TCPTP (D182) is mutated to alanine. The TCPTP D182A substrate-trapping mutants were transiently overexpressed in COS cells, and their ability to form complexes with tyrosine-phosphorylated (pTyr) proteins was assessed. No pTyr proteins formed complexes with wild-type TCPTP. In contrast, TC48-D182A formed a complex in the ER with pTyr epidermal growth factor receptor (EGFR). In response to EGF, TC45-D182A exited the nucleus and accumulated in the cytoplasm, where it bound pTyr proteins of approximately 50, 57, 64, and 180 kDa. Complex formation was disrupted by vanadate, highlighting the importance of the PTP active site in the interaction and supporting the characterization of these proteins as substrates. Of these TC45 substrates, the approximately 57- and 180-kDa proteins were identified as p52Shc and EGFR, respectively. We examined the effects of TC45 on EGFR signaling and observed that it did not modulate EGF-induced activation of p42Erk2. However, TC45 inhibited the EGF-induced association of p52Shc with Grb2, which was attributed to the ability of the PTP to recognize specifically p52Shc phosphorylated on Y239. These results indicate that TC45 recognizes not only selected substrates in a cellular context but also specific sites within substrates and thus may regulate discrete signaling events.
T细胞蛋白酪氨酸磷酸酶（TCPTP）以两种通过可变剪接产生的形式存在：一种48 kDa的内质网（ER）相关形式（TC48）和一种45 kDa的核形式（TC45）。为了鉴定TCPTP的底物，我们构建了底物捕获突变体，其中TCPTP的不变催化酸性氨基酸（D182）突变为丙氨酸。TCPTP D182A底物捕获突变体在COS细胞中瞬时过表达，并评估它们与酪氨酸磷酸化（pTyr）蛋白形成复合物的能力。没有pTyr蛋白与野生型TCPTP形成复合物。相反，TC48-D182A在内质网中与pTyr表皮生长因子受体（EGFR）形成复合物。响应表皮生长因子（EGF）时，TC45-D182A离开细胞核并积聚在细胞质中，在那里它结合了大约50、57、64和180 kDa的pTyr蛋白。复合物的形成被钒酸盐破坏，突出了PTP活性位点在相互作用中的重要性，并支持将这些蛋白鉴定为底物。在这些TC45底物中，大约57 kDa和180 kDa的蛋白分别被鉴定为p52Shc和EGFR。我们研究了TC45对EGFR信号传导的影响，观察到它不调节EGF诱导的p42Erk2激活。然而，TC45抑制了EGF诱导的p52Shc与Grb2的结合，这归因于该PTP特异性识别Y239位点磷酸化的p52Shc的能力。这些结果表明，TC45不仅在细胞环境中识别特定底物，还识别底物内的特定位点，因此可能调节离散的信号事件。
