Development of ''substrate-trapping'' mutants to identify physiological substrates of protein tyrosine phosphatases
Development of ''substrate-trapping'' mutants to identify physiological substrates of protein tyrosine phosphatases
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DOI：
10.1073/pnas.94.5.1680
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发表时间：
1997-03-04
影响因子：
11.1
通讯作者：
Tonks, NK
中科院分区：
文献类型：
--
作者：
Flint, AJ;Tiganis, T;Tonks, NK
蛋白酪氨酸磷酸酶（PTPs）底物的鉴定是全面了解该酶家族成员生理功能的重要一步。PTPs是由一个保守的催化结构域，包含27个不变的残基定义的，从我们对PTP 1B晶体结构的了解指导的这些不变残基的诱变研究中，我们发现了一个不变的催化酸（PTP 1B中的Asp-181）的突变，该突变将一个非常活跃的酶转化为底物陷阱。这种PTP 1B的D181 A突变体在COS和293细胞中的表达产生了一种酶，该酶与内源性PTP 1B竞争底物，并促进磷酸酪氨酸主要在表皮生长因子（EGF）受体以及120、80和70 kDa蛋白上的积累。PTP 1B的D181 A突变体和这些底物之间的关联是足够稳定的，以允许通过免疫沉淀分离的复合物，正如预测的PTP 1B和其底物的底物结合位点之间的相互作用，复合物被破坏钒酸盐，对于EGF受体，相互作用绝对需要受体自磷酸化。此外，从免疫荧光研究中，PTP 1B的D181 A突变体似乎保留了胞内复合物中的内源性EGF受体，这些结果表明EGF受体是体内PTP 1B的真正底物，并且PTP 1B的一个重要功能是防止内质网中新合成的EGF受体的不适当的、配体非依赖性的激活，这种必需的催化天冬氨酸残基存在于所有PTP中，并且在双特异性磷酸酶和低分子量PTP中具有结构上等同的对应物，因此我们预期这种方法可以广泛适用于促进该酶家族的其他成员的底物的鉴定。
The identification of substrates of protein tyrosine phosphatases (PTPs) is an essential step toward a complete understanding of the physiological function of members of this enzyme family. PTPs are defined by a conserved catalytic domain harboring 27 invariant residues, From a mutagenesis study of these invariant residues that was guided by our knowledge of the crystal structure of PTP1B, we have discovered a mutation of the invariant catalytic acid (Asp-181 in PTP1B) that converts an extremely active enzyme into a ''substrate trap.'' Expression of this D181A mutant of PTP1B in COS and 293 cells results in an enzyme that competes with endogenous PTP1B for substrates and promotes the accumulation of phosphotyrosine primarily on the epidermal growth factor (EGF) receptor as well as on proteins of 120, 80, and 70 kDa. The association between the D181A mutant of PTP1B and these substrates was sufficiently stable to allow isolation of the complex by immunoprecipitation, As predicted for an interaction between the substrate-binding site of PTP1B and its substrates, the complex is disrupted by vanadate and, for the EGF receptor, the interaction absolutely requires receptor autophosphorylation. Furthermore, from immunofluorescence studies, the D181A mutant of PTP1B appeared to retain the endogenous EGF receptor in an intracellular complex, These results suggest that the EGF receptor is a bona fide substrate for PTP1B in vivo and that one important function of PTP1B is to prevent the inappropriate, ligand-independent, activation of newly synthesized EGF receptor in the endoplasmic reticulum, This essential catalytic aspartate residue is present in all PTPs and has structurally equivalent counterparts in the dual-specificity phosphatases and the low molecular weight PTPs, Therefore we anticipate that this method may be widely applicable to facilitate the identification of substrates of other members of this enzyme family.