Activation of the Stt7/STN7 Kinase through Dynamic Interactions with the Cytochrome b6f Complex

Photosynthetic organisms have the ability to adapt to changes in light quality by readjusting the cross sections of the light-harvesting systems of photosystem II (PSII) and photosystem I (PSI). This process, called state transitions, maintains the redox poise of the photosynthetic electron transfer chain and ensures a high photosynthetic yield when light is limiting. It is mediated by the Stt7/STN7 protein kinase, which is activated through the cytochrome b₆f complex upon reduction of the plastoquinone pool. Its probable major substrate, the light-harvesting complex of PSII, once phosphorylated, dissociates from PSII and docks to PSI, thereby restoring the balance of absorbed light excitation energy between the two photosystems. Although the kinase is known to be inactivated under high-light intensities, the molecular mechanisms governing its regulation remain unknown. In this study we monitored the redox state of a conserved and essential Cys pair of the Stt7/STN7 kinase and show that it forms a disulfide bridge. We could not detect any change in the redox state of these Cys during state transitions and high-light treatment. It is only after prolonged anaerobiosis that this disulfide bridge is reduced. It is likely to be mainly intramolecular, although kinase activation may involve a transient covalently linked kinase dimer with two intermolecular disulfide bonds. Using the yeast two-hybrid system, we have mapped one interaction site of the kinase on the Rieske protein of the cytochrome b₆f complex.Photosynthetic organisms are subjected to constant changes in light quality and quantity and need to adapt to these changes in order to optimize, on the one hand, their photosynthetic yield, and to minimize photo-oxidative damage on the other. The photosynthetic electron transfer chain consists of photosystem II (PSII), the plastoquinone (PQ) pool, the cytochrome b₆f complex (Cyt b₆f), plastocyanin, and photosystem I (PSI). All of these complexes and components are integrated or closely associated with the thylakoid membrane. The two antenna systems of PSII and PSI capture and direct the light excitation energy to the corresponding reaction centers in which a chlorophyll dimer is oxidized and charge separation occurs across the thylakoid membrane. These processes lead to the onset of electron flow from water on the donor side of PSII to ferredoxin on the acceptor side of PSI coupled with proton translocation across the thylakoid membrane. In order to sustain optimal electron flow along this electron transfer chain, the redox poise needs to be maintained under changing environmental conditions. Several mechanisms have evolved for the maintenance of this redox balance. In the case of over-reduction of the acceptor side of PSI, excess electrons can reduce molecular oxygen through the Mehler reaction to superoxide, which is then converted to hydrogen peroxide by a plastid superoxide dismutase and ultimately to water by a peroxidase (Asada, 2000). Over-reduction of the PQ pool can be alleviated by PTOX, the plastid terminal oxidase responsible for oxidizing PQH₂ to form hydrogen peroxide, which is subsequently converted to water (Carol et al., 1999; Cournac et al., 2000; Wu et al., 1999).In addition to these electron sinks that prevent the over-reduction of the electron transfer chain, the photosynthetic apparatus is able to maintain the redox poise of the PQ pool by readjusting the relative cross sections of the light harvesting systems of PSII and PSI upon unequal excitation of the two photosystems. This readjustment can occur both in the short term through state transitions and in the long term by changing the stoichiometry between PSII and PSI (Bonaventura and Myers, 1969; Murata, 1969; Pfannschmidt, 2003). State transitions occur because of perturbations of the redox state of the PQ pool due to unequal excitation of PSII and PSI, limitations in electron acceptors downstream of PSI, and/or in CO₂ availability. Excess excitation of PSII relative to PSI leads to reduction of the PQ pool and thus favors the docking of PQH₂ to the Qo site of the Cyt b₆f complex. This process activates the Stt7/STN7 protein kinase (Vener et al., 1997; Zito et al., 1999), which is closely associated with this complex and leads to the phosphorylation of some LHCII proteins and to their detachment from PSII and binding to PSI (Depège et al., 2003; Lemeille et al., 2009). Although both Lhcb1 and Lhcb2 are phosphorylated, only the phosphorylated form of Lhcb2 is associated with PSI whereas phosphorylated Lhcb1 is excluded from this complex (Longoni et al., 2015). This state corresponds to state 2. In this way the change in the relative antenna sizes of the two photosystems restores the redox poise of the PQ pool. The process is reversible as over-excitation of PSI relative to PSII leads to the oxidation of the PQ pool and to the inactivation of the kinase. Under these conditions, phosphorylated LHCII associated with PSI is dephosphorylated by the PPH1/TAP38 phosphatase (Pribil et al., 2010; Shapiguzov et al., 2010) and returns to PSII (state 1). It should be noted, however, that a strict causal link between LHCII phosphorylation and its migration from PSII to PSI has been questioned recently by the finding that some phosphorylated LHCII remains associated with PSII supercomplexes and that LHCII serves as antenna for both photosystems under most natural light conditions (Drop et al., 2014; Wientjes et al., 2013).State transitions are important at low light but do not occur under high light because the LHCII kinase is inactivated under these conditions (Schuster et al., 1986). It was proposed that inactivation of the kinase is mediated by the ferredoxin-thioredoxin system and that a disulfide bond in the kinase rather than in the substrate may be the target site of thioredoxin (Rintamäki et al., 1997, 2000). Analysis of the Stt7/STN7 protein sequences indeed reveals the presence of two conserved Cys residues close to the N-terminal end of this kinase, which are conserved in all species examined and both are essential for kinase activity although they are located outside of the kinase catalytic domain (Fig. 1) (Depège et al., 2003; Lemeille et al., 2009). Based on protease protection studies, this model of the Stt7/STN7 kinase proposes that the N-terminal end of the kinase is on the lumen side of the thylakoid membrane separated from the catalytic domain on the stromal side by an unusual transmembrane domain containing several Pro residues (Lemeille et al., 2009). This configuration of the kinase allows its catalytic domain to act on the substrate sites of the LHCII proteins, which are exposed to the stroma. Although in this model the conserved Cys residues in the lumen are on the opposite side from the stromal thioredoxins, it is possible that thiol-reducing equivalents are transferred across the thylakoid membrane through the CcdA and Hcf164 proteins, which have been shown to operate in this way during heme and Cyt b₆f assembly (Lennartz et al., 2001; Page et al., 2004) or through the LTO1 protein (Du et al., 2015; Karamoko et al., 2011).Figure 1.Conserved Cys in the Stt7/STN7 kinase. Alignment of the sequences of the Stt7/STN protein kinase from Selaginella moelendorffii (Sm), Physcomitrella patens (Pp), Oryza sativa (Os), Populus trichocarpa (Pt), Arabidopsis thaliana (At), Chlamydomonas reinhardtii ...Here we have examined the redox state of the Stt7/STN7 kinase during state transitions and after illumination with high light to test the proposed model. We find that the Stt7/STN7 kinase contains a disulfide bridge that appears to be intramolecular and maintained not only during state transitions but also in high light when the kinase is inactive. Although these results suggest at first sight that the disulfide bridge of Stt7/STN7 is maintained during its activation and inactivation, we propose that a transient opening of this bridge occurs during the activation process followed by the formation of an intermolecular disulfide bridge and the appearance of a short-lived, covalently linked kinase dimer.     

Purification and Characterization of a Chloroplast Outer-Envelope-Bound, ATP-Dependent Protein Kinase

An ATP-dependent protein kinase was partially purified from isolated outer envelope membranes of pea (Pisum sativum L., Progress No. 9) chloroplasts. The purified kinase had a molecular weight of 70 kilodaltons, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was of the cyclic nucleotide and Ca²⁺, calmodulin-independent type. The purification involved the detergent solubilization of purified outer envelopes by 0.5% cholate and 1% octylglycoside, followed by centrifugation on a linear 6 to 25% sucrose gradient. Active enzyme fractions were further purified by affinity chromatography on histone III-S Sepharose 4B and ion exchange chromatography on diethylaminoethyl cellulose. The protein kinase eluted at 100 millimolar and 50 millimolar NaCl, respectively. The protein kinase was essentially pure as judged by Western blot analysis. The enzyme has a K_M of 450 micromolar for ATP and a V_max of 25 picomoles of ³²P incorporated into histone III-S per minute per microgram. Inhibition by ADP is competitive (K_i 150 micromolar).     

Dynamic Ubiquitination of the Mitogen-activated Protein Kinase Kinase (MAPKK) Ste7 Determines Mitogen-activated Protein Kinase (MAPK) Specificity

Ubiquitination is a post-translational modification that tags proteins for proteasomal degradation. In addition, there is a growing appreciation that ubiquitination can influence protein activity and localization. Ste7 is a prototype MAPKK in yeast that participates in both the pheromone signaling and nutrient deprivation/invasive growth pathways. We have shown previously that Ste7 is ubiquitinated upon pheromone stimulation. Here, we show that the Skp1/Cullin/F-box ubiquitin ligase SCF^Cdc4 and the ubiquitin protease Ubp3 regulate Ste7 ubiquitination and signal specificity. Using purified components, we demonstrate that SCF^Cdc4 ubiquitinates Ste7 directly. Using gene deletion mutants, we show that SCF^Cdc4 and Ubp3 have opposing effects on Ste7 ubiquitination. Although SCF^Cdc4 is necessary for proper activation of the pheromone MAPK Fus3, Ubp3 is needed to limit activation of the invasive growth MAPK Kss1. Finally, we show that Fus3 phosphorylates Ubp3 directly and that phosphorylation of Ubp3 is necessary to limit Kss1 activation. These results reveal a feedback loop wherein one MAPK limits the ubiquitination of an upstream MAPKK and thereby prevents spurious activation of a second competing MAPK.     

油菜叶绿体核糖体蛋白基因rps7的克隆和序列分析(英文)

从甘蓝型油菜 (Brassicanapuscv .H1 65)叶绿体基因组克隆得到了编码核糖体蛋白的基因rps7。经序列分析得知 ,该基因编码区包含 468个核苷酸 ,编码一个分子量为 2 0 1 0 9D、由 1 55个氨基酸组成的蛋白质。该基因的核苷酸和编码的氨基酸序列与烟草对应基因的同源性皆高达 97% ;而与水稻对应基因的同源性分别为 90 %和 84%。该基因不含内含子 ,没有典型的SD序列 ,但在 5’端 - 2 5～- 2 2位发现一个与烟草psbA基因的顺式作用元件RBS2完全相同的TGAT框。与烟草和水稻的同源序列比较 ,该基因在 3’端非编码区变异较大 ,发生了多次插入和缺失。构建了包含该基因在内的一个 1 .0kbDNA的限制性内切酶图谱。所报告的基因序列已登录GenBank。     

Analysis of Phosphorylation of the Receptor-Like Protein Kinase HAESA during Arabidopsis Floral Abscission

Receptor-like protein kinases (RLKs) are the largest family of plant transmembrane signaling proteins. Here we present functional analysis of HAESA, an RLK that regulates floral organ abscission in Arabidopsis. Through in vitro and in vivo analysis of HAE phosphorylation, we provide evidence that a conserved phosphorylation site on a region of the HAE protein kinase domain known as the activation segment positively regulates HAE activity. Additional analysis has identified another putative activation segment phosphorylation site common to multiple RLKs that potentially modulates HAE activity. Comparative analysis suggests that phosphorylation of this second activation segment residue is an RLK specific adaptation that may regulate protein kinase activity and substrate specificity. A growing number of RLKs have been shown to exhibit biologically relevant dual specificity toward serine/threonine and tyrosine residues, but the mechanisms underlying dual specificity of RLKs are not well understood. We show that a phospho-mimetic mutant of both HAE activation segment residues exhibits enhanced tyrosine auto-phosphorylation in vitro, indicating phosphorylation of this residue may contribute to dual specificity of HAE. These results add to an emerging framework for understanding the mechanisms and evolution of regulation of RLK activity and substrate specificity.     

AMPK上游激酶——肿瘤抑制因子LKB1

AMPK是哺乳动物细胞中高度保守的蛋白质,是细胞的“代谢感受器”。AMPK的活化需要上游激酶(AMPKK)对AMPKα亚基活化环上Thr172进行磷酸化来完成。最近的研究发现,肿瘤抑制因子LKB1可以磷酸化Thr172进而激活AMPK,因此认为它是AMPKK家族的成员。     

Stage-Specific State I-State II Transitions during the Cell Cycle of Euglena gracilis

In synchronized Euglena gracilis (light-dark regime of 14:10 hours) the successive formation of the photosynthetic apparatus during cell ontogeny is correlated with large changes in photosynthetic efficiency (P Brandt, B von Kessel 1983 Plant Physiol 72: 616-619; B Kohnke, P Brandt 1984 Biochim Biophys Acta 766: 156-160). This observation led us to investigate the functional association of the chlorophyll a/b light-harvesting protein complex (LHCP) with photosystem I or II, because changes in energy flow to photosystem I or II and in energy transfer between the two photosystems can be a reason for these alterations. As criterion for the association of the LHCP with photosystem I or II, state transitions were determined after 15 minutes preillumination using wave-lengths of 725 or 620 nanometers. The state transitions were determined from measurements of fluorescence induction at room temperature, and fluorescence kinetics at 77 K. According to the obtained data (a) mobile LHCP is present only between the 6th and the 10th hour of the light-time of the cell cycle and (b) this functional relation of the LHCP to photosystem I only at this stage of Euglena chloroplast development is not accompanied by a decrease in stacking. A model for the organization of the newly inserted LHCP within the photosynthetic apparatus of E. gracilis is discussed.     

Protein Synthesis in Isolated Plastids during Chloroplast Development in Wheat

Light-driven protein synthesis in isolated plastids was studiedduring the greening of etiolated wheat (Triticum aestivum L.)seedlings. The process was divided into five phases (I to V)according to the recovery of plastids from the leaf tissue.The activity was not detected in the etioplasts, but rapidlyincreased to the maximum level in phase I and remained at thislevel through phase II. During the transition from phase IIto III, the activity rapidly decreased to one-third and thencontinued to decrease slowly. The plastid polypeptides synthesizedduring the greening were analyzed by SDS-polyacrylamide gelelectrophoresis. In phase I, membrane polypeptides having molecularweights of about 21k were synthesized, while 23 k membrane polypeptidewas synthesized in phases III, IV and V. Synthesis of solublepolypeptides of 50–60 k and membrane polypeptides of 15k and 30–35 k was active in phases I and II, but decreasedbetween phases II and III. (Received October 31, 1983; Accepted May 14, 1984)     

Regulation of Plasmodium falciparum Development by Calcium-dependent Protein Kinase 7 (PfCDPK7)

Second messengers such as phosphoinositides and calcium are known to control diverse processes involved in the development of malaria parasites. However, the underlying molecular mechanisms and pathways need to be unraveled, which may be achieved by understanding the regulation of effectors of these second messengers. Calcium-dependent protein kinase (CDPK) family members regulate diverse parasitic processes. Because CDPKs are absent from the host, these kinases are considered as potential drug targets. We have dissected the function of an atypical CDPK from Plasmodium falciparum, PfCDPK7. The domain architecture of PfCDPK7 is very different from that of other CDPKs; it has a pleckstrin homology domain adjacent to the kinase domain and two calcium-binding EF-hands at its N terminus. We demonstrate that PfCDPK7 interacts with PI(4,5)P₂ via its pleckstrin homology domain, which may guide its subcellular localization. Disruption of PfCDPK7 caused a marked reduction in the growth of the blood stage parasites, as maturation of rings to trophozoites was markedly stalled. In addition, parasite proliferation was significantly attenuated. These findings shed light on an important role for PfCDPK7 in the erythrocytic asexual cycle of malaria parasites.     

水通道蛋白7对蛋白激酶B的影响

该文旨在探讨水通道蛋白7(AQP7)在3T3-L1脂肪细胞不同分化阶段的表达以及其对胰岛素信号通路中蛋白激酶B(PKB)的影响。通过培养3T3-L1前体脂肪细胞,诱导分化为成熟的脂肪细胞,用荧光定量PCR、Western blot、酶学方法分析显示,随3T3-L1脂肪细胞分化过程,AQP7与PKB磷酸化水平同步上升,同时培养基中释放的甘油浓度伴随AQP7的表达平行增加。以TNF-α处理分化成熟的脂肪细胞构建胰岛素抵抗模型,AQP7与PKB磷酸化水平均下降,转染高表达AQP7基因的重组腺病毒载体(Ad-AQP7)之后,随着AQP7表达上调,胰岛素刺激下的PKB磷酸化水平提高,并且葡萄糖代谢能力增强。由此可见,AQP7水平随3T3-L1脂肪细胞分化过程逐渐上升,其高表达可能通过增加PKB磷酸化水平改善胰岛素敏感性,提示AQP7可能成为治疗肥胖的一个重要作用靶点。     

Origins of Pressure-Induced Protein Transitions

The molecular mechanisms underlying pressure-induced protein denaturation can be analyzed based on the pressure-dependent differences in the apparent volume occupied by amino acids inside the protein and when they are exposed to water in an unfolded conformation. We present here an analysis for the peptide group and the 20 naturally occurring amino acid side chains based on volumetric parameters for the amino acids in the interior of the native state, the micelle-like interior of the pressure-induced denatured state, and the unfolded conformation modeled by N-acetyl amino acid amides. The transfer of peptide groups from the protein interior to water becomes increasingly favorable as pressure increases. Thus, solvation of peptide groups represents a major driving force in pressure-induced protein denaturation. Polar side chains do not appear to exhibit significant pressure-dependent changes in their preference for the protein interior or solvent. The transfer of nonpolar side chains from the protein interior to water becomes more unfavorable as pressure increases. We conclude that a sizeable population of nonpolar side chains remains buried inside a solvent-inaccessible core of the pressure-induced denatured state. At elevated pressures, this core may become packed almost as tightly as the interior of the native state. The presence and partial disappearance of large intraglobular voids is another driving force facilitating pressure-induced denaturation of individual proteins. Our data also have implications for the kinetics of protein folding and shed light on the nature of the folding transition state ensemble.     

Protein Phosphatase 2A and Cdc7 Kinase Regulate the DNA Unwinding Element-binding Protein in Replication Initiation

The DNA unwinding element (DUE)-binding protein (DUE-B) binds to replication origins coordinately with the minichromosome maintenance (MCM) helicase and the helicase activator Cdc45 in vivo, and loads Cdc45 onto chromatin in Xenopus egg extracts. Human DUE-B also retains the aminoacyl-tRNA proofreading function of its shorter orthologs in lower organisms. Here we report that phosphorylation of the DUE-B unstructured C-terminal domain unique to higher organisms regulates DUE-B intermolecular binding. Gel filtration analyses show that unphosphorylated DUE-B forms multiple high molecular weight (HMW) complexes. Several aminoacyl-tRNA synthetases and Mcm2–7 proteins were identified by mass spectrometry of the HMW complexes. Aminoacyl-tRNA synthetase binding is RNase A sensitive, whereas interaction with Mcm2–7 is nuclease resistant. Unphosphorylated DUE-B HMW complex formation is decreased by PP2A inhibition or direct DUE-B phosphorylation, and increased by inhibition of Cdc7. These results indicate that the state of DUE-B phosphorylation is maintained by the equilibrium between Cdc7-dependent phosphorylation and PP2A-dependent dephosphorylation, each previously shown to regulate replication initiation. Alanine mutation of the DUE-B C-terminal phosphorylation target sites increases MCM binding but blocks Cdc45 loading in vivo and inhibits cell division. In egg extracts alanine mutation of the DUE-B C-terminal phosphorylation sites blocks Cdc45 loading and inhibits DNA replication. The effects of DUE-B C-terminal phosphorylation reveal a novel S phase kinase regulatory mechanism for Cdc45 loading and MCM helicase activation.     

The Formation of Ribulose Diphosphate Carboxylase Protein during Chloroplast Development in Barley

Ribulose 1,5-diphosphate carboxylase is synthesized in barley leaves growing in the dark. Upon illumination there is a marked increase in the rate of synthesis of the enzyme. The specific activity of the enzyme expressed as cpm incorporated into phosphoglyceric acid per μg of fraction I protein, after isolation shows no change either during dark growth or greening. During early stages of illumination of 7 day dark grown leaves with 320 foot-candles the enzymic activity in the water soluble protein fraction of the leaf shows a short term decline after 15 min which lasts for 30 min. Leaves greening at 2 foot-candles show a similar decline which is shifted to a time between the fourth and eighth hr after the onset of illumination.     

Molecular Genetic Studies of the Cdc7 Protein Kinase and Induced Mutagenesis in Yeast

The Saccharomyces cerevisiae CDC7 gene encodes a protein kinase that functions in DNA replication, repair, and meiotic recombination. The sequence of several temperature-sensitive (ts) cdc7 mutations was determined and correlated with protein kinase consensus domain structure. The positions of these ts alleles suggests some general principles for predicting ts protein kinase mutations. Pedigree segregation lag analysis demonstrated that all of the mutant proteins are less active or less stable than wild-type Cdc7p. Two new mutations were constructed, one by site-directed and the other by insertional mutagenesis. All of the cdc7 mutants were assayed for induced mutagenesis in response to mutagenic agents at the permissive temperature. Some cdc7 mutants were found to be hypomutable, while others are hypermutable. The differences in mutability are observed most clearly when log phase cells are used. Both hypo- and hypermutability are recessive to wild type. Cdc7p may participate in DNA repair by phosphorylating repair enzymes or by altering chromatin structure to allow accessibility to DNA lesions.     

Physiological Phosphorylation of Protein Kinase A at Thr-197 Is by a Protein Kinase A Kinase

Phosphorylation of the catalytic subunit of cyclic AMP-dependent protein kinase, or protein kinase A, on Thr-197 is required for optimal enzyme activity, and enzyme isolated from either animal sources or bacterial expression strains is found phosphorylated at this site. Autophosphorylation of Thr-197 occurs in Escherichia coli and in vitro but is an inefficient intermolecular reaction catalyzed primarily by active, previously phosphorylated molecules. In contrast, the Thr-197 phosphorylation of newly synthesized protein kinase A in intact S49 mouse lymphoma cells is both efficient and insensitive to activators or inhibitors of intracellular protein kinase A. Using [³⁵S]methionine-labeled, nonphosphorylated, recombinant catalytic subunit as the substrate in a gel mobility shift assay, we have identified an activity in extracts of protein kinase A-deficient S49 cells that phosphorylates catalytic subunit on Thr-197. The protein kinase A kinase activity partially purified by anion-exchange and hydroxylapatite chromatography is an efficient catalyst of protein kinase A phosphorylation in terms of both a low K_m for ATP and a rapid time course. Phosphorylation of wild-type catalytic subunit by the kinase kinase activates the subunit for binding to a pseudosubstrate peptide inhibitor of protein kinase A. By both the gel shift assay and a [γ-³²P]ATP incorporation assay, the enzyme is active on wild-type catalytic subunit and on an inactive mutant with Met substituted for Lys-72 but inactive on a mutant with Ala substituted for Thr-197. Combined with the results from mutant subunits, phosphoamino acid analysis suggests that the enzyme is specific for phosphorylation of Thr-197.     

状态转换对光合作用中激发能分配的调节及其与光破坏防御的关系

高等植物和其他低等光合生物可以通过能量满溢,捕光截面变化等方式进行状态转换,有效地调节激发能在两个光系统间的分配,提高光能利用率,减轻强光对光合机构的破坏。