den Hertog J, Tracy S, Hunter T
Salk Institute, Molecular Biology and Virology Laboratory, La Jolla, CA 92037.
EMBO J. 1994 Jul 1;13(13):3020-32. doi: 10.1002/j.1460-2075.1994.tb06601.x.
Receptor protein-tyrosine phosphatase alpha (RPTP alpha) is a transmembrane protein with a short extracellular domain (123 amino acids) and two cytoplasmically localized protein-tyrosine phosphatase (PTP) domains. Here we report that RPTP alpha is constitutively phosphorylated on tyrosine in NIH 3T3 mouse fibroblasts. The in vivo tyrosine phosphorylation site was localized to the C-terminus of RPTP alpha by phosphopeptide mapping experiments using in vivo and in vitro 32P-labeled RPTP alpha. The identity of this site as Tyr789, located five residues from the C-terminus, was confirmed by site-directed mutagenesis. Transient overexpression of c-Src together with RPTP alpha in human embryonic kidney 293 cells increased phosphorylation of Tyr789, suggesting that c-Src may phosphorylate RPTP alpha in vivo. RPTP alpha had autodephosphorylation activity in vitro. When expressed in 293 cells the level of Tyr789 phosphorylation was higher in a non-functional mutant of RPTP alpha than in wild type RPTP alpha, indicating that RPTP alpha may have autodephosphorylation activity in vivo as well. The sequence on the C-terminal side of Tyr789 (YANF) fits the consensus binding site for the SH3-SH2-SH3 adaptor protein GRB2 (YXNX). We show that RPTP alpha, but not a mutant of RPTP alpha with a Tyr-->Phe mutation at position 789, bound to GRB2 in vitro. In addition, RPTP alpha co-immunoprecipitated with GRB2 from NIH 3T3 cells, demonstrating that GRB2 bound to RPTP alpha in vivo. The guanine nucleotide releasing factor for the Ras GTPase, Son of sevenless (Sos), which associates with GRB2 via its SH3 domains, was not detected in RPTP alpha immunoprecipitates. Our results suggest a role for RPTP alpha in attenuation of GRB2-mediated signaling.
受体蛋白酪氨酸磷酸酶α（RPTPα）是一种跨膜蛋白，具有一个短的细胞外结构域（123个氨基酸）和两个位于细胞质中的蛋白酪氨酸磷酸酶（PTP）结构域。在此我们报道，在NIH 3T3小鼠成纤维细胞中，RPTPα在酪氨酸上组成性磷酸化。通过使用体内和体外32P标记的RPTPα进行磷酸肽图谱实验，将体内酪氨酸磷酸化位点定位到RPTPα的C末端。通过定点诱变证实该位点为位于C末端五个残基处的Tyr789。在人胚肾293细胞中，c-Src与RPTPα一起瞬时过表达增加了Tyr789的磷酸化，表明c-Src可能在体内使RPTPα磷酸化。RPTPα在体外具有自动去磷酸化活性。当在293细胞中表达时，RPTPα的无功能突变体中Tyr789的磷酸化水平高于野生型RPTPα，这表明RPTPα在体内也可能具有自动去磷酸化活性。Tyr789（YANF）C末端一侧的序列符合SH3-SH2-SH3衔接蛋白GRB2（YXNX）的共有结合位点。我们发现，RPTPα而非在789位具有Tyr→Phe突变的RPTPα突变体在体外与GRB2结合。此外，RPTPα与来自NIH 3T3细胞的GRB2共免疫沉淀，证明GRB2在体内与RPTPα结合。在RPTPα免疫沉淀中未检测到通过其SH3结构域与GRB2结合的Ras GTP酶的鸟嘌呤核苷酸释放因子七号缺席之子（Sos）。我们的结果表明RPTPα在减弱GRB2介导的信号传导中起作用。
