PHOSPHORYLATION OF RECEPTOR PROTEIN-TYROSINE-PHOSPHATASE-ALPHA ON TYR789, A BINDING-SITE FOR THE SH3-SH2-SH3 ADAPTER PROTEIN GRB-2 IN-VIVO
PHOSPHORYLATION OF RECEPTOR PROTEIN-TYROSINE-PHOSPHATASE-ALPHA ON TYR789, A BINDING-SITE FOR THE SH3-SH2-SH3 ADAPTER PROTEIN GRB-2 IN-VIVO
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DOI：
10.1002/j.1460-2075.1994.tb06601.x
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发表时间：
1994-07-01
期刊：
影响因子：
11.4
通讯作者：
HUNTER, T
中科院分区：
文献类型：
--
作者：
DENHERTOG, J;TRACY, S;HUNTER, T
受体蛋白酪氨酸磷酸酶α（RPTP α）是具有短的胞外结构域（123个氨基酸）和两个胞质定位的蛋白酪氨酸磷酸酶（PTP）结构域的跨膜蛋白。在这里，我们报告说，RPTP α是组成性磷酸化的酪氨酸在NIH 3 T3小鼠成纤维细胞。使用体内和体外P-32标记的RPTP α，通过磷酸肽作图实验将体内酪氨酸磷酸化位点定位于RPTP α的C-末端。通过定点诱变证实了该位点为Tyr 789，位于距C-末端5个残基处。人胚肾293细胞中c-Src与RPTP α的瞬时过表达增加了Tyr 789的磷酸化，表明c-Src可能在体内磷酸化RPTP α。RPTP α在体外具有自身去磷酸化活性。当在293细胞中表达时，RPTP α的非功能突变体中Tyr 789磷酸化水平高于野生型RPTP α，表明RPTP α在体内也可能具有自身去磷酸化活性。Tyr 789（YANF）C-末端侧的序列符合SH 3-SH 2-SH 3衔接蛋白GRB 2（YXNX）的共有结合位点。我们表明，RPTP α，而不是在位置789处具有Tyr-->Phe突变的RPTP α的突变体，在体外与GRB 2结合。此外，RPTP α与NIH 3 T3细胞的GRB 2共免疫沉淀，证明GRB 2在体内与RPTP α结合。RPTP α免疫沉淀物中未检测到Ras GTP酶的鸟嘌呤核苷酸释放因子，即Sos，其通过其SH 3结构域与GRB 2结合。我们的研究结果表明RPTP α在减弱GRB 2介导的信号传导中的作用。
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 P-32-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.