MOLECULAR-CLONING OF A NOVEL MITOGEN-INDUCIBLE NUCLEAR-PROTEIN WITH ARAN GTPASE-ACTIVATING DOMAIN THAT AFFECTS CELL-CYCLE PROGRESSION

We have cloned a novel cDNA (Spa-1) which is little expressed in the q uiescent state but induced in the interleukin 2-stimulated cycling sta te of an interleukin 2-responsive murine lymphoid cell line by differe ntial hybridization. Spa-1 mRNA (3.5 kb) was induced in normal lymphoc ytes following various types of mitogenic stimulation. In normal organ s it is preferentially expressed in both fetal and adult lymphohematop oietic tissues. A Spa-1-encoded protein of 68 kDa is localized mostly in the nucleus. Its N-terminal domain is highly homologous to a human Rap1 GTPase-activating protein (GAP), and a fusion protein of this dom ain (SpanN) indeed exhibited GAP activity for Rap1/Rsr1 but not for Ra s or Rho in vitro. Unlike the human Rap1 GAP, however, SpanN also exhi bited GAP activity for Ran, so far the only known Pas-related GTPase i n the nucleus. In the presence of serum, stable Spa-1 cDNA transfectan ts of NIH 3T3 cells (NIH/Spa-1) hardly overexpressed Spa-1 (p68), and they grew as normally as did the parental cells. When NM/Spa-1 cells w ere serum starved to be arrested in the G(1)/G(0) phase of the cell cy cle, however, they, unlike the control cells, exhibited progressive Sp a-1 p68 accumulation, and following the addition of serum they showed cell death resembling mitotic catastrophes of the S phase during cell cycle progression. The results indicate 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 prematu rely expressed. Functions of the Spa-1 protein and its regulation are discussed in the context of its possible interaction with the Ran/RCC- 1 system, which is involved in the coordinated nuclear functions, incl uding cell division.