Controlling small guanine–nucleotide-exchange factor function through cytoplasmic RNA intramers

ADP-ribosylation factor (ARF) GTPases and their regulatory proteins have been implicated in the control of diverse biological functions. Two main classes of positive regulatory elements for ARF have been discovered so far: the large Sec7/Gea and the small cytohesin/ARNO families, respectively. These proteins harbor guanine–nucleotide-exchange factor (GEF) activity exerted by the common Sec7 domain. The availability of a specific inhibitor, the fungal metabolite brefeldin A, has enabled documentation of the involvement of the large GEFs in vesicle transport. However, because of the lack of such tools, the biological roles of the small GEFs have remained controversial. Here, we have selected a series of RNA aptamers that specifically recognize the Sec7 domain of cytohesin 1. Some aptamers inhibit guanine–nucleotide exchange on ARF1, thereby preventing ARF activation in vitro. Among them, aptamer M69 exhibited unexpected specificity for the small GEFs, because it does not interact with or inhibit the GEF activity of the related Gea2-Sec7 domain, a member of the class of large GEFs. The inhibitory effect demonstrated in vitro clearly is observed as well in vivo, based on the finding that M69 produces similar results as a dominant-negative, GEF-deficient mutant of cytohesin 1: when expressed in the cytoplasm of T-cells, M69 reduces stimulated adhesion to intercellular adhesion molecule-1 and results in a dramatic reorganization of F-actin distribution. These highly specific cellular effects suggest that the ARF-GEF activity of cytohesin 1 plays an important role in cytoskeletal remodeling events of lymphoid cells.

Linking osteopetrosis and pycnodysostosis: Regulation of cathepsin K expression by the microphthalmia transcription factor family

Various genetic conditions produce dysfunctional osteoclasts resulting in osteopetrosis or osteosclerosis. These include human pycnodysostosis, an autosomal recessive syndrome caused by cathepsin K mutation, cathepsin K-deficient mice, and mitf mutant rodent strains. Cathepsin K is a highly expressed cysteine protease in osteoclasts that plays an essential role in the degradation of protein components of bone matrix. Cathepsin K also is expressed in a significant fraction of human breast cancers where it could contribute to tumor invasiveness. Mitf is a member of a helix–loop–helix transcription factor subfamily, which contains the potential dimerization partners TFE3, TFEB, and TFEC. In mice, dominant negative, but not recessive, mutations of mitf, produce osteopetrosis, suggesting a functional requirement for other family members. Mitf also has been found—and TFE3 has been suggested—to modulate age-dependent changes in osteoclast function. This study identifies cathepsin K as a transcriptional target of Mitf and TFE3 via three consensus elements in the cathepsin K promoter. Additionally, cathepsin K mRNA and protein were found to be deficient in mitf mutant osteoclasts, and overexpression of wild-type Mitf dramatically up-regulated expression of endogenous cathepsin K in cultured human osteoclasts. Cathepsin K promoter activity was disrupted by dominant negative, but not recessive, mouse alleles of mitf in a pattern that closely matches their osteopetrotic phenotypes. This relationship between cathepsin K and the Mitf family helps explain the phenotypic overlap of their corresponding deficiencies in pycnodysostosis and osteopetrosis and identifies likely regulators of cathepsin K expression in bone homeostasis and human malignancy.

Activation of NF-κB is required for hypertrophic growth of primary rat neonatal ventricular cardiomyocytes

The transcription factor NF-κB regulates expression of genes that are involved in inflammation, immune response, viral infection, cell survival, and division. However, the role of NF-κB in hypertrophic growth of terminally differentiated cardiomyocytes is unknown. Here we report that NF-κB activation is required for hypertrophic growth of cardiomyocytes. In cultured rat primary neonatal ventricular cardiomyocytes, the nuclear translocation of NF-κB and its transcriptional activity were stimulated by several hypertrophic agonists, including phenylephrine, endothelin-1, and angiotensin II. The activation of NF-κB was inhibited by expression of a “supersuppressor” IκBα mutant that is resistant to stimulation-induced degradation and a dominant negative IκB kinase (IKKβ) mutant that can no longer be activated by phosphorylation. Furthermore, treatment with phenylephrine induced IκBα degradation in an IKK-dependent manner, suggesting that NF-κB is a downstream target of the hypertrophic agonists. Importantly, expression of the supersuppressor IκBα mutant or the dominant negative IKKβ mutant blocked the hypertrophic agonist-induced expression of the embryonic gene atrial natriuretic factor and enlargement of cardiomyocytes. Conversely, overexpression of NF-κB itself induced atrial natriuretic factor expression and cardiomyocyte enlargement. These findings suggest that NF-κB plays a critical role in the hypertrophic growth of cardiomyocytes and may serve as a potential target for the intervention of heart disease.

Hyaluronan Activates Cell Motility of v-Src-transformed Cells via Ras-Mitogen–activated Protein Kinase and Phosphoinositide 3-Kinase-Akt in a Tumor-specific Manner

We investigated the production of hyaluronan (HA) and its effect on cell motility in cells expressing the v-src mutants. Transformation of 3Y1 by v-src virtually activated HA secretion, whereas G2A v-src, a nonmyristoylated form of v-src defective in cell transformation, had no effect. In cells expressing the temperature-sensitive mutant of v-Src, HA secretion was temperature dependent. In addition, HA as small as 1 nM, on the other side, activated cell motility in a tumor-specific manner. HA treatment strongly activated the motility of v-Src–transformed 3Y1, whereas it showed no effect on 3Y1- and 3Y1-expressing G2A v-src. HA-dependent cell locomotion was strongly blocked by either expression of dominant-negative Ras or treatment with a Ras farnesyltransferase inhibitor. Similarly, both the MEK1 inhibitor and the kinase inhibitor clearly inhibited HA-dependent cell locomotion. In contrast, cells transformed with an active MEK1 did not respond to the HA. Finally, an anti-CD44–neutralizing antibody could block the activation of cell motility by HA as well as the HA-dependent phosphorylation of mitogen-activated protein kinase and Akt. Taken together, these results suggest that simultaneous activation of the Ras-mitogen-activated protein kinase pathway and the phosphoinositide 3-kinase pathway by the HA-CD44 interaction is required for the activation of HA-dependent cell locomotion in v-Src–transformed cells.

CrkII signals from epidermal growth factor receptor to Ras.

A rat fibroblast mutant defective in oncogenic transformation and signaling from epidermal growth factor receptor to Ras has been isolated. The mutant contains dominant negative-type point mutations in the C-terminal SH3 domain of one crkII gene. Among the adapters tested, the mutant is complemented only by crkII cDNA. Expression of the mutated crkII in parent cells generates the phenotype indistinguishable from the mutant cell. Yet overexpression or reduced expression of Grb2 in the mutant before and after complementation with crkII have little effect on its phenotype. We conclude that adapter molecules are highly specific and that the oncogenic growth signal from epidermal growth factor receptor to Ras is predominantly mediated by CrkII in rat fibroblast.

Tumor necrosis factor receptor associated factor 2 is a mediator of NF-kappa B activation by latent infection membrane protein 1, the Epstein-Barr virus transforming protein.

Latent infection membrane protein 1 (LMP1), the Epstein-Barr virus transforming protein, associates with tumor necrosis factor receptor (TNFR) associated factor 1 (TRAF1) and TRAF3. Since TRAF2 has been implicated in TNFR-mediated NF-kappa B activation, we have evaluated the role of TRAF2 in LMP1-mediated NF-kappa B activation. TRAF2 binds in vitro to the LMP1 carboxyl-terminal cytoplasmic domain (CT), coprecipitates with LMP1 in B lymphoblasts, and relocalizes to LMP1 plasma membrane patches. A dominant negative TRAF2 deletion mutant that lacks amino acids 6-86 (TRAF/ delta 6-86) inhibits NF-kappa B activation from the LMP1 CT and competes with TRAF2 for LMP1 binding. TRAF2 delta 6-86 inhibits NF-kappa B activation mediated by the first 45 amino acids of the LMP1 CT by more than 75% but inhibits NF-kappa B activation through the last 55 amino acids of the CT by less than 40%. A TRAF interacting protein, TANK, inhibits NF-kappa B activation by more than 70% from both LMP1 CT domains. These data implicate TRAF2 aggregation in NF-kappa B activation by the first 45 amino acids of the LMP1 CT and suggest that a different TRAF-related pathway may be involved in NF-kappa B activation by the last 55 amino acids of the LMP1 CT.

The role of CD4-Lck in T-cell receptor antagonism: evidence for negative signaling.

Small changes in the complex between a peptide and a molecule of the major histocompatibility complex generate ligands able to partially activate (partial agonist) or even inhibit (antagonist) T-cell functions. T-cell receptor engagement of antagonist complex results in a partial zeta chain phosphorylation without activation of the associated ZAP-70 kinase. Herein we show that, despite a strong inhibition of both inositol phospholipid hydrolysis and extracellular increasing antagonist concentrations increased the activity of the CD4-Lck kinase. Addition of anti-CD4 antibody to culture medium prevented inhibitory effects induced by antagonist ligand. We propose that CD4-Lck activation triggered by antagonist complexes may act in a dominant negative mode, thus overriding stimulatory signals coming from agonist ligand. These findings identify a new T-cell signaling profile that may explain the ability of some T-cell receptor variant ligands to inhibit specific biological activities or trigger alternative activation programs.

Spectrum of HERG K+-channel dysfunction in an inherited cardiac arrhythmia.

Long QT syndrome (LQT) is an autosomal dominant disorder that can cause sudden death from cardiac arrhythmias. We recently discovered that mutations in HERG, a K+-channel gene, cause chromosome 7-linked LQT. Heterologous expression of HERG in Xenopus oocytes revealed that HERG current was similar to a well-characterized cardiac delayed rectifier K+ current, IKr, and led to the hypothesis that mutations in HERG reduced IKr, causing prolonged myocellular action potentials. To define the mechanism of LQT, we injected oocytes with mutant HERG complementary RNAs, either singly or in combination with wild-type complementary RNA. Some mutations caused loss of function, whereas others caused dominant negative suppression of HERG function. These mutations are predicted to cause a spectrum of diminished IKr and delayed ventricular repolarization, consistent with the prolonged QT interval observed in individuals with LQT.

Truncated WT1 mutants alter the subnuclear localization of the wild-type protein.

WT1 encodes a zinc-finger protein, expressed as distinct isoforms, that is inactivated in a subset of Wilms tumors. Both constitutional and somatic mutations disrupting the DNA-binding domain of WT1 result in a potentially dominant-negative phenotype. In generating inducible cell lines expressing wild-type isoforms of WT1 and WT1 mutants, we observed dramatic differences in the subnuclear localization of the induced proteins. The WT1 isoform that binds with high affinity to a defined DNA target, WT1(-KTS), was diffusely localized throughout the nucleus. In contrast, expression of an alternative splicing variant with reduced DNA binding affinity, WT1 (+KTS), or WT1 mutants with a disrupted zinc-finger domain resulted in a speckled pattern of expression within the nucleus. Although similar in appearance, the localization of WT1 variants to subnuclear clusters was clearly distinct from that of the essential splicing factor SC35, suggesting that WT1 is not directly involved in pre-mRNA splicing. Localization to subnuclear clusters required the N terminus of WT1, and coexpression of a truncated WT1 mutant and wild-type WT1(-KTS) resulted in their physical association, the redistribution of WT1(-KTS) from a diffuse to a speckled pattern, and the inhibition of its transactivational activity. These observations suggest that different WT1 isoforms and WT1 mutants have distinct subnuclear compartments. Dominant-negative WT1 proteins physically associate with wild-type WT1 in vivo and may result in its sequestration within subnuclear structures.

Critical role of the interleukin 2 (IL-2) receptor gamma-chain-associated Jak3 in the IL-2-induced c-fos and c-myc, but not bcl-2, gene induction.

The interleukin 2 receptor (IL-2R) consists of three subunits, the IL-2R alpha, IL-2R beta c, and IL-2R gamma c chains. Two Janus family protein tyrosine kinases (PTKs), Jak1 and Jak3, were shown to associate with IL-2R beta c and IL-2R gamma c, respectively, and their PTK activities are increased after IL-2 stimulation. A Jak3 mutant with truncation of the C-terminal PTK domain lacks its intrinsic kinase activity but can still associate with IL-2R gamma c. In a hematopoietic cell line, F7, that responds to either IL-2 or IL-3, overexpression of this Jak3 mutant results in selective inhibition of the IL-2-induced activation of Jak1/Jak3 PTKs and of cell proliferation. Of the three target nuclear protooncogenes of the IL-2 signaling, c-fos and c-myc genes, but not the bcl-2 gene, were found to be impaired. On the other hand, overexpression of the dominant negative form of the IL-2R gamma c chain, which lacks most of its cytoplasmic domain, in F7 cells resulted in the inhibition of all three protooncogenes. These results provide a further molecular basis for the critical role of Jak3 in IL-2 signaling and also suggest a Jak PTK-independent signaling pathway(s) for the bcl-2 gene induction by IL-2R.

Involvement of the double-stranded-RNA-dependent kinase PKR in interferon expression and interferon-mediated antiviral activity.

The signaling mechanisms responsible for the induced expression of interferon (IFN) genes by viral infection or double-stranded RNA (dsRNA) are not well understood. Here we investigate the role of the interferon-induced dsRNA-dependent protein kinase PKR in the regulation of IFN induction. Biological activities attributed to PKR include regulating protein synthesis, mediating IFN actions, and functioning as a possible tumor suppressor. Since binding of dsRNA is required for its activation, PKR has been considered as a candidate signal transducer for regulating IFN expression. To examine this role of PKR, loss-of-function phenotypes in stable transformants of promonocytic U-937 cells were achieved by two different strategies, overexpression of an antisense PKR transcript or a dominant negative PKR mutant gene. Both types of PKR-deficient cells were more permissive for viral replication than the control U-937 cells. As the result of PKR loss, they also showed impaired induction of IFN-alpha and IFN-beta genes in response to several inducers--specifically, encephalomyocarditis virus, lipopolysaccharide, and phorbol 12-myristate 13-acetate. Interestingly, while IFN-alpha induction by dsRNA was impaired in PKR-deficient cells, IFN-beta induction remained intact. Loss of PKR function also resulted in decreased antiviral activity as elicited by IFN-alpha and, to a greater extent, by IFN-gamma. These results implicate PKR in the regulation of several antiviral activities.

Identification of four acidic amino acids that constitute the catalytic center of the RuvC Holliday junction resolvase.

Escherichia coli RuvC protein is a specific endonuclease that resolves Holliday junctions during homologous recombination. Since the endonucleolytic activity of RuvC requires a divalent cation and since 3 or 4 acidic residues constitute the catalytic centers of several nucleases that require a divalent cation for the catalytic activity, we examined whether any of the acidic residues of RuvC were required for the nucleolytic activity. By site-directed mutagenesis, we constructed a series of ruvC mutant genes with similar amino acid replacements in 1 of the 13 acidic residues. Among them, the mutant genes with an alteration at Asp-7, Glu-66, Asp-138, or Asp-141 could not complement UV sensitivity of a ruvC deletion strain, and the multicopy mutant genes showed a dominant negative phenotype when introduced into a wild-type strain. The products of these mutant genes were purified and their biochemical properties were studied. All of them retained the ability to form a dimer and to bind specifically to a synthetic Holliday junction. However, they showed no, or extremely reduced, endonuclease activity specific for the junction. These 4 acidic residues, which are dispersed in the primary sequence, are located in close proximity at the bottom of the putative DNA binding cleft in the three-dimensional structure. From these results, we propose that these 4 acidic residues constitute the catalytic center for the Holliday junction resolvase and that some of them play a role in coordinating a divalent metal ion in the active center.

Rescue of defective mitogenic signaling by D-type cyclins.

Three gene products, including Myc and the D- and E-type G1 cyclins, are rate limiting for G1 progression in mammalian fibroblasts. Quiescent mouse NIH 3T3 fibroblasts engineered to express a mutant colony-stimulating factor (CSF-1) receptor (CSF-1R 809F) fail to synthesize c-myc and cyclin D1 mRNAs upon CSF-1 stimulation and remain arrested in early G1 phase. Ectopic expression of c-myc or either of three D-type cyclin genes, but not cyclin E, resensitized these cells to the mitogenic effects of CSF-1, enabling them to proliferate continuously in liquid culture and to form colonies in agar in response to the growth factor. Rescue by cyclin D1 was enhanced by c-myc but not by cyclin E and was reversed by infecting cyclin D1-reconstituted cells with a retroviral vector encoding catalytically inactive cyclin-dependent kinase 4. Induction of cyclin D1 mRNA by CSF-1 was restored in cells forced to express c-myc, and vice versa, suggesting that expression of the two genes is interdependent. Cells reconstituted with c-myc were prevented from entering S phase when microinjected with a monoclonal antibody to cyclin D1, and conversely, those rescued by cyclin D1 were inhibited from forming CSF-1-dependent colonies when challenged with a dominant-negative c-myc mutant. Cyclin D mutants defective in binding to the retinoblastoma protein were impaired in rescuing mitogenic signaling. Therefore, Myc and D-type cyclins collaborate during the mitogenic response to CSF-1, whereas cyclin E functions in a separate pathway.

Oligomerization of CD4 is required for stable binding to class II major histocompatibility complex proteins but not for interaction with human immunodeficiency virus gp120.

Previous studies have failed to detect an interaction between monomeric soluble CD4 (sCD4) and class II major histocompatibility complex (MHC) proteins, suggesting that oligomerization of CD4 on the cell surface may be required to form a stable class II MHC binding site. To test this possibility, we transfected the F43I CD4 mutant, which is incapable of binding to class II MHC or human immunodeficiency virus (HIV) gp120, into COS-7 cells together with wild-type CD4 (wtCD4). Expression of F43I results in a dominant negative effect: no class II MHC binding is observed even though wtCD4 expression is preserved. Apparently, F43I associates with wtCD4 oligomers and interferes with the formation of functional class II MHC binding structures. In contrast, F43I does not affect the binding of gp120 to wtCD4, implying that gp120 binds to a CD4 monomer. By production and characterization of chimeric CD4 molecules, we show that domains 3 and/or 4 appear to be involved in oligomerization. Several models of the CD4-class II MHC interaction are offered, including the possibility that one or two CD4 molecules initially interact with class II MHC dimers and further associate to create larger complexes important for facilitating T-cell receptor crosslinking.

Functional domain analysis of glass, a zinc-finger-containing transcription factor in Drosophila.

The glass gene is required for proper photo-receptor differentiation during development of the Drosophila eye glass codes for a DNA-binding protein containing five zinc fingers that we show is a transcriptional activator. A comparison of the sequences of the glass genes from two species of Drosophila and a detailed functional domain analysis of the Drosophila melanogaster glass gene reveal that both the DNA-binding domain and the transcriptional-activation domain are highly conserved between the two species. Analysis of the DNA-binding domain of glass indicates that the three carboxyl-terminal zinc fingers alone are necessary and sufficient for DNA binding. We also show that a deletion mutant of glass containing only the DNA-binding domain can behave in a dominant-negative manner both in vivo and in a cell culture assay that measures transcriptional activation.