A Capillary Electrophoresis-Based Assay for Protein Kinases and Protein Phosphatases Using Peptide Substrates

A novel procedure for detection and assay of protein kinase and phosphatase activities in complex biological mixtures was developed. By means of capillary zone electrophoresis (CZE) methodology, the phosphorylated and dephosphorylated forms of the peptide Kemptide, a 46-amino-acid fragment from protein phosphatase inhibitor-1 and a peptide fragment corresponding to the R_II subunit of cAMP-dependent protein kinase (PKA), were rapidly resolved. This facilitated nonradioactive detection of PKA and protein phosphatase-2B (calcineurin) in rabbit skeletal muscle extracts. In addition, the CZE procedure enabled a site-specific assay of a 14-amino-acid peptide from the glycogen-binding subunit of protein phosphatase-1 monophosphorylated on distinct sites by PKA and casein kinase-II. These results suggest that CZE may prove to be extremely useful for the analysis of peptides that are phosphorylated at multiple sites in vivo.     

Dinucleotide Receptor Modulation by Protein Kinases (Protein Kinases A and C) and Protein Phosphatases in Rat Brain Synaptic Terminals

Abstract: The diadenosine polyphosphates, diadenosine tetraphosphate and diadenosine pentaphosphate (Ap₅A), can activate an ionotropic dinucleotide receptor that induces Ca²⁺ transients into synaptosomes prepared from rat brain. This receptor, also termed the P₄ purinoceptor, is sensitive only to adenine dinucleotides and is insensitive to ATP. Studies on the modulatory role of protein kinase A (PKA), protein kinase C (PKC), and protein phosphatases on the response of diadenosine polyphosphate receptors were performed by measuring the changes in the intracellular Ca²⁺ levels with fura-2. Activation and inhibition of PKA were carried out by means of forskolin and the PKA inhibitory peptide (PKA-IP), respectively. The Ap₅A response was inhibited by forksolin to 35% of control values, but PKA-IP induced an increase of 37%. The effect of PKC activation was similar to that observed for PKA. PKC stimulation with phorbol 12,13-dibutyrate produced an inhibition of 67%, whereas the PKC inhibitors staurosporine and PKC inhibitory peptide enhanced the responses elicited by Ap₅A to 40% in both cases. Protein phosphatase inhibitors diminished the responses elicited by Ap₅A to 17% in the case of okadaic acid, to 50% for microcystin, and to 45% in the case of cyclosporin A. Thus, the activity of dinucleotide receptors in rat brain synaptosomes appears to be modulated by phosphorylation/dephosphorylation. These processes could be of physiological significance in the control of transmitter release from neurons that are postsynaptic to nerves that release diadenosine polyphosphates.     

A Genetic Analysis of Chloroplast Division and Expansion in Arabidopsis thaliana

A nuclear recessive mutant of Arabidopsis thaliana, arc5, has been isolated in which there is no significant increase in chloroplast number during leaf mesophyll cell expansion and in which there are only 13 chloroplasts per mesophyll cell compared with 121 in wild-type cells. Mature arc5 chloroplasts in fully expanded mesophyll cells are 6-fold larger than in wild-type cells. A large proportion of arc5 chloroplasts also show some degree of central constriction, suggesting that the mutation has prevented the completion of the chloroplast division process. To examine the interaction of arc loci, a double mutant was constructed between arc1, a mutant possessing many small chloroplasts, and arc5. A second double mutant was also constructed between arc3, a previously discovered mutant also possessing few large chloroplasts per cell, and arc1. Analysis of these double mutants shows that chloroplast number per mesophyll cell is greater when arc5 and arc3 mutations are expressed in the arc1 background than when expressed alone. The cell-specific nature of arc mutants was also analyzed. The phenotypic traits characteristic of arc3 and arc5 are a reduction in chloroplast number and an increase in chloroplast size in mesophyll cells: these changes are also observed in reduced form in the epidermal and guard cell chloroplasts of arc3 and arc5 plants. Analysis of parenchyma sheath cell chloroplasts suggests that in leaves of arc1 plants the normal developmental distinction between mesophyll and parenchyma sheath chloroplasts is perturbed. The relevance of these findings to the analysis of the control of chloroplast division in mesophyll cells is discussed.     

A Putative Chloroplast Thylakoid Metalloprotease VIRESCENT3 Regulates Chloroplast Development in Arabidopsis thaliana

The chloroplast is the site of photosynthesis and many other essential plant metabolic processes, and chloroplast development is an integral part of plant growth and development. Mutants defective in chloroplast development can display various color phenotypes including the intriguing virescence phenotype, which shows yellow/white coloration at the leaf base and greening toward the leaf tip. Through large scale genetic screens, we identified a series of new virescent mutants including virescent3-1 (vir3-1), vir4-1, and vir5-1 in Arabidopsis thaliana. We showed that VIR3 encodes a putative chloroplast metalloprotease by map-based cloning. Through site-directed mutagenesis, we showed that the conserved histidine 235 residue in the zinc binding motif HEAGH of VIR3 is indispensable for VIR3 accumulation in the chloroplast. The chloroplast localization of VIR3 was confirmed by the transient expression of VIR3-GFP in leaf protoplasts. Furthermore, taking advantage of transgenic lines expressing VIR3-FLAG, we demonstrated that VIR3 is an intrinsic thylakoid membrane protein that mainly resides in the stromal lamellae. Moreover, topology analysis using transgenic lines expressing a dual epitope-tagged VIR3 indicated that both the N and C termini of VIR3 are located in the stroma, and the catalytic domain of VIR3 is probably facing the stroma. Blue native gel analysis indicated that VIR3 is likely present as a monomer or part of a small complex in the thylakoid membrane. This work not only implicates VIR3 as a new factor involved in early chloroplast development but also provides more insight into the roles of chloroplast proteases in chloroplast biogenesis.     

Regulation of Ethylene Biosynthesis and Signaling by Protein Kinases and Phosphatases

Ethylene, a gaseous plant hormone, plays critical roles in plant growth, development, and response to environment. Ethylene-regulated processes are initiated by the elevation of ethylene biosynthesis, which is under tight control by a complex signaling network. An elevated level of ethyl- ene is then perceived by ethylene receptors in local and neighboring cells, which activates signaling pathways that lead to ethylene responses. Different types of tissues/cells have differential capacities in producing ethylene and dif- ferential sensitivity to ethylene, which are crucial to the diverse functions of ethylene in plants. This report high- lights recent advances in our understanding of kinases and phosphatases in ethylene biosynthesis and signaling.     

Differential Protein Phosphorylation Regulates Chloroplast Movement in Response to Strong Light and Darkness in Arabidopsis thaliana

Phototropin-dependent chloroplast movement is essential to the photosynthetic acclimation of mesophyll cells to incident light. Chloroplast movement involves many cellular actors, such as chloroplast-associated actin filaments and proteins that mediate signalling between phototropins and chloroplast motion. In the past few years, genetic approaches have identified several key proteins but the intrinsic mechanisms of the signalling cascade, such as phosphorylation events, remain undefined. Here, we took advantage of phosphoproteomics to examine the involvement of protein phosphorylation in chloroplast movement in darkness or under high light, at different CO₂ mole fractions (100, 380 or 1,000 ppm) to vary photosynthetic activity. Amongst the 100 relevant identified phosphopeptides, 19 (corresponding to 8 proteins) were differentially phosphorylated in darkness vs. high light. There was no significant CO₂ effect on the observed phosphorylation patterns. We further characterized the phosphorylation sites in THRUMIN1, which is believed to be crucial for the attachment of chloroplast-associated actin filaments to the plasma membrane and thus for chloroplast movements. The mutant thrumin1 was complemented with a mutated protein in which phospho-sites were substituted to a phosphomimetic (Asp) or a non-phosphorylatable (Ala) residue. While the phosphomimetic substitution altered the chloroplast response in the light only, both light and dark responses were altered with the non-phosphorylatable substitution. Our data suggest a key role of protein phosphorylation, including that of THRUMIN1, in the light/dark control of chloroplast movements.     

Mitogen-Activated Protein Kinases 3 and 6 Are Required for Full Priming of Stress Responses in Arabidopsis thaliana

In plants and animals, induced resistance (IR) to biotic and abiotic stress is associated with priming of cells for faster and stronger activation of defense responses. It has been hypothesized that cell priming involves accumulation of latent signaling components that are not used until challenge exposure to stress. However, the identity of such signaling components has remained elusive. Here, we show that during development of chemically induced resistance in Arabidopsis thaliana, priming is associated with accumulation of mRNA and inactive proteins of mitogen-activated protein kinases (MPKs), MPK3 and MPK6. Upon challenge exposure to biotic or abiotic stress, these two enzymes were more strongly activated in primed plants than in nonprimed plants. This elevated activation was linked to enhanced defense gene expression and development of IR. Strong elicitation of stress-induced MPK3 and MPK6 activity is also seen in the constitutive priming mutant edr1, while activity was attenuated in the priming-deficient npr1 mutant. Moreover, priming of defense gene expression and IR were lost or reduced in mpk3 or mpk6 mutants. Our findings argue that prestress deposition of the signaling components MPK3 and MPK6 is a critical step in priming plants for full induction of defense responses during IR.     

拟南芥未知功能基因At3g61870编码蛋白的叶绿体定位研究

利用反向遗传学研究方法对1个预测的拟南芥叶绿体未知功能基因At3g61870编码蛋白进行了亚细胞定位研究.通过克隆At3g61870基因5′端长229 bp的DNA片段,与绿色荧光蛋白(GFP)基因构建重组表达载体pMON530-CP-TP-GFP,经农杆菌介导转化拟南芥.转基因植株的叶肉细胞经激光共聚焦显微镜观察,叶绿素自发荧光与GFP荧光共定位于叶绿体中.结果表明,未知功能基因At3g61870编码的蛋白质为叶绿体蛋白质.     

拟南芥叶绿体蛋白质组研究

在后基因组时代,蛋白质组学技术已成为鉴定植物蛋白质功能的有力工具之一,以模式植物拟南芥为材料进行蛋白质组学研究是大家关注的焦点.叶绿体作为重要的细胞器,在植物蛋白质组学中已有较多的研究,本文对近年来拟南芥叶绿体蛋白质组学的研究加以综述,并对未来发展趋势进行了展望.     

Select Alterations in Protein Kinases and Phosphatases During Apoptosis of Differentiated PC12 Cells

Abstract: The involvement of cell cycle-regulatory proteins in apoptosis of neuronally differentiated PC12 cells induced by the removal of nerve growth factor and serum was examined. Three major findings are presented. (1) Cdc2 kinase protein levels increased fivefold in apoptotic PC12 cells by day 3 of serum and nerve growth factor deprivation. Histone H1 kinase activity was increased significantly in p13^suc1 precipitates of apoptotic PC12 cells, which was due to increased activation and/or expression of cdc2 kinase. (2) The protein levels of cyclin-dependent kinase 4, cyclin D, and proliferating cell nuclear antigen that are normally expressed in the cell cycle were increased during neuronal PC12 cell apoptosis. (3) The levels of the catalytic subunit, but not the regulatory subunit of the calcium/calmodulin-dependent protein phosphatase 2B, decreased significantly concomitant with a significant decrease in protein phosphatase 2B activity early in the apoptotic process. Protein phosphatase 2A activity decreased slightly but significantly after 3 days of serum and nerve growth factor deprivation, and no alterations in protein phosphatase 1 were observed during the apoptotic process. These data demonstrate that certain cell cycle-regulatory proteins are inappropriately expressed and that alterations in specific phosphorylation events, as indicated by the increase in histone H1 kinase activity and the decrease in protein phosphatase 2B activity, are most likely occurring during apoptosis of PC12 cells. These observations support the hypothesis that apoptosis may be due in part to a nondividing cell's uncoordinated attempt to reenter and progress through the cell cycle.     

Brassinosteroid-regulated GSK3/Shaggy-like Kinases Phosphorylate Mitogen-activated Protein (MAP) Kinase Kinases,Which Control Stomata Development in Arabidopsis thaliana

Brassinosteroids (BRs) are steroid hormones that coordinate fundamental developmental programs in plants. In this study we show that in addition to the well established roles of BRs in regulating cell elongation and cell division events, BRs also govern cell fate decisions during stomata development in Arabidopsis thaliana. In wild-type A. thaliana, stomatal distribution follows the one-cell spacing rule; that is, adjacent stomata are spaced by at least one intervening pavement cell. This rule is interrupted in BR-deficient and BR signaling-deficient A. thaliana mutants, resulting in clustered stomata. We demonstrate that BIN2 and its homologues, GSK3/Shaggy-like kinases involved in BR signaling, can phosphorylate the MAPK kinases MKK4 and MKK5, which are members of the MAPK module YODA-MKK4/5-MPK3/6 that controls stomata development and patterning. BIN2 phosphorylates a GSK3/Shaggy-like kinase recognition motif in MKK4, which reduces MKK4 activity against its substrate MPK6 in vitro. In vivo we show that MKK4 and MKK5 act downstream of BR signaling because their overexpression rescued stomata patterning defects in BR-deficient plants. A model is proposed in which GSK3-mediated phosphorylation of MKK4 and MKK5 enables for a dynamic integration of endogenous or environmental cues signaled by BRs into cell fate decisions governed by the YODA-MKK4/5-MPK3/6 module.     

A Pseudomonas syringae ADP-Ribosyltransferase Inhibits Arabidopsis Mitogen-Activated Protein Kinase Kinases

The successful recognition of pathogen-associated molecular patterns (PAMPs) as a danger signal is crucial for plants to fend off numerous potential pathogenic microbes. The signal is relayed through mitogen-activated protein kinase (MPK) cascades to activate defenses. Here, we show that the Pseudomonas syringae type III effector HopF2 can interact with Arabidopsis thaliana MAP KINASE KINASE5 (MKK5) and likely other MKKs to inhibit MPKs and PAMP-triggered immunity. Inhibition of PAMP-induced MPK phosphorylation was observed when HopF2 was delivered naturally by the bacterial type III secretion system. In addition, HopF2 Arg-71 and Asp-175 residues that are required for the interaction with MKK5 are also necessary for blocking MAP kinase activation, PAMP-triggered defenses, and virulence function in plants. HopF2 can inactivate MKK5 and ADP-ribosylate the C terminus of MKK5 in vitro. Arg-313 of MKK5 is required for ADP-ribosylation by HopF2 and MKK5 function in the plant cell. Together, these results indicate that MKKs are important targets of HopF2.     

Chloroplast Cpn20 forms a tetrameric structure in Arabidopsis thaliana

Chloroplast chaperonin 20 (Cpn20) in higher plants is a functional homologue of the Escherichia coli GroES, which is a critical regulator of chaperonin-mediated protein folding. The cDNA for a Cpn20 homologue of Arabidopsis thaliana was isolated. It was 958 bp long, encoding a protein of 253 amino acids. The protein was composed of an N-terminal chloroplast transit peptide, and the predicted mature region comprised two distinct GroES domains that showed 42% amino acid identity to each other. The isolated cDNA was constitutively expressed in transgenic tobacco. Immunogold labelling showed that Cpn20 is accumulated in chloroplasts of transgenic tobacco. A Northern blot analysis revealed that mRNA for the chloroplast Cpn20 is abundant in leaves and is increased by heat treatment. To examine the oligomeric structure of Cpn20, a histidine-tagged construct lacking the transit peptide was expressed in E. coli and purified by affinity chromatography. Gel-filtration and cross-linking analyses showed that the expressed products formed a tetramer. The expressed products could substitute for GroES to assist the refolding of citrate synthase under non-permissive conditions. The analysis on the subunit stoichiometry of the GroEL-Cpn20 complex also revealed that the functional complex is composed of a GroEL tetradecamer and a Cpn20 tetramer.     

In vivo Studies on the Roles of Tic55-Related Proteins in Chloroplast Protein Import in Arabidopsis thaliana

The TicS5 （Translocon at the inner envelope membrane of chloroplasts, 55 kDa） protein was identified in pea as a putative regulator, possibly linking chloroplast protein import to the redox state of the photosynthetic machinery. Two Tic55 homologs have been proposed to exist in Arabidopsis： atTic55-11 and AtPTC52 （Protochlorophyllide-dependent Trans- Iocon Component, 52 kDa; has also been called atTic55-1V）. Our phylogenetic analysis shows that attic55-11 is an ortholog of psTic55 from pea （Pisum sativurn）, and that AtPTC52 is a more distant homolog of the two. AtPTC52 was included in this study to rule out possible functional links between the proteins in Arabidopsis. No detectable mutant phenotypes were found in two independent T-DNA knockout mutant plant lines for each Arabidopsis protein, when compared with wild- type： visible appearance, chlorophyll content, photosynthetic performance, and chloroplast protein import, for example, were all normal. Both wild-type and tic55-11 mutant chloroplasts exhibited deficient protein import when treated with diethylpyrocarbonate, indicating that Tic55 is not the sole target of this reagent in relation to protein import. Furthermore, ptc52 mutant chloroplasts were not defective with respect to pPORA import, which was previously reported to involve PTC52 in barley. Thus, we conclude that atTic55-11 and AtPTC52 are not strictly required for functional protein import in Arabidopsis.     

Eukaryote-Like Serine/Threonine Kinases and Phosphatases in Bacteria

Summary: Genomic studies have revealed the presence of Ser/Thr kinases and phosphatases in many bacterial species, although their physiological roles have largely been unclear. Here we review bacterial Ser/Thr kinases (eSTKs) that show homology in their catalytic domains to eukaryotic Ser/Thr kinases and their partner phosphatases (eSTPs) that are homologous to eukaryotic phosphatases. We first discuss insights into the enzymatic mechanism of eSTK activation derived from structural studies on both the ligand-binding and catalytic domains. We then turn our attention to the identified substrates of eSTKs and eSTPs for a number of species and to the implications of these findings for understanding their physiological roles in these organisms.     

拟南芥叶绿体DNA全序列微卫星分布规律的分析

为进一步以拟南芥为模板,开发果树通用的叶绿体微卫星或称简单序列重复（chloroplast simple sequence repeat, cpSSR）引物,对拟南芥叶绿体DNA全序列cpSSR进行了统计分析.结果表明,拟南芥cpSSR以单碱基重复为主,占总数的78.6%,二碱基重复占总数的19%,三碱基重复占2.4%,三碱基以上重复为零.在单碱基重复中,又以A和T重复为主,占98.5%.二碱基重复全部为AT重复.单碱基重复中的纯粹重复41个,占总数的62.1%.间断重复数为23个,占总数的34.8%.复合重复数仅为2个.