Cleavage and nuclear translocation of the caspase 3 substrate Rho GDP-dissociation inhibitor, D4-GDI, during apoptosis.
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While investigating endonucleases potentially involved in apoptosis, an antisera was raised to bovine deoxyribonuclease II, but it recognized a smaller protein of 26 kDa protein in a variety of cell lines. The 26 kDa protein underwent proteolytic cleavage to 22 kDa concomitantly with DNA digestion in cells induced to undergo apoptosis. Sequencing of the 26 kDa protein identified it as the Rho GDP-dissociation inhibitor D4-GDI. Zinc, okadaic acid, calyculin A, cantharidin, and the caspase inhibitor z-VAD-fmk, all prevented the cleavage of D4-GDI, DNA digestion, and apoptosis. The 26 kDa protein resided in the cytoplasm of undamaged cells, whereas following cleavage, the 22 kDa form translocated to the nucleus. Human D4-GDI, and D4-GDI mutated at the caspase 1 or caspase 3 sites, were expressed in Chinese hamster ovary cells which show no detectable endogenous D4-GDI. Mutation at the caspase 3 site prevented D4-GDI cleavage but did not inhibit apoptosis induced by staurosporine. The cleavage of D4-GDI could lead to activation of Jun N-terminal kinase which has been implicated as an upstream regulator of apoptosis in some systems. However, the results show that the cleavage of D4-GDI and translocation to the nucleus do not impact on the demise of the cell. (+info)
Vav, a GDP/GTP nucleotide exchange factor, interacts with GDIs, proteins that inhibit GDP/GTP dissociation.
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Vav functions as a specific GDP/GTP nucleotide exchange factor which is regulated by tyrosine phosphorylation in the hematopoietic system. Loss of the amino-terminus sequences of Vav was sufficient to control its transforming potential and its function in T cells. We report here the identification of the hematopoietic GDP dissociation inhibitor protein, Ly-GDI, as a protein that interacts with the amino-terminus of Vav. Further analysis confirmed that Vav and Ly-GDI interact both in in vitro and in in vivo assays. This association is maximal only when the amino region of Vav is intact and requires an intact carboxy-terminus of Ly-GDI. The interaction between Vav and Ly-GDI is not dependent on the tyrosine phosphorylation status of Vav. In addition, Rho-GDI, the highly homologous protein to Ly-GDI, associates with Vav as well. The contribution of the interaction between Vav and GDIs, proteins that are involved in the GDP/GTP exchange processes, to the biological function of Vav is further discussed. (+info)
Differential expression and regulation of GTPases (RhoA and Rac2) and GDIs (LyGDI and RhoGDI) in neutrophils from patients with severe congenital neutropenia.
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Severe congenital neutropenia (SCN) or Kostmann syndrome is a disorder of myelopoiesis characterized by a maturation arrest at the stage of promyelocytes or myelocytes in bone marrow and absolute neutrophil counts less than 200/microL in peripheral blood. Treatment of these patients with granulocyte colony-stimulating factor (G-CSF) leads to a significant increase in circulating neutrophils and a reduction in infection-related events in more than 95% of the patients. To date, little is known regarding the underlying pathomechanism of SCN. G-CSF-induced neutrophils of patients with SCN are functionally defective (eg, chemotaxis, superoxide anion generation, Ca(++ )mobilization). Two guanosine triphosphatases (GTPases), Rac2 and RhoA, were described to be involved in many neutrophil functions. The expression of these GTPases and their regulation in patients' neutrophils were of interest. This study determined that the guanosine diphosphate (GDP)-dissociation inhibitor RhoGDI is overexpressed at the protein level in patients' neutrophils and that overexpression is a result of G-CSF treatment. RhoA and LyGDI are expressed at similar levels, whereas Rac2 shows a decreased expression. In addition, association of Rac2 and RhoGDI or LyGDI is abrogated or not detectable based on the low Rac2 expression in patients' neutrophils. (Blood. 2000;95:2947-2953) (+info)
TNF-alpha-mediated neutrophil apoptosis involves Ly-GDI, a Rho GTPase regulator.
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We investigated intracellular signaling events involved in fibronectin-accelerated TNF-alpha-mediated PMN apoptosis by means of 2-D gel electrophoresis and western blotting. Proteins were sequenced with electrospray ionization mass spectrometry. Apoptosis was quantitated by flow cytometry. We detected a cluster of acidic, high molecular-weight proteins that were only tyrosine phosphorylated when TNF-alpha-treated PMN interacted with fibronectin. Sequence analysis revealed that one of these proteins was Ly-GDI, a regulator of Rho GTPases. Fibronectin increased the TNF-alpha-induced Ly-GDI cleavage, yielding a 23-kD fragment. At 8 h, intact Ly-GDI was decreased to 33% on fibronectin, compared with 69% on PolyHema (P<0.05). Inhibition of tyrosine phosphorylation prevented phosphorylation of Ly-GDI, fibronectin-accelerated Ly-GDI cleavage, and fibronectin-accelerated apoptosis in TNF-alpha-treated PMN. We found that Ly-GDI cleavage was dependent on caspase-3 activation and that caspase-3 inhibition decreased apoptosis. We conclude that tyrosine phosphorylation of Ly-GDI, followed by increased caspase-3-mediated Ly-GDI cleavage, is a signaling event associated with accelerated TNF-alpha-mediated apoptosis on fibronectin. (+info)
D4-GDI is cleaved by caspase-3 during daunorubicin-induced apoptosis in HL-60 cells.
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Daunorubicin, an anti-cancer drug, is known to induce apoptosis in HL-60 cells in a dose-dependent manner through the activation of caspase-3 (CPP32). Caspase-3 selective inhibitor, Ac-DEVD-CHO, prevented both the activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase (PARP). D4-GDI is a GDP dissociation inhibitor for the Ras-related Rho family GTPase in hematopoietic cells. Here we report that D4-GDI is a substrate for the caspase-3. D4-GDI was cleaved to a 23 kDa fragment by daunorubicin treatment in HL-60 cells with kinetics that parallel the onset of apoptosis. D4-GDI cleavage as well as DNA fragmentation was inhibited by treatment with Ac-DEVD-CHO but not with Ac-YVAD-CHO, a caspase-1 inhibitor. These data suggest that D4-GDI of Rho family GTPase may be regulated during apoptosis through the caspase-3 mediated cleavage of the GDI protein. (+info)
Vav1 and Ly-GDI two regulators of Rho GTPases, function cooperatively as signal transducers in T cell antigen receptor-induced pathways.
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The Rho family GTPases are pivotal for T cell signaling; however, the regulation of these proteins is not fully known. One well studied regulator of Rho GTPases is Vav1; a hematopoietic cell-specific guanine nucleotide exchange factor critical for signaling in T cells, including stimulation of the nuclear factor of activated T cells (NFAT). Surprisingly, Vav1 associates with Ly-GDI, a hematopoietic cell-specific guanine nucleotide dissociation inhibitor of Rac. Here, we studied the functional significance of the interaction between Vav1 and Ly-GDI in T cells. Upon organization of the immunological synapse, both Ly-GDI and Vav1 relocalize to T cell extensions in contact with the antigen-presenting cell. Ly-GDI is phosphorylated on tyrosine residues following T cell receptor stimulation, and it associates with the Src homology 2 region of an adapter protein, Shc. In addition, the interaction between Ly-GDI and Vav1 requires tyrosine phosphorylation. Overexpression of Ly-GDI alone is inhibitory to NFAT stimulation and calcium mobilization. However, when co-expressed with Vav1, Ly-GDI enhances Vav1 induction of NFAT activation, phospholipase Cgamma phosphorylation, and calcium mobilization. Moreover, Ly-GDI does not alter the regulation of these phenomena when coexpressed with oncogenic Vav1. Since oncogenic Vav1 does not bind Ly-GDI, this suggests that the functional cooperativity of Ly-GDI and Vav1 is dependent upon their association. Thus, our data suggest that the interaction of Vav1 and Ly-GDI creates a fine tuning mechanism for the regulation of intracellular signaling pathways leading to NFAT stimulation. (+info)
RhoGDI2 is an invasion and metastasis suppressor gene in human cancer.
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To discover novel metastasis suppressor genes that are clinically relevant in common human cancers, we used isogenic human bladder cancer cell lines and used DNA microarray technology to identify genes whose expression diminishes as a function of invasive and metastatic competence. We then evaluated the expression profile of such genes in 105 pathologically characterized tumors from seven common organ sites, and we identified one gene, RhoGDI2, whose expression was diminished as a function of primary tumor stage and grade. When RhoGDI2 was transferred back into cells with metastatic ability that lacked its expression, it suppressed experimental lung metastasis but did not affect in vitro growth, colony formation, or in vivo tumorigenicity. In addition, RhoGDI2 reconstitution in these cells blocked invasion in an organotypic assay and led to a reduction of in vitro motility. These results indicate that RhoGDI2 is a metastasis suppressor gene, a marker of aggressive human cancer, and a promising target for therapy. (+info)
Reduced expression of metastasis suppressor RhoGDI2 is associated with decreased survival for patients with bladder cancer.
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PURPOSE: RhoGDI2 was recently shown to be a metastasis suppressor gene in models of bladder cancer. We sought to further understand its importance in human cancer by determining the level of its expression and the distribution of its encoded protein in normal human tissues and cell lines and to evaluate whether its protein expression is a determinant of human bladder cancer progression. EXPERIMENTAL DESIGN: RhoGDI2 mRNA and protein expression was evaluated in cell lines and human tissues using Affymetrix and tissue microarrays, respectively. Tissue microarrays represented most human normal adult tissues and material from 51 patients that had undergone radical cystectomy for bladder cancer. In these 51 patients, the chi(2) test was used to test for associations between RhoGDI2 and stage, grade of urothelial carcinoma, histological type, and disease-specific survival status. Cox proportional hazards regression analyses were used to estimate the effect of RhoGDI2 expression level on time to development of metastatic disease and disease-specific survival time, adjusting for grade, stage, and histological type. RESULTS: In normal tissues, there was strong RhoGDI2 protein expression in WBCs, endothelial cells, and transitional epithelium. RhoGDI2 mRNA expression was inversely related to the invasive and metastatic phenotype in human bladder cancer cell lines. In patients with bladder cancer, univariate analysis indicated that reduced tumor RhoGDI2 protein expression was associated with a lower actuarial 5-year disease-free and disease-specific survival (P = 0.01). In addition, patients with tumors that had low or absent RhoGDI2 had a shorter time to disease-specific death (P +info)