Identification and subcellular localization of a putative cell-to-cell transport protein from red clover mottle virus

To investigate the mode of gene expression of red clover mottle virus (RCMV) middle component (M) RNA, we have synthesized an oligopeptide corresponding to the predicted carboxy-terminus of the RCMV counterparts of the cowpea mosaic virus (CPMV) 48K and 58K proteins. Using an antiserum raised against this synthetic oligopeptide, we have detected a 43-kDa protein in the 30,000 g pellet from extracts of RCMV-infected cowpea protoplasts. Immunogold cytochemistry further localized this protein to the plasmodesmata of RCMV-infected pea tissue. This subcellular location, taken together with other evidence, suggests that this 43-kDa protein has a role in the cell-to-cell spread of RCMV.     

Identification of a novel receptor-like protein kinase that interacts with a geminivirus nuclear shuttle protein

Despite extensive studies in plant virus-host interactions, the molecular mechanisms of geminivirus movement and interactions with host components remain largely unknown. A tomato kinase protein and its soybean homolog were found to interact specifically with the nuclear shuttle protein (NSP) of Tomato golden mosaic virus (TGMV) and Tomato crinkle leaf yellows virus (TCrLYV) through yeast two-hybrid screening and in vitro protein binding assays. These proteins, designated LeNIK (Lycopersicon esculentum NSP-Interacting Kinase) and GmNIK (Glycine max NIK), belong to the LRR-RLK (leucine rich-repeat receptor-like kinase) family that is involved in plant developmental processes and/or resistance response. As such, NIK is structurally organized into characteristic domains, including a serine/threonine kinase domain with a nucleotide binding site at the C-terminal region, an internal transmembrane segment and leucine-rich repeats (LRR) at the N-terminal portion. The potential significance of the NSP-NIK interaction is discussed.     

T细胞衔接活化因子-绿色荧光蛋白真核表达质粒的构建及其在Jurkat细胞的定位

目的:构建增强型绿色荧光蛋白(EGFP)与T细胞衔接活化因子(Linker for activated ofT cells,LAT)融合蛋白的真核表达载体,观测LAT-EGFP在Jurkat细胞中的定位表达.方法:利用RT-PCR技术提取并扩增LAT除去终止密码子外的全部序列,克隆到真核表达载体PEGFP-N3,酶切鉴定并测序.瞬时转染到Jurkat细胞中进行表达,荧光共聚焦显微镜观察LAT-EGFP在Jurkat细胞中的表达及细胞定位.提取转染后细胞总RNA,通过RT-PCR的方法检测LAT-EGFP在转录水平的表达.利用Western blot法进一步鉴定融合蛋白的表达.结果:重组载体经酶切鉴定,切出片段长度在750 bp左右与插入序列长度相符,并进一步进行测序鉴定证实连接完全正确.共聚焦显微镜观察表达的LAT-EGFP融合蛋白定位在细胞膜上,并呈点簇状聚集状态.RT-PCR扩增证实了LAT和EGFP的融合蛋白在Jurkat细胞中在转录水平的表达,Western blot分析进一步证明了LAT和EGFP融合蛋白构建成功,并在蛋白水平上有明显的融合表达.结论:成功构建真核表达载体LAT-EGFP,且融合蛋白LAT-EGFP与野生型LAT在Jurkat细胞中的定位一致,具有功能表达效应,这为后续准确研究具有棕榈酰化位点的跨膜接头蛋白的信号转导作用提供了一种良好的研究载体和方法.     

蛋白质的亚细胞定位预测研究进展

蛋白质处于特定的亚细胞位置上才能行使其功能,故研究亚细胞定位对了解蛋白质功能非常重要。随着后基因组时代的来临,蛋白质序列信息增长迅速,而利用实验手段分析蛋白亚细胞定位的不易大规模进行。近年来,通过提取蛋白质的各种特征信息,自动预测蛋白质的亚细胞定位的算法得到了较快的发展,本文拟从蛋白质特征信息的提取、预测算法和预测效果检验等方面,介绍蛋白质亚细胞定位预测领域中研究进展。     

Positive inotropic effect of interleukin-2. Role of phospholipases and protein kinase C

Recently, we have shown that soluble factors released by human lymphocytes after lectin stimulation could increase the contractile tension of rat atria “in vitro” and that interleukin-2 (IL-2) could be part of this reaction. The effect of IL-2 was potentiated by the Ca²⁺ ionophore A23187 or free arachidonic acid (AA). In this study we demonstrate that the action of IL-2 can be prevented by pre-incubation of the heart tissue with monoclonal anti-IL-2 receptor (anti-p55), suggesting that binding to the IL-2 receptor is necessary for the induction of the biologic effect. In the presence of A23187 or AA, the effect of the synthetic diacylglyceride oleoyl-acetyl-glycerol (OAG) was similar to that of IL-2. Elimination of phospholipase C activity by pre-incubation of the atria with 2-nitro-carboxyphenyl,N,N-diphenylcarbamate (NCDC) abrogated the effects of IL-2 in the presence of A23187 or AA, but was ineffective when OAG + A23187 or OAG + AA was used. Inhibition of atrial phospholipase A₂ activity with p-bromo-phenacylbromide (BPB) blocked the response of atria to either IL-2 + A23187 or OAG + A23187 but was not effective when AA was used as second signal (IL-2 + AA or OAG + AA). Both the OAG and the IL-2 positive inotropic effects could be prevented by the protein kinase C inhibitor 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine (H7) but were poorly inhibited by N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA1004), an inhibitor of the cyclic nucleotide-dependent protein kinases. The atrial cyclo-oxygenase and lipoxygenase pathways of AA metabolism were required for the development of the IL-2 or OAG effects, indicating a role for the oxidative metabolites of AA in the production of the positive inotropic effect. These observations suggest that in this experimental system, IL-2 may trigger phospholipid turnover, generating metabolites of the phospholipase A₂ and phospholipase C pathways that can in turn favor the activation of protein kinase C and the positive inotropic response of the heart.     

Pharmacological regulation of network kinetics by protein kinase C localization

Protein kinase C (PKC) is a conserved family of 11 serine/threonine kinases. Most cell types express multiple members of the family. Because the catalytic sites are homologous, and able to accommodate a broad range of substrates in vitro, specificity in function is dependent on subcellular localization of each isozyme in each cell type. Physiological stimulation can result in major changes in localization of individual PKC isozymes, mediated through binding to specific anchoring proteins. We describe data demonstrating that disruption of such translocations of PKC isozymes by pharmacological agents, peptides, or antibodies, causes profound effects on T cell functions. The pharmacological opportunity provided by distinct kinetic properties of complex assembly is also discussed.     

The Interaction between geminivirus pathogenicity proteins and adenosine kinase leads to increased expression of primary cytokinin-responsive genes

Pathogenicity proteins (AL2/C2) of begomo- and curtoviruses suppress silencing through inhibition of the methyl cycle, as a consequence of inhibiting adenosine kinase (ADK). ADK phosphorylates cytokinin nucleosides, helping maintain a pool of bioactive cytokinins through interconversion of free-bases, nucleosides and nucleotides. We provide evidence that inhibiting ADK affects expression of primary cytokinin-responsive genes. Specifically, we demonstrate increased activity of a primary cytokinin-responsive promoter in adk mutant Arabidopsis plants, and in response to silencing ADK expression or inhibiting ADK activity in transient assays. Similar changes in expression are observed in geminivirus infected tissue and when AL2/C2 are over-expressed. Increased cytokinin-responsive promoter activity may therefore be a consequence of an ADK/AL2/C2 interaction. Application of exogenous cytokinin increases susceptibility to geminivirus infection, characterized by a reduced mean latent period and enhanced viral replication. Thus, ADK appears to be a high value target of geminiviruses that includes increasing expression of primary cytokinin-responsive genes.     

Interaction of geminivirus Rep protein with replication factor C and its potential role during geminivirus DNA replication

Geminivirus DNA replication during the rolling-circle stage depends on the use of a DNA primer, a strategy poorly understood as compared with other eukaryotic viral systems that rely on RNA or protein as primers. Here we have used wheat dwarf virus (WDV) with the aim of elucidating the events leading to recruitment of cell factors at the replication origin. We have identified a novel interaction of WDV Rep, the replication initiation protein, with the large subunit of the wheat replication factor C complex (TmRFC-1). In other systems, the heteropentameric RFC clamp loader complex stimulates loading of DNA polymerase delta to the primer-template. Expression of TmRFC-1 is subjected to cell-cycle regulation, with a peak in early S-phase. We show that WDV Rep stimulates binding of recombinant TmRFC-1 to a model substrate containing a 3'-OH terminus and a WDV Rep-binding site. This was confirmed using cellular fractions enriched for wheat RFC complex, supporting the idea that, in addition to generating a 3'-OH terminus during initiation of DNA replication, WDV Rep could participate in the recruitment of RFC to the newly formed primer. We propose that this pathway may represent an initial event to facilitate the assembly of other replication factors, e.g., PCNA and/or DNA polymerase delta, a model that could also apply to other eukaryotic replicons, such as nanoviruses, circoviruses, and parvoviruses with a similar DNA replication strategy.     

Effect of exercise on protein kinase C activity and localization in human skeletal muscle

To investigate the effect of exercise on protein kinase C (PKC) activity and localization in human skeletal muscle, eight healthy men performed cycle ergometer exercise for 40 min at 76 ± 1% the peak pulmonary O₂ uptake     , with muscle samples obtained at rest and after 5 and 40 min of exercise. PKC expression, phosphorylation and activities were examined by immunoblotting and in vitro kinase assays of fractionated and whole tissue preparations. In response to exercise, total PKC activity was slightly higher at 40 min in an enriched membrane fraction, and using a pSer-PKC-substrate motif antibody it was revealed that exercise increased the serine phosphorylation of a ∼50 kDa protein. There were no changes in conventional PKC (cPKC) or PKCθ activities; however, atypical PKC (aPKC) activity was ∼70% higher at 5 and 40 min, and aPKC expression and Thr^410/403 phosphorylation were unaltered by exercise. There were no effects of exercise on the abundance of PKCα, PKCδ, PKCθ and aPKC within cytosolic or enriched membrane fractions of skeletal muscle. These data indicate that aPKC, but not cPKC or PKCθ, are activated by exercise in contracting muscle suggesting a potential role for aPKC in the regulation of skeletal muscle function and metabolism during exercise in humans.     

Differential localization of protein kinase C isotypes in equine eosinophils and neutrophils

Phorbol esters, which activate protein kinase C (PKC), stimulate equine eosinophil superoxide production and adherence. After showing that superoxide production could be inhibited by the nonselective PKC inhibitors, staurosporine and bisindolymaleimide I, the PKC isotypes in equine eosinophils were characterized, because evidence suggests that individual isotypes may play distinct roles in regulating eosinophil function. Western blots demonstrated that equine eosinophils expressed PKC alpha, beta, delta, epsilon, iota, and zeta. However, unlike the equine neutrophil, the majority of the PKC was detected in the particulate fraction of the cell. Despite this unusual location, the PKC in equine eosinophils was activatable, suggesting that it is functionally competent. The regulatory role of PKC in equine eosinophils may reflect the association of activity with the particulate fraction and the profile of isotype expression.     

Identification of a putative motif for binding of peptides to HLA-DQ2

To understand the rules determining peptide binding to the celiacdisease and type 1 diabetes mellitus associated HLA-DQ2 molecule,we have studied in detail the binding of a peptide OVA 258–276Y(IINFEKLTEWTSSNVMEERY) which exhibitsstrong binding to DQ2.First we tested a set of N- and C-terminal truncated variants,and found the core binding region to comprise residues 267–276Y.Single alanine substitution analysis of the OVA 267–276Ypeptide revealed thatreplacements of V₂₇₂, E₂₇₅ and the C-terminalY had negative effects whereas the substitution of N₂₇₁ hada positive effect. A polyalanine analogue of the OVA 267–276Ypeptide with V₂₇₂, E₂₇₅ and a C-terminal Y bound at least aswell as the original peptide. A variant peptide with a deletionof R₂₇₆ displayed decreased binding, suggesting that the anchorresidues were out of frame in this analogue. To further characterizethe residues playing a role in the binding of the OVA 267–276Ypeptide to DQ2 we tested the binding ofseveral analogues withsubstitutions for V₂₇₂, E₂₇₅ and the C-terminal Y residue. Ourresults indicate that peptides binding to DQ2 have anchor residuesin relative positions 4, 7 and 9 (P4, P7 and P9). Residues withnegatively charged or hydrophobic aliphatic but not positivelycharged side chains are preferred in P4 and P7, whereas residueswith bulky hydrophobic side chains are preferred in P9.     

Robust prediction of protein subcellular localization combining PCA and WSVMs

Automated prediction of protein subcellular localization is an important tool for genome annotation and drug discovery, and Support Vector Machines (SVMs) can effectively solve this problem in a supervised manner. However, the datasets obtained from real experiments are likely to contain outliers or noises, which can lead to poor generalization ability and classification accuracy. To explore this problem, we adopt strategies to lower the effect of outliers. First we design a method based on Weighted SVMs, different weights are assigned to different data points, so the training algorithm will learn the decision boundary according to the relative importance of the data points. Second we analyse the influence of Principal Component Analysis (PCA) on WSVM classification, propose a hybrid classifier combining merits of both PCA and WSVM. After performing dimension reduction operations on the datasets, kernel-based possibilistic c-means algorithm can generate more suitable weights for the training, as PCA transforms the data into a new coordinate system with largest variances affected greatly by the outliers. Experiments on benchmark datasets show promising results, which confirms the effectiveness of the proposed method in terms of prediction accuracy.     

Synthesis and subcellular localization of the murine coronavirus nucleocapsid protein

The synthesis and processing of the nucleocapsid protein (pp60) of the JHM strain of murine coronaviruses were examined. Pulse-chase experiments showed that pp60 was synthesized initially as a protein of approximately 57,000 in molecular weight (p57). Immunoprecipitation using mouse anti-JHMV antiserum indicated that p57 was virus specific. Immunoprecipitation with monoclonal antibodies specific for pp60 showed that p57 was antigenically related to pp60 and was not phosphorylated, while the intracellular protein that comigrated with the virion nucleocapsid protein, pp60, was phosphorylated. The p57 was found exclusively in the cytosol while the majority of pp60 was associated with the membrane fraction but pp60 was not an integral membrane protein.     

Factors influencing subcellular localization of the human papillomavirus L2 minor structural protein

Two structural proteins form the capsids of papillomaviruses. The major structural protein L1 is the structural determinant of the capsids and is present in 360 copies arranged in 72 pentamers. The minor structural protein L2 is estimated to be present in twelve copies per capsid. Possible roles for L2 in interaction with cell surface receptors and in virion uptake have been suggested. As previously reported, L2 localizes in subnuclear domains identified as nuclear domain 10 (ND10). As it was demonstrated that L2 is able to recruit viral and cellular proteins to ND10, a possible role for L2 as a mediator in viral assembly has been proposed. In this study, we determined factors influencing the localization of L2 at ND10. Under conditions of moderate L2 expression level and in the absence of heterologous viral components, we observed that, in contrast to previous reports, L2 is mainly distributed homogeneously throughout the nucleus. L2, however, is recruited to ND10 at a higher expression level or in the presence of viral components derived from vaccinia virus or from Semliki Forest virus. We observed that translocation of L2 to ND10 is not a concentration-dependent accumulation but rather seems to be triggered by yet unidentified cellular factors. In contrast to HPV 11 and 16 L2, the HPV 18 L2 protein seems to require L1 for efficient nuclear accumulation.     

The geminivirus nuclear shuttle protein is a virulence factor that suppresses transmembrane receptor kinase activity

Despite the large number of leucine-rich-repeat (LRR) receptor-like-kinases (RLKs) in plants and their conceptual relevance in signaling events, functional information is restricted to a few family members. Here we describe the characterization of new LRR-RLK family members as virulence targets of the geminivirus nuclear shuttle protein (NSP). NSP interacts specifically with three LRR-RLKs, NIK1, NIK2, and NIK3, through an 80-amino acid region that encompasses the kinase active site and A-loop. We demonstrate that these NSP-interacting kinases (NIKs) are membrane-localized proteins with biochemical properties of signaling receptors. They behave as authentic kinase proteins that undergo autophosphorylation and can also phosphorylate exogenous substrates. Autophosphorylation occurs via an intermolecular event and oligomerization precedes the activation of the kinase. Binding of NSP to NIK inhibits its kinase activity in vitro, suggesting that NIK is involved in antiviral defense response. In support of this, infectivity assays showed a positive correlation between infection rate and loss of NIK1 and NIK3 function. Our data are consistent with a model in which NSP acts as a virulence factor to suppress NIK-mediated antiviral responses.     

Geminivirus pathogenicity protein C4 interacts with Arabidopsis thaliana shaggy-related protein kinase AtSKeta, a component of the brassinosteroid signalling pathway

Beet curly top virus (BCTV) C4 interacted with two members of the shaggy-related protein kinase family (AtSKeta and AtSKzeta) and a putative leucine-rich repeat receptor-like kinase (LRR-RLK) in a yeast two-hybrid assay. Tomato golden mosaic virus (TGMV) AC4 also bound with similar efficiency to AtSKeta and AtSKzeta but was unable to interact with the LRR-RLK. BCTV C4 interaction with AtSKeta was confirmed using an in vitro binding assay. The protein kinases were capable of autophosphorylation in vitro and AtSKeta phosphorylated BCTV C4 at threonine and serine residues. AtSKeta phosphorylation of TGMV AC4 was significantly less efficient. The LRR-RLK did not efficiently phosphorylate BCTV C4. BCTV C4 localisation to the cell periphery in Nicotiana benthamiana was dependent on an intact N-terminal myristoylation motif, consistent with plasma membrane targeting. The intact motif was also required to produce the wild-type disease phenotype. Transient expression of BCTV C4 and TGMV AC4 derivatives in N. benthamiana identified additional amino acids within a central domain that contribute to the phenotype. The interaction with AtSKeta indicates that BCTV C4 interacts with the brassinosteroid signalling pathway.     

Two protein tyrosine phosphatases, Ptp2 and Ptp3, modulate the subcellular localization of the Hog1 MAP kinase in yeast

The MAP kinase Hog1 transiently accumulates in the nucleus upon activation. Although Hog1 nuclear export correlates with its dephosphorylation, we find that dephosphorylation is not necessary for export. Unexpectedly, a strain lacking the nuclear protein tyrosine phosphatase, Ptp2, showed decreased Hog1 nuclear retention, while a strain lacking the cytoplasmic Ptp3 showed prolonged Hog1 nuclear accumulation, consistent with Ptp2 being a nuclear tether for Hog1 and Ptp3 being a cytoplasmic anchor. In support of this result PTP2 overexpression sequestered Hog1 in the nucleus while PTP3 overexpression restricted Hog1 to the cytoplasm. Thus, Ptp2 and Ptp3 regulate Hog1 localization by binding Hog1.     

Expression and chromosomal localization of KIAA0369, a putative kinase structurally related to Doublecortin

Neuropathy in vertebrates can be a consequence of failure of genes involved in the nervous system to be expressed at the correct times and levels during embryonic life. Recently, a brain specific gene, Doublecortin, was cloned and was shown to have mutations in X-linked lissencephaly and double cortex syndrome. KIAA0369 is a putative kinase that is structurally related to Doublecortin. We compared the expression of KIAA0369 with that of Doublecortin, both of which were expressed specifically or predominantly in fetal brain among 20 different tissues examined. The deduced products of both genes contain a unique domain (the Doublecortin [DC] domain), but KIAA0369 also contains a calmodulin-dependent kinase (CaM kinase)-like domain following the DC domain. We found at least four splicing variants of KIAA0369: KIAA0369-AS (type A, short version), KIAA0369-AL (type A, long version), KIAA0369-BS (type B, short version), and KIAA0369-BL (type B, long version). KIAA0369-B, which lacked the DC domain and maintained the kinase domain, was expressed in adult as well as fetal brain, but the variants that included the DC domain, KIAA0369-A, were expressed predominantly in fetal brain. These results suggest that the DC domain plays an important role in the development of the nervous system. In the adult brain, KIAA0369 was expressed in all 15 different regions examined, more intensely in cerebral cortex, occipital pole, frontal lobe, amygdala, and hippocampus, and less intensely in corpus callosum and thalamus. The murine homologs of Doublecortin and KIAA0369 were not detectable in 7-day mouse embryos, but both genes were expressed extensively in 11-day embryos. Human KIAA0369 was mapped by fluorescence in situ hybridization (FISH) to chromosome 13q13–q14.1. The presence of genes related to neuropathy has been reported in this locus. Received: February 23, 1998 / Accepted: April 14, 1998