RASAL3, a novel hematopoietic RasGAP protein, regulates the number and functions of NKT cells

Abstract

Ras GTPase-activating proteins negatively regulate the Ras/Erk signaling pathway, thereby playing crucial roles in the proliferation, function, and development of various types of cells. In this study, we identified a novel Ras GTPase-activating proteins protein, RASAL3, which is predominantly expressed in cells of hematopoietic lineages, including NKT, B, and T cells. We established systemic RASAL3-deficient mice, and the mice exhibited a severe decrease in NKT cells in the liver at 8 weeks of age. The treatment of RASAL3-deficient mice with α-GalCer, a specific agonist for NKT cells, induced liver damage, but the level was less severe than that in RASAL3-competent mice, and the attenuated liver damage was accompanied by a reduced production of interleukin-4 and interferon-γ from NKT cells. RASAL3-deficient NKT cells treated with α-GalCer in vitro presented augmented Erk phosphorylation, suggesting that there is dysregulated Ras signaling in the NKT cells of RASAL3-deficient mice. Taken together, these results suggest that RASAL3 plays an important role in the expansion and functions of NKT cells in the liver by negatively regulating Ras/Erk signaling, and might be a therapeutic target for NKT-associated diseases.Keywords: α-GalCer r IL-4 r Liver injury r NKT cell r Ras r RasGAP Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionRas proteins have been shown to play pivotal roles in proliferation, differentiation, and oncogenesis by functioning as a molecular switch for intracellular signaling pathways [1,2]. In order to act as a switch, Ras has two forms, an inactive GDP-bound form and an active GTP-bound form. GDP-bound Ras is dominant under the steady-state condition. Upon agonist binding to its receptor on the cell membrane, GDP-bound Ras is converted into active GTPbound Ras, which stimulates downstream signaling cascades, such as the Raf/Erk and phosphoinositide-3 kinase/Akt pathways [3]. Such switching of Ras forms is controlled by its intrinsic GTPase activity, and a balance between Ras guanine nucleotide exchange factors (RasGEFs) and Ras GTPase-activating proteins (RasGAPs) [4]. While RasGEFs act as a positive factor for Ras signaling by stimulating the conversion of Ras-GDP into Ras-GTP, RasGAPs act as negative factors by stimulating the rate of GTP hydrolysis from Ras-GTP into Ras-GDP [4]. Ras regulates the activation, differentiation, and proliferation of lymphoid cells. For instance, p21ras (K-Ras) regulates the function and growth of T cells and thymocytes during both immune activation and T-cell development [5][6][7]. In addition, TCR activation in T cells stimulates Ras, which initiates downstream signaling pathways leading to the production of cytokines such as IL-2, and subsequent IL-2-induced proliferation [8,9]. A deficiency of the RasGRP1 gene, a RasGEF, in mice inactivates the TCR activation-induced Ras signaling in thymocytes, and the mice have reduced single-positive (CD4 + CD8 − and CD4 − CD8 + ) thymocytes [10]....

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