Otsuki Y, Tanaka M, Yoshii S, Kawazoe N, Nakaya K, Sugimura H
First Department of Pathology and First Department of Medicine, Hamamatsu University School of Medicine, 3600 Handa-cho, Hamamatsu 431-3192, Japan.
Proc Natl Acad Sci U S A. 2001 Apr 10;98(8):4385-90. doi: 10.1073/pnas.071411598. Epub 2001 Mar 27.
The putative tumor metastasis suppressor nm23H1 was originally identified in murine melanomas by subtraction cloning. It displays nucleoside diphosphate kinase activity and regulates cellular events, including growth and development. Recently nm23H1 has been reported to also act as a GTPase-activating protein of the Ras-related GTPase Rad. We attempted to determine whether nm23H1 also regulates Rho-family GTPases. Although we were unable to detect a direct association between nm23H1 and Rho-family GTPases, nm23H1 was shown to be associated with a Rac1-specific nucleotide exchange factor, Tiam1, by interaction with its amino-terminal region in extracts from the cells expressing exogenous Tiam1 and from native tissue. Overexpression of nm23H1 inhibited the Tiam1-induced production of GTP-bound Rac1 and activation of c-Jun kinase. On the other hand, forced overexpression of the wild type, but not the kinase-inactivated mutant of nm23H1, converted the GDP-bound forms of Rac1, Cdc42, and RhoA to their GTP-bound forms in vitro by its nucleoside diphosphate kinase activity, but nm23H1 alone apparently did not produce the GTP-bound form of these GTPases in vivo. These results suggest that nm23H1 negatively regulates Tiam1 and inhibits Rac1 activation in vivo. Moreover, adhesion-stimulated membrane ruffles of Rat1 fibroblasts were reduced by overexpression of nm23H1. Based on these observations, we concluded that we had identified a function of nm23H1 as a regulator of Rac1 and that it may be related to the effect of nm23H1 as a tumor metastasis suppressor.
推定的肿瘤转移抑制因子nm23H1最初是通过消减克隆在鼠黑色素瘤中鉴定出来的。它具有核苷二磷酸激酶活性，并调节包括生长和发育在内的细胞活动。最近有报道称nm23H1还可作为Ras相关GTP酶Rad的GTP酶激活蛋白。我们试图确定nm23H1是否也调节Rho家族GTP酶。尽管我们未能检测到nm23H1与Rho家族GTP酶之间的直接关联，但在表达外源性Tiam1的细胞提取物和天然组织提取物中，nm23H1通过与其氨基末端区域相互作用，显示出与Rac1特异性核苷酸交换因子Tiam1相关。nm23H1的过表达抑制了Tiam1诱导的GTP结合型Rac1的产生和c-Jun激酶的激活。另一方面，野生型nm23H1（而非激酶失活突变体）的强制过表达，通过其核苷二磷酸激酶活性在体外将Rac1、Cdc42和RhoA的GDP结合形式转化为GTP结合形式，但单独的nm23H1在体内显然不会产生这些GTP酶的GTP结合形式。这些结果表明，nm23H1在体内负向调节Tiam1并抑制Rac1激活。此外，nm23H1的过表达减少了Rat1成纤维细胞黏附刺激的膜皱褶。基于这些观察结果，我们得出结论，我们已经确定了nm23H1作为Rac1调节因子的功能，并且它可能与nm23H1作为肿瘤转移抑制因子的作用有关。
