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

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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.