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

我们已经克隆了一个新的cDNA（SPA-1），这是很少在静止状态下表达，但诱导在白细胞介素2（IL 2）刺激的循环状态的IL 2-响应小鼠淋巴细胞系的差异杂交。在正常淋巴细胞中，经各种促有丝分裂刺激后，可诱导出3.5kb的Spa-1 mRNA。在正常器官中，它优先在胎儿和成人淋巴造血组织中表达。Spa-1编码的68 kDa蛋白主要定位于细胞核。它的N-末端结构域与人Rap 1 GTP酶激活蛋白（GAP）高度同源，并且该结构域的融合蛋白（SpanN）在体外确实表现出对Rap 1/Rsr 1的GAP活性，但对Ras或Rho没有GAP活性。然而，与人类Rapl GAP不同，SpanN还表现出Ran的GAP活性，Ran是迄今为止细胞核中唯一已知的Ras相关GTdR。在血清存在下，NIH 3 T3的稳定的Spa-1 cDNA转染子（NIH/Spa-1）几乎不过度表达Spa-1（p68），并且与亲本细胞一样正常生长。然而，当NIH/Spa-1细胞被血清饥饿而被停滞在G1/0时，它们表现出与对照细胞不同的进行性Spa-1 p68积累，并且在加入血清后，它们表现出类似于细胞周期进程中S期的有丝分裂灾难的细胞死亡。结果表明，具有潜在活性的Ran GAP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有丝分裂原诱导的细胞周期进程。在可能与Ran/RCC-1系统相互作用的背景下讨论了Spa-1蛋白的功能及其调节，Ran/RCC-1系统参与协调的核功能，包括细胞分裂。

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.