Klinghoffer R A, Kazlauskas A
National Jewish Center for Immunology and Respiratory Medicine, Division of Basic Sciences, Denver, Colorado 80206, USA.
J Biol Chem. 1995 Sep 22;270(38):22208-17. doi: 10.1074/jbc.270.38.22208.
Because the protein-tyrosine phosphatase (PTP) Syp associates with the tyrosine-phosphorylated platelet-derived growth factor beta receptor (beta PDGFR), the beta PDGFR is a likely Syp substrate. We tested this hypothesis by determining whether recombinant Syp (rSyp) and a control PTP, recombinant PTP1B (rPTP1B), were able to dephosphorylate the beta PDGFR. The beta PDGFR was phosphorylated at multiple tyrosine residues in an in vitro kinase assay and then incubated with increasing concentrations of rSyp or rPTP1B. While the receptor was nearly completely dephosphorylated by high concentrations of rPTP1B, receptor dephosphorylation by rSyp plateaued at approximately 50%. Two-dimensional phosphopeptide maps of the beta PDGFR demonstrated that rSyp displayed a clear preference for certain receptor phosphorylation sites; the most efficiently dephosphorylated sites were phosphotyrosines (Tyr(P)-771 and -751, followed by Tyr(P)740, while Tyr(P)-1021 and Tyr(P)-1009 were very poor substrates. In contrast, rPTP1B displayed no selectivity for the various rPTP1B displayed no selectivity for the various beta PDGFR tyrosine phosphorylation sites and dephosphorylated all of them with comparable efficiency. A Syp construct that lacked the SH2 domains was still able to discriminate between the various receptor phosphorylation sites, although less effectively than full-length Syp. These in vitro studies predicted that Syp can dephosphorylate the receptor in vivo. Indeed, we found that a beta PDGFR mutant (F1009) that associates poorly with Syp, had a much slower in vivo rate of receptor dephosphorylation than the wild type receptor. In addition, the GTPase-activating protein of Ras (GAP) and phosphatidylinositol 3-kinase were less stably associated with the wild type beta PDGFR than with the F1009 receptor. These findings are consistent with the in vitro experiments showign that Syp prefers to dephosphorylate sites on the beta PDGFR, that are important for binding phosphatidylinositol 3-kinase (Tyr(P)-740 and Tyr(P)-751) and GAP (Tyr(P)-771). These studies reveal that Syp is a substrate-selective PTP and that both the catalytic domain and the SH2 domains contribute to Syp's ability to choose substrates. Furthermore, it appears that Syp plays a role in PDGF-dependent intracellular signal relay by selectively dephosphorylating the beta PDGFR and thereby regulating the binding of a distinct group of receptor-associated signal relay enzymes.
由于蛋白酪氨酸磷酸酶（PTP）Syp与酪氨酸磷酸化的血小板衍生生长因子β受体（βPDGFR）相关联，βPDGFR可能是Syp的底物。我们通过确定重组Syp（rSyp）和对照PTP（重组PTP1B，rPTP1B）是否能够使βPDGFR去磷酸化来验证这一假设。在体外激酶测定中，βPDGFR在多个酪氨酸残基处被磷酸化，然后与浓度不断增加的rSyp或rPTP1B一起孵育。虽然高浓度的rPTP1B可使受体几乎完全去磷酸化，但rSyp使受体去磷酸化的程度在约50%时趋于平稳。βPDGFR的二维磷酸肽图谱表明，rSyp对某些受体磷酸化位点表现出明显的偏好；最易被去磷酸化的位点是磷酸酪氨酸（Tyr(P)-771和-751，其次是Tyr(P)740，而Tyr(P)-1021和Tyr(P)-1009是非常差的底物。相比之下，rPTP1B对βPDGFR的各种酪氨酸磷酸化位点没有选择性，并且以相当的效率使所有位点去磷酸化。一个缺乏SH2结构域的Syp构建体仍然能够区分各种受体磷酸化位点，尽管其效率不如全长Syp。这些体外研究预测Syp在体内可以使受体去磷酸化。事实上，我们发现一个与Syp结合能力较差的βPDGFR突变体（F1009），其在体内受体去磷酸化的速率比野生型受体慢得多。此外，Ras的GTP酶激活蛋白（GAP）和磷脂酰肌醇3激酶与野生型βPDGFR的结合稳定性低于与F1009受体的结合稳定性。这些发现与体外实验一致，表明Syp更喜欢使βPDGFR上对结合磷脂酰肌醇3激酶（Tyr(P)-740和Tyr(P)-751）和GAP（Tyr(P)-771）很重要的位点去磷酸化。这些研究表明Syp是一种底物选择性的PTP，并且催化结构域和SH2结构域都有助于Syp选择底物的能力。此外，似乎Syp通过选择性地使βPDGFR去磷酸化，从而调节一组不同的受体相关信号转导酶的结合，在PDGF依赖性细胞内信号转导中发挥作用。
