Nagaraja Ganachari M, Kandpal Raj P
Department of Biological Sciences, Fordham University, Bronx, NY 10458, USA.
Biochem Biophys Res Commun. 2004 Jan 16;313(3):654-65. doi: 10.1016/j.bbrc.2003.12.001.
We have characterized the cDNA for a Rho GTPase activating protein (GAP) mapping to chromosome 13q12. The cDNA was characterized by determining the complete sequence of a 4.8 kb cDNA clone that represents the 5' untranslated region (UTR), the translated region, and the 3' UTR. The protein has a sterile alpha-motif (SAM), a distinct GAP domain, and a conserved START (StAR related lipid transfer) domain. The cDNA has 5 instability motifs (ATTTA) in the 3' UTR and one motif in the translated region between GAP and START domains. The RhoGAP transcript is truncated in some breast carcinoma cell lines and it has low expression in other breast cancer cell lines as compared to a normal breast cell line. We have previously observed the absence of RhoGAP transcript in a breast tumor specimen. A GST-fusion of the RhoGAP was tested for its specificity on RhoA, Cdc42, and Rac1. The protein was most active for RhoA. Transfection of RhoGAP into MCF7 cells significantly inhibited cell growth. The introduction of the RhoGAP construct into MDAMB231 cells that had previously been transfected with a p21 construct did not affect cell proliferation, indicating the involvement of p21 in Rho-mediated proliferation of cancer cells. NIH3T3 cells overexpressing RhoGAP showed considerable inhibition of stress fiber formation. Several cDNAs were identified as RhoGAP interactors by using the yeast two-hybrid assay system. These cDNAs correspond to SWI/SNF, alpha-tubulin, HMG CoA reductase, and TAX1 binding protein (TAX1BP1). The interaction with HMG CoA reductase may partially explain the growth inhibition of breast carcinoma cells by statin class of cholesterol lowering drugs. The biological significance of the interacting proteins is discussed in the context of their involvement in tumorigenesis. Our results indicate that loss of RhoGAP or its altered activity suppresses the growth of breast tumor cells. The presence of various motifs in RhoGAP and its interaction with several other proteins suggest that the protein may regulate Rho signaling in multiple ways and possibly function in a Rho-independent manner.
我们已对定位于13号染色体q12区的一种Rho GTP酶激活蛋白（GAP）的cDNA进行了特征分析。通过确定一个4.8 kb cDNA克隆的完整序列来对该cDNA进行特征分析，该克隆代表5'非翻译区（UTR）、翻译区和3'UTR。该蛋白具有一个无活性α基序（SAM）、一个独特的GAP结构域和一个保守的START（类固醇生成急性调节蛋白相关脂质转运）结构域。该cDNA在3'UTR中有5个不稳定基序（ATTTA），在GAP和START结构域之间的翻译区有一个基序。RhoGAP转录本在一些乳腺癌细胞系中被截断，与正常乳腺细胞系相比，它在其他乳腺癌细胞系中的表达较低。我们之前在一个乳腺肿瘤标本中观察到RhoGAP转录本缺失。对RhoGAP的谷胱甘肽S-转移酶（GST）融合蛋白针对RhoA、Cdc42和Rac1的特异性进行了检测。该蛋白对RhoA的活性最高。将RhoGAP转染到MCF7细胞中显著抑制细胞生长。将RhoGAP构建体导入先前已用p21构建体转染的MDAMB231细胞中并不影响细胞增殖，表明p21参与了Rho介导的癌细胞增殖。过表达RhoGAP的NIH3T3细胞显示出应力纤维形成受到显著抑制。通过酵母双杂交检测系统鉴定了几个cDNA作为RhoGAP相互作用蛋白。这些cDNA分别对应于SWI/SNF、α微管蛋白、HMG CoA还原酶和TAX1结合蛋白（TAX1BP1）。与HMG CoA还原酶的相互作用可能部分解释了他汀类降胆固醇药物对乳腺癌细胞生长的抑制作用。在相互作用蛋白参与肿瘤发生的背景下讨论了它们的生物学意义。我们的结果表明，RhoGAP的缺失或其活性改变会抑制乳腺肿瘤细胞的生长。RhoGAP中各种基序的存在及其与其他几种蛋白的相互作用表明，该蛋白可能以多种方式调节Rho信号传导，并且可能以不依赖Rho的方式发挥作用。
