1: J Virol. 1998 Aug;72(8):6777-84. Cytoplasmic forms of human T-cell leukemia virus type 1 Tax induce NF-kappaB activation. Nicot C, Tie F, Giam CZ. Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814, USA. Human T-cell leukemia virus type 1 (HTLV-1) Tax targets I-kappaB alpha and I-kappaB beta for phosphorylation, ubiquitination, and proteasome-mediated degradation, causing the nuclear translocation of NF-kappaB/Rel proteins and transcription induction of many cellular genes. The mechanism by which a nuclear protein such as Tax stimulates I-kappaB phosphorylation and degradation remains unclear. Here, we describe two cytoplasmic mutants of Tax, designated TaxDeltaN81 and TaxDeltaN109, from which the domains important for cyclic AMP response element binding factor (CREB) and serum response factor (SRF) binding and nuclear transport have been removed. These mutants were unable to trans activate from the HTLV-1 21-bp repeats or the serum response element in the c-fos promoter. In contrast, they activated NF-kappaB reporters, suggesting that activation of NF-kappaB by Tax occurs in the cytoplasm. Incorporation of the nuclear localization signal (NLS) of the simian virus 40 large T antigen into TaxDeltaN81 and TaxDeltaN109 redirected both proteins predominantly to the nucleus yet did not restore trans activation via CREB or SRF. The NLS fusion had little effect on TaxDeltaN81 but reduced NF-kappaB trans activation by TaxDeltaN109, possibly because of its proximity to the NF-kappaB-activating domain of Tax. In contrast to wild-type Tax, the cytoplasmic TaxDeltaN mutants are not cytotoxic. Stable expression of TaxDeltaN109 in HeLa cells resulted in a significant reduction in the intracellular level of I-kappaB alpha, with the constitutive presence of NF-kappaB in the nucleus and concomitant activation of the NF-kappaB enhancer. These results are suggestive of a potential application of the TaxDeltaN109-like mutants in targeting I-kappaB degradation and NF-kappaB activation. Interestingly, a Tax species with a molecular mass similar to that of TaxDeltaN109 was identified in many HTLV-1-transformed T cells, suggesting that TaxDeltaN109-like species might play a role in HTLV-1-induced leukemogenesis. PMID: 9658126 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 2: Oncogene. 1997 Nov 27;15(22):2675-85. Identification and characterization of R-ras3: a novel member of the RAS gene family with a non-ubiquitous pattern of tissue distribution. Kimmelman A, Tolkacheva T, Lorenzi MV, Osada M, Chan AM. The Derald H. Ruttenberg Cancer Center, Mount Sinai School of Medicine, New York, New York 10029, USA. Members of the Ras subfamily of GTP-binding proteins, including Ras (H-, K-, and N-), TC21, and R-ras have been shown to display transforming activity, and activating lesions have been detected in human tumors. We have identified an additional member of the Ras gene family which shows significant sequence similarity to the human TC21 gene. This novel human ras-related gene, R-ras3, encodes for a protein of 209 amino acids, and shows approximately 60-75% sequence identity in the N-terminal catalytic domain with members of the Ras subfamily of GTP-binding proteins. An activating mutation corresponding to the leucine 61 oncogenic lesion of the ras oncogenes when introduced into R-ras3, activates its transforming potential. R-ras3 weakly stimulates the mitogen-activated protein kinase (MAPK) activity, but this effect is greatly potentiated by the co-expression of c-raf-1. By the yeast two-hybrid system, R-ras3 interacts only weakly with known Ras effectors, such as Raf and RalGDS, but not with RglII. In addition, R-ras3 displays modest stimulatory effects on trans-activation from different nuclear response elements which bind transcription factors, such as SRF, ETS/TCF, Jun/Fos, and NF-kappaB/Rel. Interestingly, Northern blot analysis of total RNA isolated from various tissues revealed that the 3.8 kilobasepair (kb) transcript of R-ras3 is highly restricted to the brain and heart. The close evolutionary conservation between R-ras3 and Ras family members, in contrast to the significant differences in its biological activities and the pattern of tissue expression, raise the possibility that R-ras3 may control novel cellular functions previously not described for other GTP-binding proteins. PMID: 9400994 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 3: J Virol. 1996 Dec;70(12):8590-605. Synergistic interactions between overlapping binding sites for the serum response factor and ELK-1 proteins mediate both basal enhancement and phorbol ester responsiveness of primate cytomegalovirus major immediate-early promoters in monocyte and T-lymphocyte cell types. Chan YJ, Chiou CJ, Huang Q, Hayward GS. Department of Pharmacology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA. Cytomegalovirus (CMV) infection is nonpermissive or persistent in many lymphoid and myeloid cell types but can be activated in differentiated macrophages. We have shown elsewhere that both the major immediate-early gene (MIE) and lytic cycle infectious progeny virus expression can be induced in otherwise nonpermissive monocyte-like U-937 cell cultures infected with either human CMV (HCMV) or simian CMV (SCMV) by treatment with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Two multicopy basal enhancer motifs within the SCMV MIE enhancer, namely, 11 copies of the 16-bp cyclic AMP response element (CRE) and 3 copies of novel 17-bp serum response factor (SRF) binding sites referred to as the SNE (SRF/NFkappaB-like element), as well as four classical NFkappaB sites within the HCMV version, contribute to TPA responsiveness in transient assays in monocyte and T-cell types. The SCMV SNE sites contain potential overlapping core recognition binding motifs for SRF, Rel/NFkappaB, ETS, and YY1 class transcription factors but fail to respond to either serum or tumor necrosis factor alpha. Therefore, to evaluate the mechanism of TPA responsiveness of the SNE motifs and of a related 16-bp SEE (SRF/ETS element) motif found in the HCMV and chimpanzee CMV MIE enhancers, we have examined the functional responses and protein binding properties of multimerized wild-type and mutant elements added upstream to the SCMV MIE or simian virus 40 minimal promoter regions in the U-937, K-562, HL-60, THP-1, and Jurkat cell lines. Unlike classical NFkappaB sites, neither the SNE nor the SEE motif responded to phosphatase inhibition by okadaic acid. However, the TPA responsiveness of both CMV elements proved to involve synergistic interactions between the core SRF binding site (CCATATATGG) and the adjacent inverted ETS binding motifs (TTCC), which correlated directly with formation of a bound tripartite complex containing both the cellular SRF and ELK-1 proteins. This protein complex was more abundant in U-937, K-562, and HeLa cell extracts than in Raji, HF, BALB/c 3T3, or HL-60 cells, but the binding activity was altered only twofold after TPA treatment. A 40-fold stimulation of chloramphenicol acetyltransferase activity mediated by four tandem repeats of the SNE could be induced within 2 h (and up to 250-fold within 6 h) after addition of TPA in DNA-transfected U-937 cells, indicating that the stimulation appeared likely to be a true protein kinase C-mediated signal transduction event rather than a differentiation response. Slight differences in the sequence of the core SRF binding site compared with that of the classical c-Fos promoter serum response element, together with differences in the spacing between the SRF and ETS motifs, appear to account for the inability of the SCMV SNEs to respond to serum induction. PMID: 8970984 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 4: J Immunol. 1995 Aug 15;155(4):1972-80. Activation of IL-2 receptor alpha-chain gene by individual members of the rel oncogene family in association with serum response factor. Pierce JW, Jamieson CA, Ross JL, Sen R. Department of Biology, Brandeis University, Waltham, MA 02254, USA. Expression of the IL-2R alpha gene is regulated by members of the c-Rel/NF-kappa B family of transcription factors binding to the kappa B site in the promoter. Previous work has not defined the role of individual members of the c-Rel family in the activation of the IL-2R alpha gene. Using the COS cell system, we were able to reconstitute the regulation of the IL-2R alpha promoter by expressing cloned Rel family members with serum response factor (SRF). We found that c-rel alone activated the IL-2R alpha promoter only weakly but worked with the p50 subunit of NF-kappa B (NFKB1) to give a higher level of expression. We showed that c-rel heterodimerizes with p50 and the amount of this heterodimer correlated with the level of IL-2R alpha gene expression. Our results provide evidence that c-rel/p50 heterodimers activate gene expression in the context of a cellular promoter. We show that c-rel or p65 can cooperate with SRF in the activation of this promoter and the transactivation by c-rel with SRF was enhanced by p50. Synergistic activation required both kappa B and CArG sites, and binding studies show that these adjacent sites can be occupied simultaneously. The transactivation observed with cloned transcription factors mimics the physiologic induction of the IL-2R alpha gene since multiple sequence elements cooperate to give gene activation. The data support the model that c-rel/p50 or p65 can cooperate with SRF to specifically target the expression of the IL-2R alpha gene in activated T cells. PMID: 7636248 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------