Analysis of a lympho-hematopoietic specific nuclear protein with homology to RaplGAP

我们克隆了一个新的基因SpA-1，该基因在白介素2(IL-2)刺激的小鼠淋巴样细胞系的静止期几乎不表达，但在白介素2(IL-2)刺激的周期状态下被诱导。正常淋巴细胞经不同有丝分裂刺激后，均可表达SPA-1mRNA(3.5kb)。在正常器官中，它在胎儿和成人淋巴造血组织中优先表达。SpA-1编码的68 kDa蛋白主要定位于细胞核。其N末端结构域与人RAPL GTP酶激活蛋白(GAP)高度同源，该结构域的融合蛋白SPANN在体外对RAPL/Rsr1具有GAP活性，但对Ras或Rho不具有GAP活性。然而，与人类RAPL GAP不同的是，SPANN也显示了RAN的GAP活性，这是迄今为止唯一已知的细胞核中与RAS相关的GTP酶。在血清存在下，稳定表达Spa-1基因的NIH3T3细胞(NIH/Spa-1)几乎不过表达Spa-1(P68)，并与亲本细胞一样正常生长。然而，当NIH/SpA-1细胞在血清饥饿后被滞留在G1/0期时，与对照组细胞不同，它们表现出进行性的Spa-1p68积聚，并且在加入血清后，细胞死亡类似于细胞周期进程中S期的有丝分裂灾难。结果表明，具有潜在活性的RAN间隙结构域的新型核蛋白Spa-1在异常和/或过早表达时严重阻碍了丝裂原诱导的细胞周期进程。本文从SpA-1蛋白与RAN/RCC-1系统相互作用的角度讨论了SpA-1蛋白的功能及其调控，RAN/RCC-1系统参与了包括细胞分裂在内的核协调功能。

We have cloned a novel cDNA (Spa-1) which was little expressed in the quiescent state but induced in the interleukin 2 (IL2) -stimulated cycling state of an IL2-responsive murine lymphoid cell line by differential hybridization. Spa-1 mRNA (3.5kb) was induced in the normal lymphocytes following various mitogenic stimulation. In normal organs it was preferentially expressed in both fetal and adult lymphohematopoietic tissues. A Spa-1-coded protein of 68 kDa was localized mostly in the nucleus. Its N-terminal domain is highly homologous to a human Rapl GTPase activating protein (GAP) , and a fusion protein of this domain (SpanN) indeed exhibited GAP activity for Rapl/Rsrl but not for Ras or Rho in vitro. Unlike the human Rapl GAP,however, SpanN also exhibited GAP activity for Ran, so far the only known Ras-related GTPase in the nucleus. In the presence of serum, stable Spa-1 cDNA transfectants of NIH3T3 (NIH/Spa-1) hardly overexpressed Spa-1 (p68) and grew normally as the parental cells. When NIH/Spa-1 cells were serum-starved to be arrested in G1/0, however, they exhibited progressive Spa-1 p68 accumulation unlike the control cells, and following the addition of serum they showed cell death resembling mitotic catastrophes of S phase during cell cycle progression. The results indicates that the novel nuclear protein, Spa-1, with a potentially active Ran GAP domain severely hampers the mitogen-induced cell cycle progression when abnormally and/or prematurely expressed. Functions of the Spa-1 protein and its regulation are discussed in the context of its possible interaction with Ran/RCC-1 system, which is involved in the coordinated nuclear functions including cell division.