IntroductionRap1, a Ras-family G protein, mediates diverse functions that are highly dependent on the contexts of the cell types, such as proliferation, differentiation, cell survival, cell adhesion, polarity, and movements. 1,2 Rap1 is activated to Rap1GTP by specific guanine nucleotide exchange factors (GEFs) coupled with diverse receptors, whereas specific GTPase-activating proteins (GAPs) hydrolyze Rap1GTP to Rap1GDP. 3 We previously reported that mice deficient for SPA-1, a principal Rap1GAP in hematopoietic progenitor cells (HPCs), developed a spectrum of myeloid leukemia or B1-cell leukemia of long latency. 4,5 The majority of myeloid leukemia in SPA-1 Ϫ/Ϫ mice resembled human chronic myelogenous leukemia and often was associated with blast crisis of myeloid, erythroid, or B-cell lineage, which strongly suggested a disorder of multipotent HPCs. 6 Although the results suggested that SPA-1 functioned as a leukemia suppressor in HPCs, the long latency implied that additional factors might be needed for development of overt leukemia. On the other hand, a Rap1GEF, CalDAG-GEF1, has been reported as a proto-oncogene in BXH-2 myeloid leukemia. 7 We also reported that human bcr-abl fusion gene product (Bcr-Abl) was a potent Rap1 activator, 8 and that Bcr-Abl ϩ SPA-1 Ϫ/Ϫ HPCs showed aggravated chronic myelogenous leukemia phenotypes compared with Bcr-Abl ϩ wild-type HPCs in a mouse model, including prolonged survival of leukemic progenitors and rapid blast crisis in vivo. 9 Thus, proto-oncogenes capable of activating Rap1 signal may be potential collaborative factors for leukemia in SPA-1 deficiency.In the present study, we investigated the effects of C3G expression in normal and SPA-1 Ϫ/Ϫ HPCs in vivo. C3G is a ubiquitous Rap1GEF translocated to the plasma membrane by a wide variety of signals via Crk-family adaptor proteins. 10,11 Farnesylated C3G (C3G-F) shows facilitated recruitment to the plasma membrane to activate endogenous Rap1, 10 although the activity can be counteracted by SPA-1. 12 Results showed that transplantation of normal HPCs expressing C3G-F resulted in a marked expansion of blastic thymocytes with unusual phenotypes, namely, CD4/CD8 double-positive (DP) CD3/TCR Ϫ , in the irradiated recipients. SPA-1 Ϫ/Ϫ HPCs expressing C3G-F caused a more aggressive thymic DP cell expansion with higher levels of Rap1 activation than C3G-F ϩ wild-type HPCs, eventually resulting in lethal T-cell acute lymphoblastic leukemia (T-ALL). Leukemic C3G-F ϩ thymocytes exhibited markedly augmented expression of Notch and the target genes. Furthermore, all the clonal C3G-F ϩ SPA-1 Ϫ/Ϫ T-ALL cell lines revealed characteristic mutations in Notch1, resulting in C-terminal truncation, which is similar to mutations reported in the majority of T-ALL in humans as well as in genetically predisposed animal models. [13][14][15][16] The proliferation was completely inhibited in the presence of a ␥-secretase inhibitor. The current results suggest a functional cross talk of endogenous Rap1 G protein and Notch signaling in thymocyte d...
The Spen protein family is found in worms, flies, and mammals, and is implicated in diverse biological processes from embryogenesis to aging. Spen proteins have three N-terminal RNA recognition motifs and a C-terminal SPOC domain. The mammalian Spen proteins Mint and its human ortholog SHARP interact with the Notch-signaling mediator RBP-J as well as Msx2 and several unliganded nuclear hormone receptors, and impart transcription-repressing activity to these molecules by recruiting corepressors through the SPOC domain. Despite these in vitro findings, Mint/SHARP's physiological role is largely unknown, because Mint germline knockouts are embryonic lethal. To analyze Mint/SHARP function in postnatal mice, we created Mint-floxed mice that allow the Cre/loxP-mediated conditional knockout of Mint. We analyzed Mint and RBP-J epistasis during Notch-dependent splenic B-lymphocyte development, and found that Mint suppresses Notch signaling through RBP-J. In addition, Mint deficiency caused severe hypoplasia in postnatal brain, suggesting it may regulate neuronal cell survival.
SPA-1 is a negative regulator of Rap1 signal in hematopoietic cells, and SPA-1-deficient mice develop myeloproliferative disorders (MPD) of long latency. In the present study, we showed that the MPDs in SPA-1
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