Villa-Moruzzi E, Puntoni F, Bardelli A, Vigna E, De Rosa S, Comoglio P M
Department of Experimental Pathology, University of Pisa, Via Roma 55, 56126 Pisa, Italy.
Biochem J. 1998 Nov 15;336 ( Pt 1)(Pt 1):235-9. doi: 10.1042/bj3360235.
We reported previously that a protein tyrosine phosphatase (PTP) activity is associated with the immunoprecipitated hepatocyte growth factor (HGF) receptor, also known as Met. The activity increased reversibly when Met was stimulated by HGF and decreased when Met was inactivated by PMA. To identify the PTP-binding region, we used deletion mutants of the receptor beta-subunit. The PTP activity did not associate with Tpr-Met, a construct containing residues 1010-1390 of Met fused to Tpr. In contrast, PTP activity was present when the expressed protein contained the full juxtamembrane region (residues 956-1390 of Met) or part of this region (residues 957-1390 or 995-1390), indicating that the PTP-binding region is between residues 995 and 1009. This region includes Tyr1003, a site involved in Met downstream signalling. Incubation of Met immunoprecipitated from GTL-16 cells with an 8-mer phosphopeptide derived from residues 1003-1010 induced a marked decrease in the associated PTP activity, suggesting that the peptide reproduced the PTP-binding region. Mutation of Glu, Asp or Arg at positions -4, -1 or +1 respectively relative to Tyr1003 in a 9-mer peptide (residues 999-1007) abolished the ability of the peptide to decrease the PTP activity associated with Met. Phosphorylation of Tyr1003 was not required for PTP binding, since: (1) both phospho- and dephospho-peptides on a solid bead bound PTP activity from a GTL-16 cell extract, and (2) PTP activity was associated with a Met deletion mutant lacking residues 1-955 in which Tyr1003 had been changed into Phe. In order to partially purify the PTP from the GTL-16 cell extract, an affinity column was prepared using the Met-derived peptide comprising residues 998-1007. Less than 0.1% of the total cellular PTP was retained by the column, and was eluted with low salt concentrations. Using antibodies, this PTP was identified as PTP-S, a soluble PTP present in epithelial cells and fibroblasts.
我们之前报道过，一种蛋白酪氨酸磷酸酶（PTP）活性与免疫沉淀的肝细胞生长因子（HGF）受体（也称为Met）相关。当Met受到HGF刺激时，该活性可逆性增加；而当Met被佛波酯（PMA）失活时，活性降低。为了鉴定PTP结合区域，我们使用了受体β亚基的缺失突变体。PTP活性与Tpr-Met不相关，Tpr-Met是一种构建体，包含与Tpr融合的Met的1010 - 1390位残基。相反，当表达的蛋白包含完整的近膜区域（Met的956 - 1390位残基）或该区域的一部分（957 - 1390位或995 - 1390位残基）时，PTP活性存在，这表明PTP结合区域在995和1009位残基之间。该区域包括Tyr1003，这是一个参与Met下游信号传导的位点。用源自1003 - 1010位残基的8聚体磷酸肽孵育从GTL - 16细胞免疫沉淀的Met，可使相关的PTP活性显著降低，这表明该肽重现了PTP结合区域。在9聚体肽（999 - 1007位残基）中，相对于Tyr1003分别在 - 4、 - 1或 + 1位的Glu、Asp或Arg突变消除了该肽降低与Met相关的PTP活性的能力。PTP结合不需要Tyr1003的磷酸化，因为：（1）固定在珠子上的磷酸化和去磷酸化肽都能结合来自GTL - 16细胞提取物的PTP活性，并且（2）PTP活性与一个缺失1 - 955位残基的Met缺失突变体相关，其中Tyr1003已被突变为Phe。为了从GTL - 16细胞提取物中部分纯化PTP，使用包含998 - 1007位残基的Met衍生肽制备了亲和柱。柱上保留的细胞总PTP不到0.1%，并用低盐浓度洗脱。使用抗体，该PTP被鉴定为PTP - S，一种存在于上皮细胞和成纤维细胞中的可溶性PTP。
