Ishibashi T, Bottaro D P, Michieli P, Kelley C A, Aaronson S A
Laboratory of Cellular and Molecular Biology, NCI, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1994 Nov 25;269(47):29897-902.
To identify new members of a family of protein-tyrosine phosphatases (PTPs), of which VH1 is prototype, we screened a B5/589 human mammary epithelial cell cDNA library by low stringency hybridization with probes for the catalytic domains of the human VHR and mouse 3CH134 phosphatases. Two overlapping clones of 1.8 and 2.5 kilobase pairs were detected by 3CH134 but not VHR probes. Sequence analysis of the largest clone, B23, revealed a 2470-nucleotide open reading frame encoding a novel protein. Within the 397 amino acid sequence, the HCXAGXXR signature sequence for PTPs was located at positions 261-268. The closest similarities were to 3CH134, its human homolog CL100, and PAC-1, PTPs induced as early response genes to mitogen stimulation. Less relatedness was observed with VHR and VH1 dual specificity phosphatases of human and vaccinia virus, respectively. A bacterially expressed recombinant protein containing the catalytic domain of B23 showed significant but consistently lower activity than VHR in vitro. Among the substrates tested, B23 displayed the highest relative activity toward phosphorylated extracellular signal regulated kinase-1, suggesting that it may be a target for B23 activity in vivo. The B23 transcript was detected in a wide variety of normal human tissues, with relatively high expression in pancreas and brain. B23 was induced by serum stimulation of human fibroblasts as well as by heat shock with similar kinetics to those observed with CL100. Thus, B23 is a new human protein phosphatase which appears to be regulated in response to mitogenic signaling and at least some forms of stress.
为了鉴定以VH1为原型的蛋白酪氨酸磷酸酶（PTP）家族的新成员，我们用人类VHR和小鼠3CH134磷酸酶催化结构域的探针进行低严谨度杂交，筛选了一个B5/589人乳腺上皮细胞cDNA文库。用3CH134探针检测到两个重叠的1.8和2.5千碱基对的克隆，但用VHR探针未检测到。对最大的克隆B23进行序列分析，发现一个2470个核苷酸的开放阅读框，编码一种新蛋白。在397个氨基酸序列中，PTP的HCXAGXXR特征序列位于261-268位。与3CH134、其人类同源物CL100以及作为有丝分裂原刺激的早期反应基因诱导的PTP PAC-1最为相似。分别与人VHR和痘苗病毒VH1双特异性磷酸酶的相关性较低。一种细菌表达的含有B23催化结构域的重组蛋白在体外显示出显著但始终低于VHR的活性。在所测试的底物中，B23对磷酸化的细胞外信号调节激酶-1显示出最高的相对活性，表明它可能是体内B23活性的靶点。在多种正常人体组织中检测到B23转录本，在胰腺和脑中表达相对较高。B23在人成纤维细胞的血清刺激以及热休克诱导下，其动力学与CL100相似。因此，B23是一种新的人类蛋白磷酸酶，似乎受有丝分裂信号和至少某些形式的应激反应调节。
