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Molecular cloning of a novel mitogen-inducible nuclear protein with a Ran GTPase-activating domain that affects cell
M Hattori1, N Tsukamoto, M S Nur-e-Kamal
1Department of Immunology and Cell Biology, Faculty of Medicine, Kyoto University, Japan.
Abstract:
We have cloned a novel cDNA (Spa-1) which is little expressed in the quiescent state but induced in the interleukin 2-stimulated cycling state of an interleukin 2-responsive murine lymphoid cell line by differential hybridization. Spa-1 mRNA (3.5 kb) was induced in normal lymphocytes following various types of mitogenic stimulation. In normal organs it is preferentially expressed in both fetal and adult lymphohematopoietic 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 domain (SpanN) indeed exhibited GAP activity for Rap1/Rsr1 but not for Ras or Rho in vitro. Unlike the human Rap1 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 NIH 3T3 cells (NIH/Spa-1) hardly overexpressed Spa-1 (p68), and they grew as normally as did the parental cells. When NIH/Spa-1 cells were serum starved to be arrested in the G1/G0 phase of the cell cycle, however, they, unlike the control cells, exhibited progressive Spa-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 prematurely 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, including cell division.
Insights
We identified a novel nuclear protein, Spa-1, that hinders cell cycle progression when abnormally expressed. Spa-1 acts as a GTPase-activating protein (GAP) for Ran, impacting cell division.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- The regulation of cell cycle progression is crucial for normal development and tissue homeostasis.
- Interleukin 2 (IL-2) signaling plays a key role in lymphocyte activation and proliferation.
- GTPase-activating proteins (GAPs) are essential regulators of small GTPases involved in various cellular processes.
Purpose of the Study:
- To clone and characterize a novel cDNA, Spa-1, induced during cell cycle progression.
- To investigate the function of the Spa-1 protein in cell cycle regulation.
- To explore the enzymatic activity and cellular localization of Spa-1.
Main Methods:
- Differential hybridization to identify novel cDNAs.
- mRNA expression analysis in lymphocytes and normal organs.
- Protein expression, purification, and in vitro enzymatic assays.
- Stable transfection of NIH 3T3 cells and cell cycle analysis.
Main Results:
- Spa-1 cDNA was cloned and found to be expressed in lymphohematopoietic tissues.
- The Spa-1 protein (68 kDa) localizes to the nucleus and exhibits GTPase-activating protein (GAP) activity for Rap1 and Ran.
- Overexpression of Spa-1 in NIH 3T3 cells, particularly during serum starvation, leads to cell cycle arrest and mitotic catastrophe upon serum re-addition.
Conclusions:
- Spa-1 is a novel nuclear protein with GAP activity towards Ran, a key regulator of nuclear transport and cell division.
- Abnormal or premature expression of Spa-1 can disrupt mitogen-induced cell cycle progression.
- Spa-1 may play a critical role in coordinating nuclear functions and cell division, potentially through interaction with the Ran/RCC-1 system.