Interferon gamma signals via a high-affinity multisubunit receptor complex that contains two types of polypeptide chain.

Signaling by interferon gamma (IFN-gamma) requires two structurally related cell surface proteins: a ligand-binding polypeptide, known as the IFN-gamma receptor (IFN-gamma R), and an accessory factor. However, it is not known whether IFN-gamma forms a ternary complex with the IFN-gamma R and accessory factor to initiate signaling. Here we demonstrate complex formation between IFN-gamma and the two proteins, both in solution and at the cell surface. We observe complexes containing ligand, two molecules of IFN-gamma R (designated the IFN-gamma R alpha chain), and one or two molecules of accessory factor (designated the IFN-gamma R beta chain). Transfected cells expressing both IFN-gamma R chains bind IFN-gamma with higher affinity than do cells expressing alpha chain alone. Anti-beta-chain antibodies prevent the beta chain from participating in the ligand-receptor complex, reduce the affinity for IFN-gamma, and block signaling. Soluble alpha- or beta-chain extracellular domains also inhibit function. These results demonstrate that IFN-gamma signals via a high-affinity multisubunit complex that contains two types of receptor chain and suggest a potential approach to inhibiting specific actions of IFN-gamma by blocking the association of receptor subunits.

In silico detection of control signals: mRNA 3′-end-processing sequences in diverse species

We have investigated mRNA 3′-end-processing signals in each of six eukaryotic species (yeast, rice, arabidopsis, fruitfly, mouse, and human) through the analysis of more than 20,000 3′-expressed sequence tags. The use and conservation of the canonical AAUAAA element vary widely among the six species and are especially weak in plants and yeast. Even in the animal species, the AAUAAA signal does not appear to be as universal as indicated by previous studies. The abundance of single-base variants of AAUAAA correlates with their measured processing efficiencies. As found previously, the plant polyadenylation signals are more similar to those of yeast than to those of animals, with both common content and arrangement of the signal elements. In all species examined, the complete polyadenylation signal appears to consist of an aggregate of multiple elements. In light of these and previous results, we present a broadened concept of 3′-end-processing signals in which no single exact sequence element is universally required for processing. Rather, the total efficiency is a function of all elements and, importantly, an inefficient word in one element can be compensated for by strong words in other elements. These complex patterns indicate that effective tools to identify 3′-end-processing signals will require more than consensus sequence identification.

Transduction of Basolateral-to-Apical Signals across Epithelial Cells: Ligand-stimulated Transcytosis of the Polymeric Immunoglobulin Receptor Requires Two Signals

Transcytosis of the polymeric immunoglobulin receptor (pIgR) is stimulated by binding of its ligand, dimeric IgA (dIgA). During this process, dIgA binding at the basolateral surface of the epithelial cell transmits a signal to the apical region of the cell, which in turn stimulates the transport of dIgA–pIgR complex from a postmicrotubule compartment to the apical surface. We have previously reported that the signal of stimulation was controlled by a protein-tyrosine kinase (PTK) activated upon dIgA binding. We now show that this signal of stimulation moves across the cell independently of pIgR movement or microtubules and acts through the tyrosine kinase activity by releasing Ca++ from inositol trisphosphate–sensitive intracellular stores. Surprisingly we have found that a second independent signal is required to achieve dIgA-stimulated transcytosis of pIgR. This second signal depends on dIgA binding to the pIgR solely at the basolateral surface and the ability of pIgR to dimerize. This enables pIgR molecules that have bound dIgA at the basolateral surface to respond to the signal of stimulation once they reach the postmicrotubule compartment. We propose that the use of two signals may be a general mechanism by which signaling receptors maintain specificity along their signaling and trafficking pathways.

A Novel Fluorescence-based Genetic Strategy Identifies Mutants of Saccharomyces cerevisiae Defective for Nuclear Pore Complex Assembly

Nuclear pore complexes (NPCs) are large proteinaceous portals for exchanging macromolecules between the nucleus and the cytoplasm. Revealing how this transport apparatus is assembled will be critical for understanding the nuclear transport mechanism. To address this issue and to identify factors that regulate NPC formation and dynamics, a novel fluorescence-based strategy was used. This approach is based on the functional tagging of NPC proteins with the green fluorescent protein (GFP), and the hypothesis that NPC assembly mutants will have distinct GFP-NPC signals as compared with wild-type (wt) cells. By fluorescence-activated cell sorting for cells with low GFP signal from a population of mutagenized cells expressing GFP-Nup49p, three complementation groups were identified: two correspond to mutant nup120 and gle2 alleles that result in clusters of NPCs. Interestingly, a third group was a novel temperature-sensitive allele of nup57. The lowered GFP-Nup49p incorporation in the nup57-E17 cells resulted in a decreased fluorescence level, which was due in part to a sharply diminished interaction between the carboxy-terminal truncated nup57pE17 and wt Nup49p. Interestingly, the nup57-E17 mutant also affected the incorporation of a specific subset of other nucleoporins into the NPC. Decreased levels of NPC-associated Nsp1p and Nup116p were observed. In contrast, the localizations of Nic96p, Nup82p, Nup159p, Nup145p, and Pom152p were not markedly diminished. Coincidentally, nuclear import capacity was inhibited. Taken together, the identification of such mutants with specific perturbations of NPC structure validates this fluorescence-based strategy as a powerful approach for providing insight into the mechanism of NPC biogenesis.

The Saccharomyces cerevisiae Homologue of Human Wiskott–Aldrich Syndrome Protein Las17p Interacts with the Arp2/3 Complex

Yeast Las17 protein is homologous to the Wiskott–Aldrich Syndrome protein, which is implicated in severe immunodeficiency. Las17p/Bee1p has been shown to be important for actin patch assembly and actin polymerization. Here we show that Las17p interacts with the Arp2/3 complex. LAS17 is an allele-specific multicopy suppressor of ARP2 and ARP3 mutations; overexpression restores both actin patch organization and endocytosis defects in ARP2 temperature-sensitive (ts) cells. Six of seven ARP2 ts mutants and at least one ARP3 ts mutant are synthetically lethal with las17Δ ts confirming functional interaction with the Arp2/3 complex. Further characterization of las17Δ cells showed that receptor-mediated internalization of α factor by the Ste2 receptor is severely defective. The polarity of normal bipolar bud site selection is lost. Las17-gfp remains localized in cortical patches in vivo independently of polymerized actin and is required for the polarized localization of Arp2/3 as well as actin. Coimmunoprecipitation of Arp2p with Las17p indicates that Las17p interacts directly with the complex. Two hybrid results also suggest that Las17p interacts with actin, verprolin, Rvs167p and several other proteins including Src homology 3 (SH3) domain proteins, suggesting that Las17p may integrate signals from different regulatory cascades destined for the Arp2/3p complex and the actin cytoskeleton.

In situ detection of activated Bruton’s tyrosine kinase in the Ig signaling complex by phosphopeptide-specific monoclonal antibodies

Bruton’s tyrosine kinase (Btk) is a critical transducer of signals originating from the B cell antigen receptor (BCR). Dosage, sequential phosphorylation, and protein interactions are interdependent mechanisms influencing Btk function. Phosphopeptide-specific mAbs recognizing two distinct phosphotyrosine modifications were used to quantify Btk activation by immunofluorescent techniques during B cell stimulation. In a population of cultured B cells stimulated by BCR crosslinking and analyzed by flow cytometry, transient phosphorylation of the regulatory Btk tyrosine residues (551Y and 223Y) was detected. The kinetics of phosphorylation of the residues were temporally distinct. Tyrosine 551, a transactivating substrate site for Src-family kinases, was maximally phosphorylated within ≈30 seconds of stimulation as monitored by flow cytometry. Tyrosine 223, an autophosphorylation site within the SH3 domain, was maximally phosphorylated at ≈5 minutes. Btk returned to a low tyrosine phosphorylation level within 30 minutes, despite persistent elevation of global tyrosine phosphorylation. Colocalization of activated Btk molecules with the crosslinked BCR signaling complex was observed to coincide with the period of maximal Btk tyrosine phosphorylation when stimulated B cells were analyzed with confocal microscopy. The results of these in situ temporal and spatial analyses imply that Btk signaling occurs in the region of the Ig receptor signaling complex, suggesting a similar location for downstream targets of its activity.

Evidence for specific nucleocytoplasmic transport pathways used by leucine-rich nuclear export signals

Various proteins with different biological activities have been observed to be translocated from the nucleus to the cytoplasm in an energy- and signal-dependent manner in eukaryotic cells. This nuclear export is directed by nuclear export signals (NESs), typically characterized by hydrophobic, primarily leucine, amino acid residues. Moreover, it has been shown that CRM1/exportin 1 is an export receptor for leucine-rich NESs. However, additional NES-interacting proteins have been described. In particular, eukaryotic initiation factor 5A (eIF-5A) has been shown to be a critical cellular cofactor for the nuclear export of the HIV type 1 (HIV-1) Rev trans-activator protein. In this study we compared the nuclear export activity of NESs of different origin. Microinjection of export substrates into the nucleus of somatic cells in combination with specific inhibitors indicated that specific nuclear export pathways exist for different NES-containing proteins. In particular, inhibition of eIF-5A blocked the nuclear export of NESs derived from the HIV-1 Rev and human T cell leukemia virus type I Rex trans-activators, whereas nucleocytoplasmic translocation of the protein kinase inhibitor-NES was unaffected. In contrast, however, inhibition of CRM1/exportin 1 blocked the nuclear export of all NES-containing proteins investigated. Our data confirm that CRM1/exportin 1 is a general export receptor for leucine-rich NESs and suggest that eIF-5A acts either upstream of CRM1/exportin 1 or forms a complex with the NES and CRM1/exportin 1 in the nucleocytoplasmic translocation of the HIV-1 Rev and human T cell leukemia virus type I Rex RNA export factors.

Generation of mucosal cytotoxic T cells against soluble protein by tissue-specific environmental and costimulatory signals

We compared peripheral and mucosal primary CD8 T cell responses to inflammatory and noninflammatory forms of antigen in a T cell-adoptive transfer system. Immunization with the soluble antigen, ovalbumin (ova), administered i.p. or orally without adjuvant, activated nonmucosal CD8 T cells but did not induce cytotoxic activity. However, after activation, the transferred cells entered the intestinal mucosa and became potent antigen-specific killers. Thus, exogenous intact soluble protein entered the major histocompatibility complex class I antigen presentation pathway and induced mucosal cytotoxic T lymphocytes. Moreover, distinct costimulatory requirements for activation of peripheral versus mucosal T cells were noted in that the CD28 ligand, B7-1, was critical for activated mucosal T cell generation but not for activation of peripheral CD8 T cells. The costimulator, B7-2, was required for optimum activation of both populations. Infection with a new recombinant vesicular stomatitis virus encoding ovalbumin induced lytic activity in mucosal as well as peripheral sites, demonstrating an adjuvant effect of inflammatory mediators produced during virus infection. Generation of antiviral cytotoxic T lymphocytes was also costimulation-dependent. The results indicated that induction of peripheral tolerance via antigen administration may not extend to mucosal sites because of distinct costimulatory and inflammatory signals in the mucosa.

A response regulator of cyanobacteria integrates diverse environmental signals and is critical for survival under extreme conditions

Microorganisms must sense their environment and rapidly tune their metabolism to ambient conditions to efficiently use available resources. We have identified a gene encoding a response regulator, NblR, that complements a cyanobacterial mutant unable to degrade its light-harvesting complex (phycobilisome), in response to nutrient deprivation. Cells of the nblR mutant (i) have more phycobilisomes than wild-type cells during nutrient-replete growth, (ii) do not degrade phycobilisomes during sulfur, nitrogen, or phosphorus limitation, (iii) cannot properly modulate the phycobilisome level during exposure to high light, and (iv) die rapidly when starved for either sulfur or nitrogen, or when exposed to high light. Apart from regulation of phycobilisome degradation, NblR modulates additional functions critical for cell survival during nutrient-limited and high-light conditions. NblR does not appear to be involved in acclimation responses that occur only during a specific nutrient limitation. In contrast, it controls at least some of the general acclimation responses; those that occur during any of a number of different stress conditions. NblR plays a pivotal role in integrating different environmental signals that link the metabolism of the cell to light harvesting capabilities and the activities of the photosynthetic apparatus; this modulation is critical for cell survival.

Altered thymic positive selection and intracellular signals in Cbl-deficient mice

Cbl is the product of the protooncogene c-cbl and is involved in T cell antigen receptor (TCR)-mediated signaling. To understand the role of Cbl for immune system development and function, we generated a Cbl-deficient mouse strain. In Cbl-deficient mice, positive selection of the thymocytes expressing major histocompatibility complex class II-restricted transgenic TCR was significantly enhanced. Two factors may have contributed to the altered thymic selection. First, Cbl deficiency markedly up-regulated the activity of ZAP-70 and mitogen-activated protein kinases. The mitogen-activated protein kinase pathway was shown previously to be involved in thymic positive selection. Second, Cbl-deficient thymocytes expressed CD3 and CD4 molecules at higher levels, which consequently may increase the avidity of TCR/major histocompatibility complex/coreceptor interaction. Thus, Cbl plays a novel role in modulating TCR-mediated multiple signaling pathways and fine-tunes the signaling threshold for thymic selection.

Signals and signs in the nervous system: The dynamic anatomy of electrical activity is probably information-rich

The dichotomy between two groups of workers on neuroelectrical activity is retarding progress. To study the interrelations between neuronal unit spike activity and compound field potentials of cell populations is both unfashionable and technically challenging. Neither of the mutual disparagements is justified: that spikes are to higher functions as the alphabet is to Shakespeare and that slow field potentials are irrelevant epiphenomena. Spikes are not the basis of the neural code but of multiple codes that coexist with nonspike codes. Field potentials are mainly information-rich signs of underlying processes, but sometimes they are also signals for neighboring cells, that is, they exert influence. This paper concerns opportunities for new research with many channels of wide-band (spike and slow wave) recording. A wealth of structure in time and three-dimensional space is different at each scale—micro-, meso-, and macroactivity. The depth of our ignorance is emphasized to underline the opportunities for uncovering new principles. We cannot currently estimate the relative importance of spikes and synaptic communication vs. extrasynaptic graded signals. In spite of a preponderance of literature on the former, we must consider the latter as probably important. We are in a primitive stage of looking at the time series of wide-band voltages in the compound, local field, potentials and of choosing descriptors that discriminate appropriately among brain loci, states (functions), stages (ontogeny, senescence), and taxa (evolution). This is not surprising, since the brains in higher species are surely the most complex systems known. They must be the greatest reservoir of new discoveries in nature. The complexity should not deter us, but a dose of humility can stimulate the flow of imaginative juices.

Adenylate kinase phosphotransfer communicates cellular energetic signals to ATP-sensitive potassium channels

Transduction of energetic signals into membrane electrical events governs vital cellular functions, ranging from hormone secretion and cytoprotection to appetite control and hair growth. Central to the regulation of such diverse cellular processes are the metabolism sensing ATP-sensitive K+ (KATP) channels. However, the mechanism that communicates metabolic signals and integrates cellular energetics with KATP channel-dependent membrane excitability remains elusive. Here, we identify that the response of KATP channels to metabolic challenge is regulated by adenylate kinase phosphotransfer. Adenylate kinase associates with the KATP channel complex, anchoring cellular phosphotransfer networks and facilitating delivery of mitochondrial signals to the membrane environment. Deletion of the adenylate kinase gene compromised nucleotide exchange at the channel site and impeded communication between mitochondria and KATP channels, rendering cellular metabolic sensing defective. Assigning a signal processing role to adenylate kinase identifies a phosphorelay mechanism essential for efficient coupling of cellular energetics with KATP channels and associated functions.

p300 is a component of an estrogen receptor coactivator complex.

The estrogen receptor (ER) is a ligand-dependent transcription factor that regulates expression of target genes in response to estrogen in concert with other cellular signaling pathways. This suggests that the mechanism by which ER transmits an activating signal to the general transcription machinery may include factors that integrate these diverse signals. We have previously characterized the estrogen receptor-associated protein, ERAP160, as a factor that complexes with ER in an agonist-dependent manner. We have now found that the transcriptional coactivator p300 associates with agonist bound ER and augments ligand-dependent activation by ER. Our studies show that an ER coactivator complex involves a direct hormone-dependent interaction between ER and ERAP160, resulting in the recruitment of p300. In addition, antibodies directed against the cloned steroid receptor coactivator 1 (SRC1) recognize ERAP160. The known role of p300 in multiple signal transduction pathways, including those involving the second messenger cAMP, suggests p300 functions as a point of integration between ER and these other pathways.

Selective response of ternary complex factor Sap1a to different mitogen-activated protein kinase subgroups.

Mitogenic and stres signals results in the activation of extracellular signal-regulated kinases (ERKs) and stress-activated protein kinase/c-Jun N-terminal kinases (SAPK/JNKs), respectively, which are two subgroups of the mitogen-activated protein kinases. A nuclear target of mitogen-activated protein (MAP) kinases is the ternary complex factor Elk-1, which underlies its involvement in the regulation of c-fos gene expression by mitogenic and stress signals. A second ternary complex factor, Sap1a, is coexpressed with Elk-1 in several cell types and shares attributes of Elk-1, the significance of which is not clear. Here we show that Sap1a is phosphorylated efficiently by ERKs but not by SAPK/JNKs. Serum response factor-dependent ternary complex formation by Sap1a is stimulated by ERK phosphorylation but not by SAPK/JNKs. Moreover, Sap1a-mediated transcription is activated by mitogenic signals but not by cell stress. These results suggest that Sap1a and Elk-1 have distinct physiological functions.

A complexity measure for selective matching of signals by the brain.

We have previously derived a theoretical measure of neural complexity (CN) in an attempt to characterize functional connectivity in the brain. CN measures the amount and heterogeneity of statistical correlations within a neural system in terms of the mutual information between subsets of its units. CN was initially used to characterize the functional connectivity of a neural system isolated from the environment. In the present paper, we introduce a related statistical measure, matching complexity (CM), which reflects the change in CN that occurs after a neural system receives signals from the environment. CM measures how well the ensemble of intrinsic correlations within a neural system fits the statistical structure of the sensory input. We show that CM is low when the intrinsic connectivity of a simulated cortical area is randomly organized. Conversely, CM is high when the intrinsic connectivity is modified so as to differentially amplify those intrinsic correlations that happen to be enhanced by sensory input. When the input is represented by an individual stimulus, a positive value of CM indicates that the limited mutual information between sensory sheets sampling the stimulus and the rest of the brain triggers a large increase in the mutual information between many functionally specialized subsets within the brain. In this way, a complex brain can deal with context and go "beyond the information given."