C3G is a guanine nucleotide exchange factor for Rap1 and is activated by the expression of Crk adaptor proteins. We found that expression of CrkI in COS cells induced significant tyrosine phosphorylation of C3G. To understand the mechanism by which C3G is phosphorylated and activated by Crk, we constructed a series of deletion mutants. Deletion of the amino terminus of C3G to amino acid 61 did not remarkably affect either tyrosine phosphorylation or Crk-dependent activation of C3G. When C3G was truncated to amino acid 390, C3G was still phosphorylated on tyrosine but was not effectively activated by CrkI. Deletion of the amino terminus of C3G to amino acid 579 significantly reduced the Crkdependent tyrosine phosphorylation of C3G and increased GTP-bound Rap1 irrespective of the presence of CrkI. We substituted all seven tyrosine residues in this region, amino acids 391-579, for phenylalanine for identification of the phosphorylation site. Among the substitution mutants, the C3G-Y504F mutant, in which tyrosine 504 was substituted by phenylalanine, was remarkably less activated and phosphorylated than the wild type. All the other substitution mutants were activated and tyrosyl-phosphorylated by the expression of CrkI. Thus, CrkI activates C3G by the phosphorylation of tyrosine 504, which represses the cis-acting negative regulatory domain outside the catalytic region.

The degradation of ornithine decarboxylase (ODC) catalyzed by the 26 S proteasome is accelerated by antizyme, an ODC inhibitory protein induced by polyamines. Previously, we have found another possible regulatory protein of ODC degradation, antizyme inhibitor. Antizyme inhibitor binds to the antizyme with a higher affinity than that of ODC, releasing ODC from ODCantizyme complex. We report here the cDNA sequence of rat heart antizyme inhibitor. The deduced sequence of the protein is highly similar to, but distinct from, sequences of ODCs from various species. Antizyme inhibitor contains amino acid residues required for formation of active sites of ODC, but it completely lacks ODC activity. Antizyme inhibitor has no homology with peptide sequence in the mammalian ODC carboxyl terminus, which is needed for rapid turnover of ODC. It inhibits antizyme-dependent ODC degradation, but, unlike ODC, its degradation is not accelerated by antizyme.Ornithine decarboxylase (ODC) 1 is a key enzyme in polyamine biosynthesis pathway (1, 2). The turnover of ODC is very rapid and highly regulated (3, 4). The degradation of ODC catalyzed by the 26 S proteasome is accelerated by ODC antizyme (5, 6), an inhibitory protein induced by polyamines (7). Strict regulation of ODC appears to be important for cell growth, because overproduction of ODC is associated with neoplastic transformation (8, 9), whereas overproduction of antizyme inhibits cell growth (10, 11). We previously found in rat liver and heart another possible regulator of ODC degradation, antizyme inhibitor (12). Antizyme inhibitor binds to the antizyme with a higher affinity than that of ODC and releases ODC from the ODC-antizyme complex (12)(13)(14). The physiological fluctuation of antizyme inhibitor in vivo suggested that it is another regulatory protein that stabilizes ODC by trapping antizyme (14). However, the possibility that antizyme inhibitor is a post-translationally modified product of ODC could not be ruled out. In this report, we describe the cloning and expression of antizyme inhibitor and show that the sequence of antizyme inhibitor is closely related to, but distinct from, that of ODC. EXPERIMENTAL PROCEDUREScDNA Cloning-Oligo(dT)-primed cDNA was synthesized from poly(A) ϩ RNA from the hearts of isoproterenol-treated Wistar rats (10 mg/kg, 2 h) and inserted into ZAPII vector (Stratagene) through EcoRI adaptors to construct a library. One positive clone was selected from 10 5 recombinants by screening with a monoclonal antibody to rat heart antizyme inhibitor (14) as a probe. This monoclonal antibody does not react with rat ODC (14). The selected clone, A1, which carried a cDNA insert of about 1.9 kb in length was purified and sequenced. Two more positive clones were selected by plaque hybridization with a probe of the partial length cDNA A1. These clones, A2 and A3, carried cDNA inserts of about 2.2 and 4 kb, respectively, and were sequenced. All the DNA sequences were determined from both strands.Northern Blot Analysis of Antizyme Inhibitor mRNA-Poly(A) ϩ ...

Crk is an adaptor protein that consists almost entirely of SH2 and SH3 domains. We have previously demonstrated, by using in vivo and in vitro systems, that C3G, which was identified as a Crk SH3 domain-binding guanine nucleotide exchange factor, specifically activates Rap1. C3G also binds to other adaptor proteins, including CrkL and Grb2. In the present study, we analyzed the effect of Crk, CrkL, and Grb2 on the C3G-Rap1 pathway. Expression of Crk, CrkL, and Grb2 with C3G in Cos1 cells significantly increased the ratio of GTP/GDP bound to Rap1. Both the SH2 and SH3 domains of Crk were required for this activity. However, Crk did not stimulate the guanine nucleotide exchange activity of C3G for Rap1 in vitro, suggesting that Crk does not activate C3G by an allosteric mechanism. The requirement of the SH2 domain of Crk for the enhancement of guanine nucleotide exchange activity for Rap1 could be compensated for by the addition of a farnesylation signal to Crk, indicating that Crk enhanced the guanine nucleotide exchange activity of C3G by membrane recruitment of C3G. These results demonstrate that Crk, CrkL, and Grb2 positively modulate the C3G-Rap1 pathway primarily by recruiting C3G to the cell membrane.

The present analyses clearly indicate that OMBC is a distinct subgroup with long-term prognosis superior to MBC, with reasonable provability for clinical cure. Further prospective studies to better characterize OMBC are warranted to improve prognosis in MBC.

The liver, skin, and gastrointestinal tract are major target organs of acute graftversus-host disease (GVHD), the major complication of allogeneic bone marrow transplantation (BMT). In order to gain a better understanding of acute GVHD in the liver, we compared the gene expression profiles of livers after experimental allogeneic and syngeneic BMT using oligonucleotide microarray. At 35 days after allogeneic BMT when hepatic GVHD was histologically evident, genes related to cellular effectors and acute-phase proteins were up-regulated, whereas genes largely related to metabolism and endocrine function were down-regulated. At day 7 after BMT before the development of histologic changes in the liver, interferon ␥ ( IntroductionAllogeneic bone marrow transplantation (BMT) is the treatment of choice for a number of malignant conditions as well as hematologic and metabolic disorders. Acute graft-versus-host disease (GVHD), a major complication of allogeneic BMT, is a rapidly progressing systemic illness characterized by tissue injury in various organs, including the liver, skin, gut, and lymphoid tissues. 1,2 Acute GVHD is initiated by donor T cells in response to host antigen-presenting cells (APCs). 3 Damage to the intestinal mucosa by the conditioning regimen of BMT, such as irradiation or chemotherapy, allows the translocation of lipopolysaccharide (LPS) into the systemic circulation, which activates host APCs and stimulates monocytes and macrophages to secrete inflammatory cytokines. 4 This cytokine cascade synergies with cellular effectors such as cytotoxic T-lymphocytes (CTLs) and natural killer (NK) cells, and results in the amplification of tissue injury. 5 In the liver, the histologic signature of acute GVHD is inflammatory cell infiltration into portal areas, bile duct epithelial damage, cholestasis, and endothelialitis. 6 We hypothesized that the global quantitative analysis of gene expression would help to illuminate the mechanisms of the complex and multifactorial process of acute GVHD in the liver, particularly early during the disease process when histology is rarely evaluable because of the morbidity of hepatic biopsies early after transplantation. We therefore analyzed hepatic tissue at 2 different time points after BMT in a well-characterized murine model to evaluate the kinetics of changes in gene expression. Materials and methods MiceFemale C57BL/6 (B6, H-2 b ) and B6D2F1 (H-2 b/d ) mice were purchased from the Jackson Laboratories (Bar Harbor, ME). The age of mice used as BM transplant recipients ranged between 9 and 15 weeks, while the age of donors was 9 to 11 weeks. BMT and isolation of the liverMice underwent transplantation according to a standard protocol described previously. 7 On day 0, B6D2F1 mice received 13 Gy total body irradiation (TBI; 137 Cs source), split into 2 doses separated by 3 hours to minimize gastrointestinal toxicity. After TBI, 5 ϫ 10 6 bone marrow cells (BMCs) and 2 ϫ 10 6 nylon wool-purified splenic donor T cells from allogeneic B6 donors or syngeneic B6D2F1 donors we...