Expression of the Armadillo family member p120 cas 1B in Xenopus embryos affects head differentiation but not axis formation

 The Armadillo family is formed by proteins which possess an Arm domain comprising multiple copies of a 42-amino-acid motif, the Arm repeat, initially described for the Drosophila segment polarity gene product Armadillo. The Arm domain serves in protein-protein interactions which are required for the family members Armadillo, β-catenin and plakoglobin to mediate cell-cell adhesion and Wnt/Wingless signalling. Similarily, p120 ^cas , the Arm domain containing src substrate, also binds to cadherins and becomes tyrosine phosphorylated in response to a variety of stimuli. However, a putative function of p120 ^cas in adhesion or signalling has not yet been demonstrated. It has also not been shown until now that an Arm domain is a common signal transduction motif. Using Xenopus embryos we show by expression of murine p120 ^cas 1B (mp120 ^cas 1B) in ventral blastomeres that this catenin cannot replace β-catenin function in dorsal axis formation. Thus, the presence of an Arm domain per se is not sufficient to activate the Wnt/Wg pathway. Indeed, injection of mp120 ^cas 1B into dorsal blastomeres led instead to delayed blastopore closure and posteriorized phenotypes with malformed head structures indicative of disturbed gastrulation movements. Because neither convergent extension behaviour nor adhesion to fibronectin was altered in the injected embryos we assume that mp120 ^cas 1B influences motility or orientation of migrating mesodermal cells. Received: 29 September 1997 / Accepted: 15 November 1997     

The role of pseudokinases in cancer

Kinases play a critical role in regulating many cellular functions including development, differentiation and proliferation. To date, over 518 proteins with kinase activity, comprising ~2-3% of total cellular proteins, have been identified from within the human kinome. Interestingly, approximately 10% of kinases are categorised as pseudokinases since they lack one or more conserved catalytic residues within their kinase domain and were originally thought to have no enzymatic activity. Recently, there has been strong evidence to suggest that some pseudokinsases can not only function as scaffold proteins, but may also possess kinase activity leading to modulation of cell signalling pathways. Altered activity of these pseudokinases can result in impaired cellular function, particularly in malignancies. In this review we are discussing recent evidence that apart from a scaffolding role, pseudokinases also orchestrate cellular processes as active kinases per se in signalling pathways of malignant cells.     

Hsp90-binding immunophilins in plants: the protein movers

Studies of cytoplasmic-nuclear trafficking of the glucocorticoid receptor in mammalian cells suggest that the hsp90/hsp70-based chaperone system and the hsp90-binding immunophilin FKBP52 are involved in targeted movement of the receptor along microtubule tracts. Over the past few years, plant cells have been found to possess a similar multiprotein chaperone machinery. Plant cells also contain high molecular weight FKBPs that bind to plant hsp90 via a conserved protein interaction involving tetratricopeptide repeat domains. The hsp90/hsp70-based machinery and the plant FKBPs might be used to target the trafficking of signalling proteins in plants.     

The IRS-signalling system during insulin and cytokine action

The discovery of the first intracellular substrate for insulin, IRS-1, redirected the field of diabetes research and has led to many important advances in our understanding of insulin action. Detailed analysis of IRS-1 demonstrates structure/function relationships for this modular docking molecule, including mechanisms of substrate recognition and signal propagation. Recent work has also identified other structurally similar molecules, including IRS-2, the Drosophila protein, DOS, and the Grb2-binding protein, Gab1, suggesting that this intracellular signalling strategy is conserved evolutionarily and is utilized by an expanding number of receptor systems. In fact, IRS-1 itself has been shown to be important in other growth factor and cytokine signalling systems, including growth hormone and several interleukins. Analysis of mice lacking IRS-1 confirms an important physiological role for this protein in glucose metabolism and general cell growth in the intact animal. Disregulation of the signalling pathways integrated by the IRS proteins may contribute to the pathophysiology of non-insulin-dependent diabetes mellitus or other diseases.     

Mammalian NLR proteins; discriminating foe from friend

Eukaryotic organisms of the plant and animal kingdoms have developed evolutionarily conserved systems of defence against microbial pathogens. These systems depend on the specific recognition of microbial products or structures by molecules of the host innate immune system. The first mammalian molecules shown to be involved in innate immune recognition of, and defence against, microbial pathogens were the Toll-like receptors (TLRs). These proteins are predominantly but not exclusively located in the transmembrane region of host cells. Interestingly, mammalian hosts were subsequently found to also harbour cytosolic proteins with analogous structures and functions to plant defence molecules. The members of this protein family exhibit a tripartite domain structure and are characterized by a central nucleotide-binding oligomerization domain (NOD). Moreover, in common with TLRs, most NOD proteins possess a C-terminal leucine-rich repeat (LRR) domain, which is required for the sensing of microbial products and structures. Recently, the name 'nucleotide-binding domain and LRR' (NLR) was coined to describe this family of proteins. It is now clear that NLR proteins play key roles in the cytoplasmic recognition of whole bacteria or their products. Moreover, it has been demonstrated in animal studies that NLRs are important for host defence against bacterial infection. This review will particularly focus on two subfamilies of NLR proteins, the NODs and 'NALPs', which specifically recognize bacterial products, including cell wall peptidoglycan and flagellin. We will discuss the downstream signalling events and host cell responses to NLR recognition of such products, as well as the strategies that bacterial pathogens employ to trigger NLR signalling in host cells. Cytosolic recognition of microbial factors by NLR proteins appears to be one mechanism whereby the innate immune system is able to discriminate between pathogenic bacteria ('foe') and commensal ('friendly') members of the host microflora.     

Molecular insights into the biochemical functions and signalling mechanisms of plant NLRs

Plant intracellular immune receptors known as NLR (nucleotide-binding leucine-rich repeat) proteins confer immunity and cause cell death. Plant NLR proteins that directly or indirectly recognize pathogen effector proteins to initiate immune signalling are regarded as sensor NLRs. Some NLR protein families function downstream of sensor NLRs to transduce immune signalling and are known as helper NLRs. Recent breakthrough studies on plant NLR protein structures and biochemical functions greatly advanced our understanding of NLR biology. Comprehensive and detailed knowledge on NLR biology requires future efforts to solve more NLR protein structures and investigate the signalling events between sensor and helper NLRs, and downstream of helper NLRs.     

Bacterial signal transduction network in a genomic perspective

Bacterial signalling network includes an array of numerous interacting components that monitor environmental and intracellular parameters and effect cellular response to changes in these parameters. The complexity of bacterial signalling systems makes comparative genome analysis a particularly valuable tool for their studies. Comparative studies revealed certain general trends in the organization of diverse signalling systems. These include (i) modular structure of signalling proteins; (ii) common organization of signalling components with the flow of information from N-terminal sensory domains to the C-terminal transmitter or signal output domains (N-to-C flow); (iii) use of common conserved sensory domains by different membrane receptors; (iv) ability of some organisms to respond to one environmental signal by activating several regulatory circuits; (v) abundance of intracellular signalling proteins, typically consisting of a PAS or GAF sensor domains and various output domains; (vi) importance of secondary messengers, cAMP and cyclic diguanylate; and (vii) crosstalk between components of different signalling pathways. Experimental characterization of the novel domains and domain combinations would be needed for achieving a better understanding of the mechanisms of signalling response and the intracellular hierarchy of different signalling pathways.     

Regulation of intracellular signalling through cysteine oxidation by reactive oxygen species

Reactive oxygen species (ROS) have been regarded as harmful molecules that damage various molecules inside cells by oxidation and are responsible for ageing and various human diseases. However, recent studies have revealed an opposite aspect of ROS that these are actively generated in cells and mediate physiological intracellular signalling as second messengers. Several proteins have been shown to function as effectors for ROS, which are sensitively and reversibly oxidized by ROS. Such ROS-effector proteins commonly possess a highly reactive cysteine (Cys) residue, of which oxidation changes the protein function, thus enabling signal transmission to downstream targets. Among the ROS effectors, protein tyrosine phosphatase (PTP), thioredoxin (TRX) and peroxiredoxin (PRX) family proteins possess special domains/motifs to maintain the reactivity of Cys and utilize them to respond to ROS. Progressively advancing identification of ROS-effector proteins reveals the pleiotropic functions of ROS in physiological and pathological cell biology.     

Search for new cyclic AMP-binding proteins

Today, there is evidence that the cAMP-dependent kinases (PKA) are not the only intracellular receptors involved in intracellular cAMP signalling in eukaryotes. Other cAMP-binding proteins have been recently identified, including some cyclic nucleotide-gated channels and Epac (exchange protein directly activated by cAMP) proteins. All these proteins bind cAMP through conserved cyclic nucleotide monophosphate-binding domains. However, all putative cAMP-binding proteins having such domains, as revealed by computer analysis, do not necessarily bind cAMP, indicating that their presence is not a sufficient criteria to predict cAMP-binding property for a protein.     

Tribbles: a family of kinase-like proteins with potent signalling regulatory function

The recent identification of tribbles as regulators of signal processing systems and physiological processes, including development, together with their potential involvement in diabetes and cancer, has generated considerable interest in these proteins. Tribbles have been reported to regulate activation of a number of intracellular signalling pathways with roles extending from mitosis and cell activation to apoptosis and modulation of gene expression. The current review summarises our current understanding of interactions between tribbles and various other proteins. Since our understanding on the molecular basis of tribbles function is far from complete, we also describe a bioinformatic analysis of various segments of tribbles proteins, which has revealed a number of highly conserved peptide motifs with potentially important functional roles.     

Conserved regions in the Epstein-Barr virus leader protein define distinct domains required for nuclear localization and transcriptional cooperation with EBNA2

Epstein-Barr virus (EBV) EBNA-LP is a latent protein whose function is not fully understood. Recent studies have shown that EBNA-LP may be an important EBNA2 cofactor by enhancing EBNA2 stimulation of the latency C and LMP-1 promoters. To further our understanding of EBNA-LP function, we have introduced a series of mutations into evolutionarily conserved regions and tested the mutant proteins for the ability to enhance EBNA2 stimulation of the latency C and LMP-1 promoters. Three conserved regions (CR1 to CR3) are located in the repeat domains that are essential for the EBNA2 cooperativity function. In addition, three serine residues are also well conserved in the repeat domains. Clustered alanine mutations were introduced into CR1 to CR3, and the conserved serines were also changed to alanine residues in an EBNA-LP with two repeats, which is the minimal protein able to cooperate with EBNA2. Mutations introduced into CR1a had no effect on EBNA-LP function, while mutations introduced into CR1b resulted in EBNA-LP with slightly decreased activity. Mutations in CR1c and CR2 resulted in proteins that no longer localized exclusively to the nucleus and also had no EBNA2 cooperation activity. Mutations introduced into conserved serines S5/71 resulted in proteins with slightly higher activity, while mutations introduced into conserved serines S35/101 or in CR3 (which contains S60/126) resulted in EBNA-LP proteins with substantially reduced activity. The potential karyophilic signals within EBNA-LP CR1c and CR2 were also examined by introducing oligonucleotides encoding these positively charged amino acid groupings into a cytoplasmic test protein, herpes simplex virus DeltaIE175, and by examining the intracellular localization of the resulting proteins. This assay identified a strong nuclear localization signal between EBNA-LP amino acids 43 and 50 (109 to 117 in the second W repeat) comprising CR2, while EBNA-LP amino acids 29 to 36 (91 to 98 in the second W repeat) were unable to function independently as a nuclear localization signal. However, a combination of amino acids 29 to 50 resulted in more efficient nuclear localization than with amino acids 43 to 50 alone. These results indicate that EBNA-LP has a bipartite nuclear localization signal and that efficient nuclear localization is essential for EBNA2 cooperativity function. Interestingly, EBNA-LP with only a single repeat localized exclusively to the cytoplasm, providing an explanation for why this isoform has no activity. In addition, two conserved serine residues which are distinct from nuclear import functions are important for EBNA2 cooperativity function.     

Impaired sucrose induction1 encodes a conserved plant-specific protein that couples carbohydrate availability to gene expression and plant growth

To identify the molecular mechanisms underlying carbohydrate allocation to storage processes, we have isolated mutants in which the sugar induction of starch biosynthetic gene expression was impaired. Here we describe the IMPAIRED SUCROSE INDUCTION1 (ISI1) gene, which encodes a highly conserved plant-specific protein with structural similarities to Arm repeat proteins. ISI1 is predominantly expressed in the phloem of leaves following the sink-to-source transition during leaf development, but is also sugar-inducible in mesophyll cells. Soil-grown isi1 mutants show reduced plant growth and seed set compared to wild-type Arabidopsis. This growth reduction is not due to reduced carbohydrate availability or a defect in sucrose export from mature leaves, suggesting that isi1 mutant plants do not utilize available carbohydrate resources efficiently. ISI1 interacts synergistically with, but is genetically distinct from, the abscisic acid (ABA) signalling pathway controlling sugar responses via ABI4. Our data show that ISI1 couples the availability of carbohydrates to the control of sugar-responsive gene expression and plant growth.     

Human immunodeficiency virus type 1 Nef binds directly to Lck and mitogen-activated protein kinase, inhibiting kinase activity.

It is now well established that human immunodeficiency virus type I (HIV-1) Nef contributes substantially to disease pathogenesis by augmenting virus replication and markedly perturbing T-cell function. The effect of Nef on host cell activation could be explained in part by its interaction with specific cellular proteins involved in signal transduction, including at least a member of the src family kinase, Lck, and the serine/threonine kinase, mitogen-activated protein kinase (MAPK). Recombinant Nef directly interacted with purified Lck and MAPK in coprecipitation experiments and binding assays. A proline-rich repeat sequence [(Pxx)4] in Nef occurring between amino acid residues 69 to 78 is highly conserved and bears strong resemblance to a defined consensus sequence identified as an SH3 binding domain present in several proteins which can interact with the SH3 domain of various signalling and cytoskeletal proteins. Binding and coprecipitation assays with short synthetic peptides corresponding to the proline-rich repeat sequence [(Pxx)4] of Nef and the SH2, SH3, or SH2 and SH3 domains of Lck revealed that the interaction between these two proteins is at least in part mediated by the proline repeat sequence of Nef and the SH3 domain of Lck. In addition to direct binding to full-length Nef, MAPK was also shown to bind the same proline repeat motif. Nef protein significantly decreased the in vitro kinase activity of Lck and MAPK. Inhibition of key members of signalling cascades, including those emanating from the T-cell receptor, by the HIV-1 Nef protein undoubtedly alters the ability of the infected T cell to respond to antigens or cytokines, facilitating HIV-1 replication and contributing to HIV-1-induced disease pathogenesis.     

ZFWD: a novel subfamily of plant proteins containing a C3H zinc finger and seven WD40 repeats

We describe a new subfamily of WD repeat proteins characterised by the presence of a C3H zinc finger at the N-terminal part of the protein associated with seven WD40 repeats. We have identified four members of this subfamily in Arabidopsis thaliana, one of them with associated expressed sequence tags (ESTs). We have also identified homologous ESTs in rice, cotton, maize, poplar, pine tree and the ice plant. We do not observe animal homologues, suggesting that this subfamily could be specific for plants. Our data suggest an important role for these proteins. Based on the high sequence conservation within the conserved domains, we suggest that these proteins could have a regulatory function.     

A large complement of the predicted Arabidopsis ARM repeat proteins are members of the U-box E3 ubiquitin ligase family

The Arabidopsis genome was searched to identify predicted proteins containing armadillo (ARM) repeats, a motif known to mediate protein-protein interactions in a number of different animal proteins. Using domain database predictions and models generated in this study, 108 Arabidopsis proteins were identified that contained a minimum of two ARM repeats with the majority of proteins containing four to eight ARM repeats. Clustering analysis showed that the 108 predicted Arabidopsis ARM repeat proteins could be divided into multiple groups with wide differences in their domain compositions and organizations. Interestingly, 41 of the 108 Arabidopsis ARM repeat proteins contained a U-box, a motif present in a family of E3 ligases, and these proteins represented the largest class of Arabidopsis ARM repeat proteins. In 14 of these U-box/ARM repeat proteins, there was also a novel conserved domain identified in the N-terminal region. Based on the phylogenetic tree, representative U-box/ARM repeat proteins were selected for further study. RNA-blot analyses revealed that these U-box/ARM proteins are expressed in a variety of tissues in Arabidopsis. In addition, the selected U-box/ARM proteins were found to be functional E3 ubiquitin ligases. Thus, these U-box/ARM proteins represent a new family of E3 ligases in Arabidopsis.