Murphy G A, Solski P A, Jillian S A, Pérez de la Ossa P, D'Eustachio P, Der C J, Rush M G
Department of Biochemistry, New York University Medical Center, New York 10016, USA.
Oncogene. 1999 Jul 1;18(26):3831-45. doi: 10.1038/sj.onc.1202758.
The small Ras-related GTPase, TC10, has been classified on the basis of sequence homology to be a member of the Rho family. This family, which includes the Rho, Rac and CDC42 subfamilies, has been shown to regulate a variety of apparently diverse cellular processes such as actin cytoskeletal organization, mitogen-activated protein kinase (MAPK) cascades, cell cycle progression and transformation. In order to begin a study of TC10 biological function, we expressed wild type and various mutant forms of this protein in mammalian cells and investigated both the intracellular localization of the expressed proteins and their abilities to stimulate known Rho family-associated processes. Wild type TC10 was located predominantly in the cell membrane (apparently in the same regions as actin filaments), GTPase defective (75L) and GTP-binding defective (31N) mutants were located predominantly in cytoplasmic perinuclear regions, and a deletion mutant lacking the carboxyl terminal residues required for post-translational prenylation was located predominantly in the nucleus. The GTPase defective (constitutively active) TC10 mutant: (1) stimulated the formation of long filopodia; (2) activated c-Jun amino terminal kinase (JNK); (3) activated serum response factor (SRF)-dependent transcription; (4) activated NF-kappaB-dependent transcription; and (5) synergized with an activated Raf-kinase (Raf-CAAX) to transform NIH3T3 cells. In addition, wild type TC10 function is required for full H-Ras transforming potential. We demonstrate that an intact effector domain and carboxyl terminal prenylation signal are required for proper TC10 function and that TC10 signals to at least two separable downstream target pathways. In addition, TC10 interacted with the actin-binding and filament-forming protein, profilin, in both a two-hybrid cDNA library screen, and an in vitro binding assay. Taken together, these data support a classification of TC10 as a member of the Rho family, and in particular, suggest that TC10 functions to regulate cellular signaling to the actin cytoskeleton and processes associated with cell growth.
小的Ras相关GTP酶TC10，根据序列同源性被归类为Rho家族的成员。这个家族包括Rho、Rac和CDC42亚家族，已被证明可调节多种明显不同的细胞过程，如肌动蛋白细胞骨架组织、丝裂原活化蛋白激酶（MAPK）级联反应、细胞周期进程和细胞转化。为了开始对TC10生物学功能的研究，我们在哺乳动物细胞中表达了该蛋白的野生型和各种突变形式，并研究了表达蛋白的细胞内定位及其刺激已知Rho家族相关过程的能力。野生型TC10主要位于细胞膜（显然与肌动蛋白丝在同一区域），GTP酶缺陷型（75L）和GTP结合缺陷型（31N）突变体主要位于细胞质核周区域，而缺乏翻译后异戊二烯化所需羧基末端残基的缺失突变体主要位于细胞核。GTP酶缺陷型（组成型激活）TC10突变体：（1）刺激长丝状伪足的形成；（2）激活c-Jun氨基末端激酶（JNK）；（3）激活血清反应因子（SRF）依赖性转录；（4）激活NF-κB依赖性转录；（5）与激活的Raf激酶（Raf-CAAX）协同作用以转化NIH3T3细胞。此外，完整的H-Ras转化潜能需要野生型TC10功能。我们证明完整的效应结构域和羧基末端异戊二烯化信号是TC10正常功能所必需的，并且TC10向至少两条可分离的下游靶途径发出信号。此外，在双杂交cDNA文库筛选和体外结合试验中，TC10都与肌动蛋白结合和丝状形成蛋白profilin相互作用。综上所述，这些数据支持将TC10归类为Rho家族的成员，特别是表明TC10的功能是调节细胞向肌动蛋白细胞骨架的信号传导以及与细胞生长相关的过程。
