Specific localization of the catalytic subunits of protein kinase CK2 at the centrosomes

The protein kinase CK2 holoenzyme is composed of two regulatory β subunits and two catalytic α or α' subunits. Although experimental evidence for involvement of the enzyme in the regulation of cell proliferation is accumulating, the exact mechanism of its action is still unclear. The subcellular localization of the enzyme may be a key to its function. We have recently shown that the CK2 holoenzyme is tightly associated with the Golgi complex and the endoplasmic reticulum. Centrosomes, which organize spindle formation during the cell cycle and microtubule cytoskeleton formation and, thereby, the location and orientation of different organelles in the cell, are in close vicinity to the Golgi complex. Because several kinases and phosphatases have been described to regulate the functions of the centrosome, we analysed the association of CK2 with these organelles. Using biochemical cell fractionation and coimmunoprecipitation, we never found the holoenzyme but only the catalytic asubunits associated with the centrosome. These data were confirmed by immunoelectron microscopy. Thus, the present data point to a particular role of the catalytic α and α' subunit of protein kinase CK2, which may be different from their roles in the holoenzyme. Received 2 August 2002; received after revision 2 October 2002; accepted 22 October 2002 RID="*" ID="*"Corresponding author.     

Signalling pathways involved in ribonuclease-7 expression

Antimicrobial peptides are host defence molecules that play a potential role in preventing infection at the epithelial surfaces. Ribonuclease (RNase)-7 has been shown to possess a broad spectrum of microbicidal activity against various pathogens. Here, we demonstrate that RNase-7 protein is localised to the superficial layers of ocular surface cells and increased in response to interleukin (IL)-1β, suggesting an active role during inflammation related to ocular surface infection. Signal transduction pathways involved in RNase-7 expression are unknown. Involvement of transforming growth factor β-activated kinase-1 (TAK-1) activated nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathway molecules [c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38] were studied because of their importance in infection and inflammation. Blocking the MAPKs resulted in inhibition of RNase-7 expression in response to IL-1β. However, RNase-7 induction by IL-1β was not affected by inhibiting the NF-κB signalling pathway. In conclusion, our results indicate that RNase-7 expression is specifically mediated via MAPKs but not NF-κB signalling pathways.     

Novel regulation and function of Src tyrosine kinase

Src tyrosine kinase is a critical signal transducer that modulates a wide variety of cellular functions. Misregulation of Src leads to cell transformation and cancer. Heterotrimeric guanine-nucleotide-binding proteins (G proteins) are another group of signaling molecules that transduce signals from cell-surface receptors to generate physiological responses. Recently, it was discovered that Gαs and Gαi could directly stimulate Src family tyrosine kinase activity. This novel regulation of Src tyrosine kinase by G proteins provides insights into the adenylyl cyclase-independent signaling mechanisms involved in ligand-induced receptor desensitization, internalization and other physiological processes. Received 17 August 2001; received after revision 22 October 2001; accepted 24 October 2001     

Protein kinases: which one is the memory molecule?

Encoding of new experiences is likely to induce activity-dependent modifications in the brain. Studies in organisms far apart on the phylogenetic scale have shown that similar, sometimes identical, signal transduction pathways subserve plasticity in neuronal systems, and they may play pivotal roles in the formation of long-term memories. It has become evident that phosphorylation/dephosphorylation reactions are critical for the initiation of cellular mechanisms that embody, retain and modify information in neural circuits. Although physiological investigations on synaptic plasticity have had a major impact, we have concentrated our review on behavioural studies that provide direct or indirect evidence for a role of kinases in mechanisms underlying memory formation. From these, it appears that the learning event induces activation of a variety of kinases with specific time courses. For instance, the calcium/calmodulin-dependent protein kinase II seems to participate in an early phase of memory formation. Apparently, activation of both protein tyrosine kinases and mitogen-activated protein kinases is required for much longer and may thus have a particular function during transformation from short-term into long-term memory. Quite different time courses appear for protein kinase C (PKC) and protein kinase A (PKA), which may function at two different time points, shortly after training and again much later. This suggests that PKC and PKA might play a role at early and late stages of memory formation. However, we have considered some examples showing that these signalling pathways do not function in isolation but rather interact in an intricate intracellular network. This is indicative of a more complex contribution of each kinase to the fine tuning of encoding and information processing. To decipher this complexity, pharmacological, biochemical and genetic investigations are more than ever necessary to unravel the role of each kinase in the syntax of learning and memory formation.     

Protein kinases mediate nitric oxide-induced apoptosis in the insect cell line IPLB-LdFB

The involvement of protein kinases (PKA, PKC and PKB) in nitric oxide (NO)-induced apoptosis with sodium nitroprusside plus N-acetyl-L-cysteine in the IPLB-LdFB cell line from the insect Lymantria dispar was investigated. The presence of protein kinase-like molecules was demonstrated by western blot analysis. The role of the kinases in programmed cell death was analysed in cytofluorimetric experiments by incubating the insect cells with H-89 (a specific inhibitor of PKA), calphostin C (an inhibitor of PKC) or wortmannin (an inhibitor of phosphatidylinositol 3-kinase). The results show that PKA is correlated with the induction and PKC and PKB with the prevention of NO-induced insect cell death. Moreover, NO-induced apoptosis involves the release of cytochrome c. Received 15 March 2002; accepted 25 March 2002     

Melatonin regulation of antioxidant enzyme gene expression

Antioxidant enzymes (AOEs) are part of the primary cellular defense against free radicals induced by toxins and/or spontaneously formed in cells. Melatonin (MLT) has received much attention in recent years due to its direct free radical scavenging and antioxidant properties. In the present work we report that MLT, at physiological serum concentrations (≈ 1 nM), increases the mRNA of both superoxide dismutases (SODs) and glutathione peroxidase (GPx) in two neuronal cell lines. The MLT effect on both SODs and GPx mRNA was mediated by a de novo synthesized protein. MLT alters mRNA stability for Cu-Zn SOD and GPx. Experiments with a short time treatment (pulse action) of MLT suggest that the regulation of AOE gene expression is likely to be receptor mediated, because 1-h treatment with MLT results in the same response as a 24-h treatment. Received 18 June 2002; received after revision 5 August 2002; accepted 27 August 2002 RID="*" ID="*"Corresponding author.     

MAP kinases in plant signal transduction

Mitogen-activated protein kinase (MAPK) pathways are modules involved in the transduction of extracellular signals to intracellular targets in all eukaryotes. Distinct MAPK pathways are regulated by different extracellular stimuli and are implicated in a wide variety of biological processes. In plants there is evidence for MAPKs playing a role in the signaling of abiotic stresses, pathogens and plant hormones. The large number and divergence of plant MAPKs indicates that this ancient mechanism of bioinformatics is extensively used in plants and may provide a new molecular handle on old questions.     

Isoforms of soluble alpha-tubulin in oocytes and brain of the frog (genus Rana): changes during oocyte maturation

Rana oocytes have previously been shown to contain much more soluble tubulin than does the brain, suggesting different assembly and disassembly dynamics of frog oocyte tubulin compared to that in brain. By using centrifugation, SDS-PAGE, two-dimensional gel electrophoresis and Western blots, probed with anti-α-tubulin monoclonal antibodies, polymorphic α-tubulins (isoforms) were compared in brains and follicle-enclosed oocytes of northern (Rana pipiens) and southern (R. berlandieri) frogs. Oocyte tubulin in both species had isoforms with greater ranges of isoelectric point (pI) than those of brain tubulins; in particular, the oocyte tubulin pIs ranged further into the acidic region of the isoelectric-focusing gels than corresponding brain tubulin. This difference may, in part, be responsible for the previously reported assembly differences between oocyte tubulin (undetectable assembly) and brain tubulin (high assembly). Isoforms of α-tubulin with relat ively acidic pI were more abundant in northern frog brain and oocyte soluble extracts than in analogous extracts from southern frogs. Furthermore, additional acidic α-tubulin isoforms were found in progesterone-treated oocytes (i.e., eggs), indicating increased heterogeneity of acidic a-tubulin isoforms during oocyte meiotic maturation. Among northern frog oocyte soluble components fractionated on Superose-6b columns, tubulin complexes with apparent molecular mass of about 1800 kDa were found to contain acidic α-tubulin isoforms while the putative oligomeric tubulins with an apparent molecular mass of about 250 kDa contained an additional relatively basic α-tubulin isoform. The acidic α-tubulin isoforms, therefore, are proposed to be associated with cold-adaptable cells of brain and oocytes, and may also be involved in stabilization of large soluble tubulin complexes in oocytes of the northern frog. Received 1 October 2002; accepted 9 October 2002 RID="*" ID="*"Corresponding author.     

Glycoconjugates of the intestinal goblet cells of four cyprinids

The aim of this work was to show differences in the terminal and subterminal sugar composition of carbohydrate chains of glycoconjugates produced by the goblet cells of the intestines of four cyprinids. We analysed intestines of two herbivorous species – sneep and grass carp – and two omnivorous ones – chub and common carp. We compared four intestinal regions of every studied species. In every region, the presence of neutral and acidic glycoconjugates was confirmed. The smallest amount of acidic glycoconjugates was present in the second region of sneep intestine. Sulphated glycoconjugates were absent in the third and fourth region of chub intestine. Lectin histochemistry provided evidence for the presence of β-D-galactose, α-N-acetylgalactosamine, β-N-acetylglucosamine and sialic acids. Additionally, the occurrence of α-L-fucose in the goblet cells of chub, grass carp and sneep was confirmed. We tried to correlate the patern of glycoconjugate glycosylation with feeding habits of the studied fishes. Received 1 July 2002; received after revision 8 August 2002; accepted 19 August 2002 RID="*" ID="*"Corresponding author.     

Circular proteoglycans from sponges: first members of the spongican family

Species-specific cell adhesion in marine sponges is mediated by a new family of modular proteoglycans whose general supramolecular structure resembles that of hyalectans. However, neither their protein nor their glycan moieties have significant sequence homology to other proteoglycans, despite having protein subunits equivalent to link proteins and to proteoglycan monomer core proteins, and glycan subunits equivalent to hyaluronan and to the glycosaminoglycans of hyalectans. In some species, these molecular components are assembled into a structure with a circular core formed by the link protein- and hyaluronan-like subunits. Besides their involvement in cell adhesion, these sponge proteoglycans, for which we propose the term spongicans, participate in signal transduction processes and are suspected to play a role in sponge self-nonself recognition. Their in vivo roles and the mild methods used to purify large amounts of functionally active spongicans make them ideal models to study the functions and possible new applications of proteoglycans in biomedical research. Received 21 May 2002; received after revision 5 July 2002; accepted 10 July 2002 RID="*" ID="*"Corresponding author.     

Oligo-1,6-glucosidase and neopullulanase enzyme subfamilies from the alpha-amylase family defined by the fifth conserved sequence region

The α-amylase enzyme family is the largest family of glycoside hydrolases. It contains almost 30 different enzyme specificities covering hydrolases, transferases and isomerases. Some of the enzyme specificities from the family are closely related, others less so. This study, based on the analysis of 79 amino acid sequences, postulates two subfamilies in the framework of the α-amylase family: the oligo-1,6-glucosidase subfamily and the neopullulanase subfamily. The specific sequence in the fifth conserved sequence region of the family served as the basis for defining the subfamilies: QpDln for the oligo-1,6-glucosidase subfamily and MPKln for the neopullulanase subfamily. This conserved sequence region is proposed to be the selection marker that enables one to distinguish between the two subfamilies. The 'intermediary' sequence MPDLN can be characteristic of the so-called intermediary group with a mixed enzyme specificity of α-amylase, cyclomaltodextrinase and neopullulanase. The evolutionary trees clearly supported the proposed definition of the two subfamilies. Received 12 July 2002; received after revision 28 August 2002; accepted 24 September 2002 RID="*" ID="*"Corresponding author.     

Structural features of prions explored by sequence analysis. II. A PrP<Superscript>Sc</Superscript> model

Prion diseases are neurodegenerative disorders associated with a conformational conversion of the prion PrP protein, in which the β-strand content increases and that of the α helix decreases. However, the structure of the pathogenous form PrP^Sc, occurring after conformational conversion of the normal cellular form PrP^C, is not yet known. From sequence analysis, we have previously proposed that helix H2 of the prion PrP^C structure might be a key region for this structural conversion. More recently, we identified the TATA box-binding protein fold as a putative scaffold that may locally satisfy the predicted secondary-structure organisation of PrP^Sc. In the present analysis, we detail the schematic construction of PrP^Sc monomeric and dimeric models, based on this hypothesis. These models are globally compatible with available data and therefore may provide further insights into the structurally and functionally elusive PrP protein. Some comments are also devoted to a comparison of the yeast Ure2p prion and animal prions. Received 29 July 2002; received after revision 24 October 2002; accepted 24 October 2002 RID="*" ID="*"Corresponding author.     

Protein kinase C-dependent NF-κB activation is altered in T cells by chronic stress

Chronic stress has been associated with impaired immune function. In this work we studied the effect of chronic mild stress (CMS) exposure on the early intracellular pathways involved in T cells after stimulation with mitogen. We found that mitogen stimulation of T lymphocytes from CMS-exposed mice resulted in a reduction of the intracellular [Ca²⁺] rise, an impairment of growth-promoting protein kinase C (PKC) activation, a lower NF-κB activation and an increase in the inhibitory cAMP-protein kinase A (PKA) pathway activity with respect to those found in control lymphocytes. However, T cell activation with the direct PKC activator phorbol 12-myristate 13-acetate plus calcium ionophore led to a similar proliferative response in both CMS and control lymphocytes, indicating that signals downstream of PKC would not be affected by stress. In summary, our results show that chronic stress induced an alteration in T cell early transduction signals that result in an impairment of the proliferative response.Received 11 February 2005; received after revision 20 May 2005; accepted 6 June 2005     

Ectoprotein kinase-mediated phosphorylation of FAT/CD36 regulates palmitate uptake by human platelets

Glycoprotein IV (FAT/CD36) has been shown to be phosphorylated by a cAMP-dependent, platelet membrane-bound ectokinase. In this study, we demonstrate that ectophosphorylation of FAT/CD36 regulates initial palmitate uptake. This is the first time that short-term regulation of the activity of a long-chain fatty acid carrier could be shown. Phosphorylation of FAT/CD36 was paralleled by a significant decrease in initial palmitate uptake by morphologically and functionally intact platelets. Maximum inhibition of palmitate uptake was achieved at 0.5 nM extracellular ATP, being significantly decreased to 72% compared to the control. Inhibition of palmitate uptake was abolished by co-incubation with the specific protein kinase A inhibitor peptide PKI or with β,γ-methylene-ATP, and was reversible upon addition of alkaline phosphatase. An extracellular ATP concentration above 5 μM completely prevented the ectophosphorylation-mediated inhibition of palmitate uptake. We conclude that FAT/CD36-mediated palmitate uptake by human platelets is short-term regulated via cAMP-dependent ectophosphorylation of FAT/CD36. Received 18 July 2002; received after revision 29 August 2002; accepted 19 September 2002 RID="*" ID="*"Corresponding author.     

Inhibition of proliferation by 1-8U in interferon-α-responsive and non-responsive cell lines

Interferon (IFN)-inducible proteins of the 1-8 gene family mediate homotypic adhesion and transduction of antiproliferative signals. Their induction correlates with inhibition of cell growth while they are often repressed in the course of malignant transformation and tumor development. Ras-mediated transformation of mouse mast cells is associated with downregulation of 1-8U expression and interferon-α (IFN-α) treatment reverts the proliferation rate to normal levels together with induction of 1-8U. Conversely, the antiproliferative responses of IFN-α in sensitive human melanoma cells are accompanied by 1-8U induction. Here we provide direct evidence that recombinant expression of 1-8U in human cell lines is sufficient to block cell proliferation. Based on the abundant expression and subcellular localization to the plasma membrane and exosome-like structures, we propose a model capable of explaining the pleiotropic functions of 1-8 family proteins in tumor cells and during normal development. Received 15 January 2003; received after revision 21 March 2003; accepted 25 March 2003 RID="*" ID="*"Corresponding author.     

Functional interactions of protein kinase A and C in signalling networks: a recapitulation

On the basis of evidence collected from the literature, we propose a general model by which protein kinase (PK) A and the different PKC isoforms can inversely affect cell growth. Molecular switches, which are able to direct the signal towards antiproliferative or mitogenic pathways, are the different isoforms of Raf and PKC. Conflicting data are also reported and discussed in an attempt to reconcile them. Received 10 November 2005; received after revision 28 December 2005; accepted 3 January 2006     

HLA-G protein processing and transport to the cell surface

Data are presented on the intracellular trafficking of HLA-G protein, taking the unique features of this non-classical molecule into consideration: the existence of seven isoforms resulting from alternative splicing (HLA-G1 to G7), and reduced tail length compared with HLA class I antigens. Biochemical studies and analysis of viral strategies for escaping the host immune system led to the demonstration that (i) both the membrane-bound (HLA-G1) and the soluble (HLA-G5) forms of the molecule require peptide association for cell surface expression, using TAP-dependent or TAP-independent pathways; (ii) peptide loading onto the HLA-G protein plays a critical role in controlling the quality of the molecule reaching the cell surface; (iii) surface expression of truncated HLA-G molecules is possible, and (iv) HLA-G expression may be restricted to soluble HLA-G5. These data reveal that HLA-G presents specific cell trafficking pathways and strongly support the contention that the primary function of HLA-G is as of an inhibitor ligand for immune-competent cells. Received 4 June 2002; accepted 2 July 2002 RID="*" ID="*"Corresponding author.     

The complexity of parathyroid hormone-related proteinsignalling

Our understanding of the mode of action of parathyroid hormone-related protein (PTHrP) has changed profoundly during the last decade. Most PTHrP activities are mediated by membrane receptors through autocrine/paracrine pathways. However, both endogenous and exogenous PTHrP also appear to have intracrine effects through translocation into the nucleus. The present review proposes unconventional PTHrP signalling, based on novel clues. First, PTHrP binding to its membrane receptor triggers internalization of the whole complex, mediated by beta-arrestin. There is growing evidence that the receptor and arrestin are the effectors of biological responses, rather than the ligand (or in addition to the ligand). Second, the existence of putative PTHrP targets within the cytoplasm is beginning to be supported. Recent findings of interactions between a COOH-terminus of PTHrP and beta-arrestin and between the PTHrP receptor and 14-3-3 proteins represent the starting point for identification of intracellular partners of both the hormone and its receptor.Received 19 June 2003; received after revision 10 July 2003; accepted 21 July 2003     

Immunomodulatory properties of cystatins

Cystatins are natural tight-binding reversible inhibitors of cysteine proteases. Because these cysteine proteases exist in all living organisms and because they are involved in various biological and pathological processes, the control of these protease functions by cystatins is of cardinal importance. Cystatins are found in mammals but cystatin-like molecules are also present in mammals and parasites. In the immune system, cystatins modulate cathepsin activities and antigen presentation. They also induce tumor necrosis factor α and interleukin 10 synthesis, and they stimulate nitric oxide production by interferon γ-activated murine macrophages. In turn, nitric oxide has inhibitory activity on cysteine proteases, especially those from parasitic protozoa. Cystatins isolated from parasitic nematodes also have immunomodulatory activities that are distinguishable from those induced by lipopolysacharide-like molecules from endosymbiotic bacteria. On the whole, cystatins and cystatin-like molecules belong to a new category of immunomodulatory molecules. Doubtless increasing data will improve our knowledge of this property, leading to practical applications in immunotherapy. Received 11 April 2002; accepted 18 April 2002 RID="*" ID="*"Corresponding author.     

Structure to function relationships in ceruloplasmin: a 'moonlighting' protein

Specialised copper sites have been recruited during evolution to provide long-range electron transfer reactivity and oxygen binding and activation in proteins destined to cope with oxygen reactivity in different organisms. Ceruloplasmin is an ancient multicopper oxidase evolved to insure a safe handling of oxygen in some metabolic pathways of vertebrates. The presently available knowledge of its structure provides a glimpse of its plasticity, revealing a multitude of binding sites that point to an elaborate mechanism of multifunctional activity. Ceruloplasmin represents an example of a 'moonlighting' protein that overcomes the one gene-one structure-one function concept to follow the changes of the organism in its physiological and pathological conditions. Received 19 February 2002; received after revision 29 March 2002; accepted 2 April 2002 RID="*" ID="*"Corresponding author.