Analysis of a novel human ras GAP-like gene

我们从人类心脏文库中克隆了一种新的与rasgap相关的分子nGAP。在先前描述的RasGAP中，nGAP与突触特异性RasGAP SynGAP和秀丽隐杆线虫RasGAP GAP-2关系最为密切。这三种分子在分子中间都有高度保守的gap相关结构域，并在其羧基端附近预测了螺旋状结构。RT-PCR检测nGAP表达的组织特异性，发现nGAP在所有组织中均有表达。在酿酒酵母中，nGAP的表达弥补了Ira2功能缺失，表明nGAP作为RasGAP对酵母Ras蛋白起作用。然而，重组GST-nGAP和重组GST-Ha-Ras在体外进行的RasGAP实验没有显示出GAP活性。当nGAP在HeLa细胞中过表达时，血清刺激的MAP激酶激活不受影响。这些结果提出了nGAP靶向其他Ras类低分子量G蛋白的可能性，而不是Ras或nGAP需要一定的条件或辅助因子才能发挥活性。对正常人类中期染色体的FISH分析和辐射杂交分析都将nGAP基因定位在人类染色体1q25上，该位点似乎包含一种遗传性前列腺癌易感基因HPC1。考虑到nGAP充当RasGAP的可能性，该赋值提出了nGAP是提议的HPC1的可能性。

We have cloned a novel RasGAP-related molecule, nGAP, from human heart library. Among previously described RasGAPs, nGAP is most closely related to the synapse-specific RasGAP, SynGAP, and a Caenorhabditis elegans RasGAP, GAP-2. All these three molecules shere highly-conserved GAP-related domain in the middle of the molecules and predicted coiled-coil structure near their carboxyl termini.Tissue specificity of nGAP expression was examined by RT-PCR analysis to show the ubiquitous expression in all tissues examined.Expression of nGAP in Saccharomyces cerevisiae defective in one of two RasGAPs, Ira2, complemented the loss of the Ira2 function, indicating that nGAP functions as a RasGAP against yeast Ras proteins.RasGAP assay performed in vitro using recombinant GST-nGAP and recombinant GST-Ha-Ras did not show GAP activity, however. When nGAP was over-expressed in HeLa cell, serum-stimulated MAP kinase activation was not affected. These result raises the possibilities that nGAP targets other Ras-like low molecular weight G proteins than Ras or nGAP requires cirtain conditions or co-factors for the activity.Both FISH analysis on normal human metaphase chromosomes and radiation-hybrid analysis assigned the nGAP gene to the human chromosome 1q25, the locus that appears to contain a hereditary prostate cancer susceptible gene, HPC1. Considering the possibility that nGAP acts as RasGAP, this assignment raises a possibility that nGAP is the proposed HPC1.