Nuclear-cytoplasmic shuttling of C-ABL tyrosine kinase

The ubiquitously expressed nonreceptor tyrosine kinase c-Abl contains three nuclear localization signals, however, it is found in both the nucleus and the cytoplasm of proliferating fibroblasts. A rapid and transient loss of c-Abl from the nucleus is observed upon the initial adhesion of fibroblasts onto a fibronectin matrix, suggesting the possibility of nuclear export [Lewis, J., Baskaran, R., Taagepera, S., Schwartz, M. & Wang, J. (1996) Proc. Natl. Acad. Sci. USA 93, 15174–15179]. Here we show that the C terminus of c-Abl does indeed contain a functional nuclear export signal (NES) with the characteristic leucine-rich motif. The c-Abl NES can functionally complement an NES-defective HIV Rev protein (RevΔ3NI) and can mediate the nuclear export of glutathione-S-transferase. The c-Abl NES function is sensitive to the nuclear export inhibitor leptomycin B. Mutation of a single leucine (L1064A) in the c-Abl NES abrogates export function. The NES-mutated c-Abl, termed c-Abl NES(−), is localized exclusively to the nucleus. Treatment of cells with leptomycin B also leads to the nuclear accumulation of wild-type c-Abl protein. The c-Abl NES(−) is not lost from the nucleus when detached fibroblasts are replated onto fibronectin matrix. Taken together, these results demonstrate that c-Abl shuttles continuously between the nucleus and the cytoplasm and that the rate of nuclear import and export can be modulated by the adherence status of fibroblastic cells.

A cytosolic activity distinct from Crm1 mediates nuclear export of protein kinase inhibitor in permeabilized cells

The leucine-rich nuclear export signal (NES) is used by a variety of proteins to facilitate their delivery from the nucleus to the cytoplasm. One of the best-studied examples, protein kinase inhibitor (PKI), binds to the catalytic subunit of protein kinase A in the nucleus and mediates its rapid export to the cytoplasm. We developed a permeabilized cell assay that reconstitutes nuclear export mediated by PKI, and we used it to characterize the cytosolic factors required for this process. The two-step assay involves an import phase and an export phase, and quantitation is achieved by digital fluorescence microscopy. During the import phase, a fluorescent derivative of streptavidin is imported into the nuclei of digitonin-permeabilized HeLa cells. During the export phase, biotinylated PKI diffuses into the nucleus, binds to fluorescent streptavidin, and mediates export of the complex to the cytoplasm. Nuclear export of the PKI complex is cytosol dependent and can be stimulated by addition of the purified NES receptor, Crm1. HeLa cell cytosol treated with N-ethylmaleimide (NEM) or phenyl-Sepharose to inactivate or deplete Crm1, respectively, is still fully active in the PKI export assay. Significantly, the export activity can be depleted from cytosol by preadsorption with a protein conjugate that contains a functional NES. These data indicate that cytosol contains an export activity that is distinct from Crm1 and is likely to correspond to an NES receptor.

Functional Dissection and Hierarchy of Tubulin-folding Cofactor Homologues in Fission Yeast

We describe the isolation of fission yeast homologues of tubulin-folding cofactors B (Alp11) and E (Alp21), which are essential for cell viability and the maintenance of microtubules. Alp11B contains the glycine-rich motif (the CLIP-170 domain) involved in microtubular functions, whereas, unlike mammalian cofactor E, Alp21E does not. Both mammalian and yeast cofactor E, however, do contain leucine-rich repeats. Immunoprecipitation analysis shows that Alp11B interacts with both α-tubulin and Alp21E, but not with the cofactor D homologue Alp1, whereas Alp21E also interacts with Alp1D. The cellular amount of α-tubulin is decreased in both alp1 and alp11 mutants. Overproduction of Alp11B results in cell lethality and the disappearance of microtubules, which is rescued by co-overproduction of α-tubulin. Both full-length Alp11B and the C-terminal third containing the CLIP-170 domain localize in the cytoplasm, and this domain is required for efficient binding to α-tubulin. Deletion of alp11 is suppressed by multicopy plasmids containing either alp21+ or alp1+, whereas alp21 deletion is rescued by overexpression of alp1+ but not alp11+. Finally, the alp1 mutant is not complemented by either alp11+ or alp21+. The results suggest that cofactors operate in a linear pathway (Alp11B-Alp21E-Alp1D), each with distinct roles.

Defining Extracellular Integrin α-Chain Sites That Affect Cell Adhesion and Adhesion Strengthening without Altering Soluble Ligand Binding

It was previously shown that mutations of integrin α4 chain sites, within putative EF-hand-type divalent cation-binding domains, each caused a marked reduction in α4β1-dependent cell adhesion. Some reports have suggested that α-chain “EF-hand” sites may interact directly with ligands. However, we show here that mutations of three different α4 “EF-hand” sites each had no effect on binding of soluble monovalent or bivalent vascular cell adhesion molecule 1 whether measured indirectly or directly. Furthermore, these mutations had minimal effect on α4β1-dependent cell tethering to vascular cell adhesion molecule 1 under shear. However, EF-hand mutants did show severe impairments in cellular resistance to detachment under shear flow. Thus, mutation of integrin α4 “EF-hand-like” sites may impair 1) static cell adhesion and 2) adhesion strengthening under shear flow by a mechanism that does not involve alterations of initial ligand binding.

Transfected Drosophila cells as a probe for defining the minimal requirements for stimulating unprimed CD8+ T cells

Stimulation of naive T cells by antigen-presenting cells (APC) is thought to involve two qualitatively different signals: signal one results from T-cell receptor (TCR) recognition of antigenic peptides bound to major histocompatibility complex (MHC) molecules, whereas signal two reflects contact with one or more costimulatory molecules. The requirements for stimulating naive T cells were studied with MHC class I-restricted CD8+ T cells from a T-cell receptor transgenic line, with defined peptides as antigen and transfected Drosophila cells as APC. Three main findings are reported. First, stimulation of naive T cells via signal one alone (MHC plus peptide) was essentially nonimmunogenic; thus T cells cultured with peptides presented by MHC class I-transfected Drosophila APC lacking costimulatory molecules showed little or no change in their surface phenotype. Second, cotransfection of two costimulatory molecules, B7-1 and intercellular adhesion molecule 1 (ICAM-1), converted class I+ Drosophila cells to potent APC capable of inducing strong T-proliferative responses and cytokine (interleukin 2) production. Third, B7-1 and ICAM-1 acted synergistically, indicating that signal two is complex; synergy between B7-1 and ICAM-1 varied from moderate to extreme and was influenced by both the dose and affinity of the peptide used and the parameter of T-cell activation studied. Transfected Drosophila cells are thus a useful tool for examining the minimal APC requirements for naive T cells.

Molecular recognition of pathogen attack occurs inside of plant cells in plant disease resistance specified by the Arabidopsis genes RPS2 and RPM1

The Arabidopsis thaliana disease resistance genes RPS2 and RPM1 belong to a class of plant disease resistance genes that encode proteins that contain an N-terminal tripartite nucleotide binding site (NBS) and a C- terminal tandem array of leucine-rich repeats. RPS2 and RPM1 confer resistance to strains of the bacterial phytopathogen Pseudomonas syringae carrying the avirulence genes avrRpt2 and avrB, respectively. In these gene-for-gene relationships, it has been proposed that pathogen avirulence genes generate specific ligands that are recognized by cognate receptors encoded by the corresponding plant resistance genes. To test this hypothesis, it is crucial to know the site of the potential molecular recognition. Mutational analysis of RPS2 protein and in vitro translation/translocation studies indicated that RPS2 protein is localized in the plant cytoplasm. To determine whether avirulence gene products themselves are the ligands for resistance proteins, we expressed the avrRpt2 and avrB genes directly in plant cells using a novel quantitative transient expression assay, and found that expression of avrRpt2 and avrB elicited a resistance response in plants carrying the corresponding resistance genes. This observation indicates that no bacterial factors other than the avirulence gene products are required for the specific resistance response as long as the avirulence gene products are correctly localized. We propose that molecular recognition of P. syringae in RPS2- and RPM1-specified resistance occurs inside of plant cells.

Enzyme plus light therapy to repair DNA damage in ultraviolet-B-irradiated human skin

Ultraviolet-B (UVB) (290–320 nm) radiation-induced cyclobutane pyrimidine dimers within the DNA of epidermal cells are detrimental to human health by causing mutations and immunosuppressive effects that presumably contribute to photocarcinogenesis. Conventional photoprotection by sunscreens is exclusively prophylactic in nature and of no value once DNA damage has occurred. In this paper, we have therefore assessed whether it is possible to repair UVB radiation-induced DNA damage through topical application of the DNA-repair enzyme photolyase, derived from Anacystis nidulans, that specifically converts cyclobutane dimers into their original DNA structure after exposure to photoreactivating light. When a dose of UVB radiation sufficient to induce erythema was administered to the skin of healthy subjects, significant numbers of dimers were formed within epidermal cells. Topical application of photolyase-containing liposomes to UVB-irradiated skin and subsequent exposure to photoreactivating light decreased the number of UVB radiation-induced dimers by 40–45%. No reduction was observed if the liposomes were not filled with photolyase or if photoreactivating exposure preceded the application of filled liposomes. The UVB dose administered resulted in suppression of intercellular adhesion molecule-1 (ICAM-1), a molecule required for immunity and inflammatory events in the epidermis. In addition, in subjects hypersensitive to nickel sulfate, elicitation of the hypersensitivity reaction in irradiated skin areas was prevented. Photolyase-induced dimer repair completely prevented these UVB radiation-induced immunosuppressive effects as well as erythema and sunburn-cell formation. These studies demonstrate that topical application of photolyase is effective in dimer reversal and thereby leads to immunoprotection.

Interaction of the poliovirus receptor with poliovirus

The structure of the extracellular, three-domain poliovirus receptor (CD155) complexed with poliovirus (serotype 1) has been determined to 22-Å resolution by means of cryo-electron microscopy and three-dimensional image-reconstruction techniques. Density corresponding to the receptor was isolated in a difference electron density map and fitted with known structures, homologous to those of the three individual CD155 Ig-like domains. The fit was confirmed by the location of carbohydrate moieties in the CD155 glycoprotein, the conserved properties of elbow angles in the structures of cell surface molecules with Ig-like folds, and the concordance with prior results of CD155 and poliovirus mutagenesis. CD155 binds in the poliovirus “canyon” and has a footprint similar to that of the intercellular adhesion molecule-1 receptor on human rhinoviruses. However, the orientation of the long, slender CD155 molecule relative to the poliovirus surface is quite different from the orientation of intercellular adhesion molecule-1 on rhinoviruses. In addition, the residues that provide specificity of recognition differ for the two receptors. The principal feature of receptor binding common to these two picornaviruses is the site in the canyon at which binding occurs. This site may be a trigger for initiation of the subsequent uncoating step required for viral infection.

The Arabidopsis thaliana RPM1 disease resistance gene product is a peripheral plasma membrane protein that is degraded coincident with the hypersensitive response

Disease resistance in plants is often controlled by a gene-for-gene mechanism in which avirulence (avr) gene products encoded by pathogens are specifically recognized, either directly or indirectly, by plant disease resistance (R) gene products. Members of the NBS-LRR class of R genes encode proteins containing a putative nucleotide binding site (NBS) and carboxyl-terminal leucine-rich repeats (LRRs). Generally, NBS-LRR proteins do not contain predicted transmembrane segments or signal peptides, suggesting they are soluble cytoplasmic proteins. RPM1 is an NBS-LRR protein from Arabidopsis thaliana that confers resistance to Pseudomonas syringae expressing either avrRpm1 or avrB. RPM1 protein was localized by using an epitope tag. In contrast to previous suggestions, RPM1 is a peripheral membrane protein that likely resides on the cytoplasmic face of the plasma membrane. Furthermore, RPM1 is degraded coincident with the onset of the hypersensitive response, suggesting a negative feedback loop controlling the extent of cell death and overall resistance response at the site of infection.

An isolated, surface-expressed I domain of the integrin αLβ2 is sufficient for strong adhesive function when locked in the open conformation with a disulfide bond

We introduced disulfide bonds to lock the integrin αLβ2 I domain in predicted open, ligand binding or closed, nonbinding conformations. Transfectants expressing αLβ2 heterodimers containing locked-open but not locked-closed or wild-type I domains constitutively adhered to intercellular adhesion molecule-1 (ICAM-1) substrates. Locking the I domain closed abolished constitutive and activatable adhesion. The isolated locked-open I domain bound as well as the activated αLβ2 heterodimer, and binding was abolished by reduction of the disulfide. Lovastatin, which binds under the conformationally mobile C-terminal α-helix of the I domain, inhibited binding to ICAM-1 by αLβ2 with wild-type, but not locked-open I domains. These data establish the importance of conformational change in the αL I domain for adhesive function and show that this domain is sufficient for full adhesive activity.

Locking in alternate conformations of the integrin αLβ2 I domain with disulfide bonds reveals functional relationships among integrin domains

We used integrin αLβ2 heterodimers containing I domains locked open (active) or closed (inactive) with disulfide bonds to investigate regulatory interactions among domains in integrins. mAbs to the αL I domain and β2 I-like domain inhibit adhesion of wild-type αLβ2 to intercellular adhesion molecule-1. However, with αLβ2 containing a locked open I domain, mAbs to the I domain were subdivided into subsets (i) that did not inhibit, and thus appear to inhibit by favoring the closed conformation, and (ii) that did inhibit, and thus appear to bind to the ligand binding site. Furthermore, αLβ2 containing a locked open I domain was completely resistant to inhibition by mAbs to the β2 I-like domain, but became fully susceptible to inhibition after disulfide reduction with DTT. This finding suggests that the I-like domain indirectly contributes to ligand binding by regulating opening of the I domain in wild-type αLβ2. Conversely, locking the I domain closed partially restrained conformational change of the I-like domain by Mn2+, as measured with mAb m24, which we map here to the β2 I-like domain. By contrast, locking the I domain closed or open did not affect constitutive or Mn2+-induced exposure of the KIM127 epitope in the β2 stalk region. Furthermore, locked open I domains, in αLβ2 complexes or expressed in isolation on the cell surface, bound to intercellular adhesion molecule-1 equivalently in Mg2+ and Mn2+. These results suggest that Mn2+ activates αLβ2 by binding to a site other than the I domain, most likely the I-like domain of β2.

Nerve growth factor control of neuronal expression of angiogenetic and vasoactive factors

In postnatal tissues, angiogenesis occurs in nontumoral conditions on appropriate stimuli. In the nervous tissue, hypoxia, neural graft, increased neural function, and synaptic activity are associated with neoangiogenesis. We have investigated the occurrence of neoangiogenesis in the superior cervical ganglia (scg) of newborn rats treated for 8–21 days with 6-hydroxy-dopamine (6-OHDA), nerve growth factor (NGF), or 6-OHDA + NGF. The two latter treatments induced a significant increase in scg size. However, the increase after combined treatment far exceeded that of NGF alone. Similarly, histological and histochemical analysis revealed neuronal hypertrophy and endothelial cell hyperplasia associated with stromal hypertrophy (as described by laminin immunostaining) and increased vascular bed (as revealed by platelet/endothelial cell adhesion molecule-1 immunostaining) in 6-OHDA + NGF-treated pups. NGF, either alone or associated with 6-OHDA, also induced a significant up-regulation of NADPH diaphorase, neuronal nitric oxide synthase, and vascular endothelial growth factor expression in scg neurons. The present investigation suggests that the increase of scg size induced by NGF and 6-OHDA + NGF is associated with neoangiogenesis, and that the induction of vasoactive and angiogenic factors in neurons represents a further and previously undisclosed effect of NGF.

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.

Reversibly locking a protein fold in an active conformation with a disulfide bond: Integrin αL I domains with high affinity and antagonist activity in vivo

The integrin αLβ2 has three different domains in its headpiece that have been suggested to either bind ligand or to regulate ligand binding. One of these, the inserted or I domain, has a fold similar to that of small G proteins. The I domain of the αM and α2 subunits has been crystallized in both open and closed conformations; however, the αL I domain has been crystallized in only the closed conformation. We hypothesized that the αL domain also would have an open conformation, and that this would be the ligand binding conformation. Therefore, we introduced pairs of cysteine residues to form disulfides that would lock the αL I domain in either the open or closed conformation. Locking the I domain open resulted in a 9,000-fold increase in affinity to intercellular adhesion molecule-1 (ICAM-1), which was reversed by disulfide reduction. By contrast, the affinity of the locked closed conformer was similar to wild type. Binding completely depended on Mg2+. Orders of affinity were ICAM-1 > ICAM-2 > ICAM-3. The kon, koff, and KD values for the locked open I domain were within 1.5-fold of values previously determined for the αLβ2 complex, showing that the I domain is sufficient for full affinity binding to ICAM-1. The locked open I domain antagonized αLβ2-dependent adhesion in vitro, lymphocyte homing in vivo, and firm adhesion but not rolling on high endothelial venules. The ability to reversibly lock a protein fold in an active conformation with dramatically increased affinity opens vistas in therapeutics and proteomics.

Genetic complexity of pathogen perception by plants: The example of Rcr3, a tomato gene required specifically by Cf-2

Genetic analysis of plant–pathogen interactions has demonstrated that resistance to infection is often determined by the interaction of dominant plant resistance (R) genes and dominant pathogen-encoded avirulence (Avr) genes. It was postulated that R genes encode receptors for Avr determinants. A large number of R genes and their cognate Avr genes have now been analyzed at the molecular level. R gene loci are extremely polymorphic, particularly in sequences encoding amino acids of the leucine-rich repeat motif. A major challenge is to determine how Avr perception by R proteins triggers the plant defense response. Mutational analysis has identified several genes required for the function of specific R proteins. Here we report the identification of Rcr3, a tomato gene required specifically for Cf-2-mediated resistance. We propose that Avr products interact with host proteins to promote disease, and that R proteins “guard” these host components and initiate Avr-dependent plant defense responses.