Phytochrome phosphorylation modulates light signaling by influencing the protein-protein interaction

Plant photoreceptor phytochromes are phosphoproteins, but the question as to the functional role of phytochrome phosphorylation has remained to be elucidated. We investigated the functional role of phytochrome phosphorylation in plant light signaling using a Pfr-specific phosphorylation site mutant, Ser598Ala of oat (Avena sativa) phytochrome A (phyA). The transgenic Arabidopsis thaliana (phyA-201 background) plants with this mutant phyA showed hypersensitivity to light, suggesting that phytochrome phosphorylation at Serine-598 (Ser598) in the hinge region is involved in an inhibitory mechanism. The phosphorylation at Ser598 prevented its interaction with putative signal transducers, Nucleoside Diphosphate Kinase-2 and Phytochrome-Interacting Factor-3. These results suggest that phosphorylation in the hinge region of phytochromes serves as a signal-modulating site through the protein-protein interaction between phytochrome and its putative signal transducer proteins.     

Multifaceted roles of Arabidopsis PP6 phosphatase in regulating cellular signaling and plant development

Reversible protein phosphorylation catalyzed by kinases and phosphatases is a major form of posttranslational regulation that plays a central role in regulating many signaling pathways. While large families of both protein kinases and protein phosphatases have been identified in plants, kinases outnumber phosphatases. This raises the question of how a relatively limited number of protein phosphatases can maintain protein phosphorylation homeostasis in a cell. Recent studies have shown that Arabidopsis FyPP1 (Phytochrome-associated serine/threonine protein phosphatase 1) and FyPP3 encode the catalytic subunits of protein phosphatase 6 (PP6), and that they directly binds to the A subunits of protein phosphatase 2A (PP2AA proteins), and SAL (SAPS domain-like) proteins to form the heterotrimeric PP6 holoenzyme complex. Emerging evidence is suggesting that PP6, acts in opposition with multiple classes of kinases, to regulate the phosphorylation status of diverse substrates and subsequently numerous developmental processes and responses to environmental stimuli.     

Phosphopeptide mapping of Avena phytochrome phosphorylated by protein kinases in vitro

We previously demonstrated that protein kinases are useful probes of conformational changes that occur upon photoconversion of phytochrome [Wong, Y.-S., Cheng, H.-C., Walsh, D. A., & Lagarias, J. C. (1986) J. Biol. Chem. 261, 12089-12097]. Here we present phosphopeptide analyses of oat phytochrome phosphorylated by three mammalian protein kinases and by a polycation-stimulated, phytochrome-associated protein kinase. Phosphorylation of the Pr form by the cAMP-dependent protein kinase occurs predominantly on Ser17 while Ser598 is the preferred phosphorylation site on Pfr. The cGMP-dependent and Ca2(+)-activated, phospholipid-dependent protein kinases, which phosphorylate only the Pr form of phytochrome, recognize the same region on the phytochrome polypeptide as the cAMP-dependent protein kinase. Polycation-stimulated phytochrome phosphorylation reveals that, in contrast to the mammalian enzymes, the plant kinase recognizes the serine-rich, blocked N-terminus of phytochrome. The potential regulatory role of phytochrome phosphorylation, particularly in the structurally conserved serine/threonine-rich N-terminal region of the phytochrome polypeptide, is suggested by these results.     

Autophosphorylation desensitizes phytochrome signal transduction

Plant red/far-red photoreceptor phytochromes are known as autophosphorylating serine/threonine kinases. However, the functional roles of autophosphorylation and kinase activity of phytochromes are largely unknown. We recently reported that the autophosphorylation of phytochrome A (phyA) plays an important role in regulating plant phytochrome signaling by controlling phyA protein stability. Two serine residues in the N-terminal extension (NTE) region were identified as autophosphorylation sites, and phyA mutant proteins with serine-to-alanine mutations were degraded in plants at a significantly slower rate than the wild-type under light conditions, resulting in transgenic plants with hypersensitive light responses. In addition, the autophosphorylation site phyA mutants had normal protein kinase activities. Collectively, our results suggest that phytochrome autophosphorylation provides a mechanism for signal desensitization in phytochrome-mediated light signaling by accelerating the degradation of phytochrome A.Key words: phytochrome, autophosphorylation, phosphorylation, protein kinase, protein degradation, light signaling, signal desensitizationHigher plants continually adapt to their light environments to promote photosynthesis for optimal growth and development. Natural light conditions are monitored by various plant photoreceptors, including red (R)/far-red (FR) photoreceptor phytochromes.^1,2 Phytochromes are dimeric chromoproteins covalently linked to tetrapyrrole chromophore phytochromobilin, and exist as two photo-interconvertible species, red-light absorbing Pr and far-red-light absorbing Pfr forms. Phytochromes are biosynthesized as the Pr form in the dark, and are transformed to the Pfr form upon exposure to red light. This photoactivation of phytochromes induces a highly regulated signaling network for photomorphogenesis in plants.^3,4 Recently, phosphorylation and dephosphorylation have been suggested to play important roles in phytochrome-mediated light signaling;^5,6 for instance, a few phytochrome-associated protein phosphatases have been shown to act as positive regulators of phytochrome signaling.^7–9 However, the functional roles of phytochrome phosphorylation remain to be explored.     

Bottom-up Assembly of the Phytochrome Network

Plants have developed sophisticated systems to monitor and rapidly acclimate to environmental fluctuations. Light is an essential source of environmental information throughout the plant’s life cycle. The model plant Arabidopsis thaliana possesses five phytochromes (phyA-phyE) with important roles in germination, seedling establishment, shade avoidance, and flowering. However, our understanding of the phytochrome signaling network is incomplete, and little is known about the individual roles of phytochromes and how they function cooperatively to mediate light responses. Here, we used a bottom-up approach to study the phytochrome network. We added each of the five phytochromes to a phytochrome-less background to study their individual roles and then added the phytochromes by pairs to study their interactions. By analyzing the 16 resulting genotypes, we revealed unique roles for each phytochrome and identified novel phytochrome interactions that regulate germination and the onset of flowering. Furthermore, we found that ambient temperature has both phytochrome-dependent and -independent effects, suggesting that multiple pathways integrate temperature and light signaling. Surprisingly, none of the phytochromes alone conferred a photoperiodic response. Although phyE and phyB were the strongest repressors of flowering, both phyB and phyC were needed to confer a flowering response to photoperiod. Thus, a specific combination of phytochromes is required to detect changes in photoperiod, whereas single phytochromes are sufficient to respond to light quality, indicating how phytochromes signal different light cues.     

Phytochrome phosphorylation in plant light signaling.

Reversible protein phosphorylation is a switching mechanism used in eukaryotes to regulate various cellular signalings. In plant light signaling, sophisticated photosensory receptor systems operate to modulate growth and development. The photoreceptors include phytochromes, cryptochromes and phototropins. Despite considerable progresses in defining the photosensory roles of these photoreceptors, the primary biochemical mechanisms by which the photoreceptor molecules transduce the perceived light signals into cellular responses remain to be elucidated. The signal-transducing photoreceptors in plants are all phosphoproteins and/or protein kinases, suggesting that light-dependent protein phosphorylation and dephosphorylation play important roles in the function of the photoreceptors. This review focuses on the role of phytochromes' reversible phosphorylation involved in the light signal transduction in plants.     

A novel protein phosphatase indirectly regulates phytochrome-interacting factor 3 via phytochrome

Light signal transduction in plants involves an intricate series of pathways which is finely regulated by interactions between specific signalling proteins, as well as by protein modifications such as phosphorylation and ubiquitination. The identification of novel phytochrome-interacting proteins and the precise signalling mechanisms that they mediate is still ongoing. In our present study, we show that the newly identified putative phytochrome-associated protein, PAPP2C (phytochrome-associated protein phosphatase type 2C), interacts in the nucleus with phyA (phytochrome A) and phyB, both in vitro and in vivo. Moreover, the phosphatase activity of PAPP2C and its association with phytochromes were found to be enhanced by red light, indicating that it plays a role in mediating phytochrome signalling. In particular, PAPP2C specifically binds to the N-terminal PHY domain of the phytochromes. We thus speculate that this interaction reflects a unique regulatory function of this phosphatase toward established phytochrome-associated proteins. We also show that PAPP2C effectively dephosphorylates phytochromes in vitro. Interestingly, PAPP2C indirectly mediates the dephosphorylation of PIF3 (phytochrome-interacting factor 3) in vitro. Taken together, we suggest that PAPP2C functions as a regulator of PIF3 by dephosphorylating phytochromes in the nucleus.     

Novel light-activated protein kinases as key regulators of plant growth and development

Plants have evolved highly sensitive sensory photoreceptor systems to regulate various aspects of their growth and development. Many responses such as seed germination, flowering and dormancy are controlled by red and far-red regions of the solar spectrum through the phytochrome family of photoreceptors. However, several other responses such as stem growth inhibition, phototropism and opening of stomata are controlled by blue and/or ultraviolet light absorbing photoreceptors called cryptochromes and phototropin. Despite their central role in plant biology, the mode of action of these photoreceptors has been shrouded in mystery. Even the biochemical isolation of a photoreceptor, as in the case of phytochrome was accomplished decades ago, did not help in elucidating the mechanism of action. Nevertheless, due to advances in recombinant DNA technology, generation of extensive databanks and the capability to predict function by base sequence analysis, a breakthrough has now come about. It is clear that certain phytochromes, at least in the cyanobacteria and algae which represent the simplest plants, are hybrid photoreceptor-cum-kinases. These novel kinases utilize captured photons rather than conventional ligands to trigger conformational change and in consequence enzyme activity. The kinases apparently, then, cause phosphorylation of many other types of target molecules, leading eventually to various developmental changes. There is suggestive evidence that in higher plants, too, at least some phytochromes may operate as kinases. As compared to work on phytochromes, the blue light photoreceptors have begun to be studied only recently. However, the exciting discovery has been made of at least one photoactive kinase that is critically required for phototropism. This article summarizes the above discoveries from the perspective of general biology. Dedicated to the memory of Drs Harry Borthwick, Sterling Hendricks and James Bonner whose classical studies paved the way for modern researches on mechanism of action of plant photoreceptors and whom the senior author was previleged to know.     

光色素信号通路中磷酸化修饰研究进展

光是植物的唯一能量来源, 植物在进化过程中产生不同的光敏色素来感知光信号。光信号通路中元件通常被特异翻译后修饰调节。光敏色素是一种自磷酸化的丝氨酸/苏氨酸蛋白激酶, 可以被一些蛋白磷酸酶去磷酸化。通过对光敏色素A (phyA)和光敏色素B (phyB)的自磷酸化位点研究, 发现自磷酸化对光敏色素的功能及其介导的信号通路起着非常重要的作用。光激活的光敏色素诱导光敏色素作用因子(PIF)磷酸化, 这对于PIF的正常降解及光形态建成的起始是必需的。该文主要介绍了光敏色素信号通路磷酸化修饰的最新进展, 以期为深入研究光敏色素信号转导机制提供参考。     

Arabidopsis phytochromes C and E have different spectral characteristics from those of phytochromes A and B

The red/far-red light absorbing phytochromes play a major role as sensor proteins in photomorphogenesis of plants. In Arabidopsis the phytochromes belong to a small gene family of five members, phytochrome A (phyA) to E (phyE). Knowledge of the dynamic properties of the phytochrome molecules is the basis of phytochrome signal transduction research. Beside photoconversion and destruction, dark reversion is a molecular property of some phytochromes. A possible role of dark reversion is the termination of signal transduction. Since Arabidopsis is a model plant for biological and genetic research, we focussed on spectroscopic characterization of Arabidopsis phytochromes, expressed in yeast. For the first time, we were able to determine the relative absorption maxima and minima for a phytochrome C (phyC) as 661/725 nm and for a phyE as 670/724 nm. The spectral characteristics of phyC and E are strictly different from those of phyA and B. Furthermore, we show that both phyC and phyE apoprotein chromophore adducts undergo a strong dark reversion. Difference spectra, monitored with phycocyanobilin and phytochromobilin as the apoprotein's chromophore, and in vivo dark reversion of the Arabidopsis phytochrome apoprotein phycocyanobilin adducts are discussed with respect to their physiological function.     

Protein kinase activity in Cucumis sativus cotyledons: effect of calcium and light

Light signals received by phytochromes in plants may be transduced through protein phosphorylation. Ca(2+) as second messenger was involved in phytochrome-mediated cellular events. Our experiments with Cucumis sativus cotyledons, treated with red (R) and far-red (FR) light, showed a stimulatory effect on in vitro protein phosphorylation of histone, added as exogenous substrate to the cotyledon extracts, and also modified the phosphorylation of endogenous polypeptides. The effect of light treatments was mimicked by the addition of Ca(2+) to the phosphorylation buffer, indicating phytochrome- and Ca(2+)-dependence on activity of some protein kinases (PKs). In-gel kinase assays were performed to characterize the PKs involved at the cotyledon stage of cucumber plants. Three proteins of about 75, 57 and 47kDa with PK activity were detected between M(r) markers of 94 and 45kDa. All three were able to phosphorylate histone and undergo autophosphorylation. However, only the 75 and 57kDa proteins autophosphorylated and phosphorylated the substrate in a Ca(2+)-dependent manner, and were inhibited when calmodulin (CaM) antagonists were added to the incubation buffer. Western-blot analysis with polyclonal antibodies directed against calcium-dependent protein kinase of rice (OsCDPK11) or Arabidopsis (AtCPK2) recognised 57 and 75kDa polypeptides, respectively. These results indicate the presence in cucumber cotyledons of at least two proteins (ca. 75 and 57kDa) with activity of PKs that could be calcium-dependent protein kinases (CDPKs). Both CDPKs could be modulated by phytochromes throughout FR-HIR and VLFR responses.     

Solid-phase tyrosine-specific protein kinase assay in multiwell substrate-immobilized polyacrylamide gel

Since tyrosine-specific protein kinase (TPK) is much less abundant than Ser/Thr-specific kinases in cells, determination of TPK activity in crude cell extracts or column chromatography eluates has been difficult. This is compounded by the absence of a rapid, economical method for the separation of high endogenous protein phosphorylation background from exogenously added tyrosine-containing substrates. We have developed a new solid-phase assay, which provides high sensitivity and efficiency at a low cost for assaying the TPK activity of crude enzyme preparations. This assay utilizes immobilized tyrosine-containing synthetic polymers such as (Glu:Tyr, 4:1)n in polyacrylamide gels. The kinase reaction is started by adding crude enzyme solutions and [tau-32P]ATP-metal ion mixtures into microtiter-size wells made in the gels. After the phosphorylation reaction, the reaction mixtures are removed and the gels are prewashed in water followed by electrophoresis to completely remove free radioactive ATP. 32P incorporation into the immobilized TPK-specific substrate can be detected by autoradiography and quantitated by cutting the gel pieces and counting them with a liquid scintillation counter. The simple, rapid method should facilitate screening of TPK inhibitors and activators as well as examining the substrate specificity of TPKs. Other enzymes, including Ser/Thr-specific protein kinases, can also be analyzed by this technique.     

Phosphorylation of JAK2 at serine 523: a negative regulator of JAK2 that is stimulated by growth hormone and epidermal growth factor

The tyrosine kinase JAK2 is a key signaling protein for at least 20 receptors in the cytokine/hematopoietin receptor superfamily and is a component of signaling for multiple receptor tyrosine kinases and several G-protein-coupled receptors. In this study, phosphopeptide affinity enrichment and mass spectrometry identified serine 523 (Ser523) in JAK2 as a site of phosphorylation. A phosphoserine 523 antibody revealed that Ser523 is rapidly but transiently phosphorylated in response to growth hormone (GH). MEK1 inhibitor UO126 suppresses GH-dependent phosphorylation of Ser523, suggesting that extracellular signal-regulated kinases (ERKs) 1 and/or 2 or another kinase downstream of MEK1 phosphorylate Ser523 in response to GH. Other ERK activators, phorbol 12-myristate 13-acetate and epidermal growth factor, also stimulate phosphorylation of Ser523. When Ser523 in JAK2 was mutated, JAK2 kinase activity as well as GH-dependent tyrosyl phosphorylation of JAK2 and Stat5 was enhanced, suggesting that phosphorylation of Ser523 inhibits JAK2 kinase activity. We hypothesize that phosphorylation of Ser523 in JAK2 by ERKs 1 and/or 2 or other as-yet-unidentified kinases acts in a negative feedback manner to dampen activation of JAK2 in response to GH and provides a mechanism by which prior exposure to environmental factors that regulate Ser523 phosphorylation might modulate the cell's response to GH.     

Flowering responses to altered expression of phytochrome in mutants and transgenic lines of Arabidopsis thaliana (L.) Heynh.

The long-day plant Arabidopsis thaliana (L.) Heynh. flowers early in response to brief end-of-day (EOD) exposures to far-red light (FR) following a fluorescent short day of 8 h. FR promotion of flowering was nullified by subsequent brief red light (R) EOD exposure, indicating phytochrome involvement. The EOD response to R or FR is a robust measure of phytochrome action. Along with their wild-type (WT) parents, mutants deficient in either phytochrome A or B responded similarly to the EOD treatments. Thus, neither phytochrome A nor B exclusively regulated flowering, although phytochrome B controlled hypocotyl elongation. Perhaps a third phytochrome species is important for the EOD responses of the mutants and/or their flowering is regulated by the amount of the FR-absorbing form of phytochrome, irrespective of the phytochrome species. Overexpression of phytochrome A or phytochrome B resulted in differing photoperiod and EOD responses among the genotypes. The day-neutral overexpressor of phytochrome A had an EOD response similar to all of the mutants and WTs, whereas R EOD exposure promoted flowering in the overexpressor of phytochrome B and FR EOD exposure inhibited this promotion. The comparisons between relative flowering times and leaf numbers at flowering of the over-expressors and their WTs were not consistent across photoperiods and light treatments, although both phytochromes A and B contributed to regulating flowering of the transgenic plants.     

Changes in photoperiod or temperature alter the functional relationships between phytochromes and reveal roles for phyD and phyE

The phytochromes are one of the means via which plants obtain information about their immediate environment and the changing seasons. Phytochromes have important roles in developmental events such as the switch to flowering, the timing of which can be crucial for the reproductive success of the plant. Analysis of phyB mutants has revealed that phyB plays a major role in this process. We have recently shown, however, that the flowering phenotype of the phyB monogenic mutant is temperature dependent. A modest reduction in temperature to 16 degrees C was sufficient to abolish the phyB mutant early-flowering phenotype present at 22 degrees C. Using mutants null for one or more phytochrome species, we have now shown that phyA, phyD, and phyE, play greater roles with respect to phyB in the control of flowering under cooler conditions. This change in the relative contributions of individual phytochromes appears to be important for maintaining control of flowering in response to modest alterations in ambient temperature. We demonstrate that changes in ambient temperature or photoperiod can alter the hierarchy and/or the functional relationships between phytochrome species. These experiments reveal new roles for phyD and phyE and provide valuable insights into how the phytochromes help to maintain development in the natural environment.     

Unchanged beta-adrenergic stimulation of cardiac L-type calcium channels in Ca v 1.2 phosphorylation site S1928A mutant mice

Phosphorylation of serine 1928 (Ser(1928)) of the cardiac Ca(v)1.2 subunit of L-type Ca(2+) channels has been proposed as the mechanism for regulation of L-type Ca(2+) channels by protein kinase A (PKA). To test this directly in vivo, we generated a knock-in mouse with targeted mutation of Ser(1928) to alanine. This mutation did not affect basal L-type current characteristics or regulation of the L-type current by PKA and the beta-adrenergic receptor, whereas the mutation abolished phosphorylation of Ca(v)1.2 by PKA. Therefore, our data show that PKA phosphorylation of Ser(1928) of Ca(v)1.2 is not functionally involved in beta-adrenergic stimulation of Ca(v)1.2-mediated Ca(2+) influx into the cardiomyocyte.     

Ser/Thr-phosphoprotein phosphatases in chondrogenesis: neglected components of a two-player game

Protein phosphorylation plays a determining role in the regulation of chondrogenesis in vitro. While signalling pathways governed by protein kinases including PKA, PKC, and mitogen-activated protein kinases (MAPK) have been mapped in great details, published data relating to the specific role of phosphoprotein phosphatases (PPs) in differentiating chondroprogenitor cells or in mature chondrocytes is relatively sparse. This review discusses the known functions of Ser/Thr-specific PPs in the molecular signalling pathways of chondrogenesis. PPs are clearly equally important as protein kinases to counterbalance the effect of reversible protein phosphorylation. Of the main Ser/Thr PPs, some of the functions of PP1, PP2A and PP2B have been characterised in the context of chondrogenesis. While PP1 and PP2A appear to negatively regulate chondrogenic differentiation and maintenance of chondrocyte phenotype, calcineurin is an important stimulatory mediator during chondrogenesis but becomes inhibitory in mature chondrocytes. Furthermore, PPs are implicated to be mediators during the pathogenesis of osteoarthritis that makes them potential therapeutic targets to be exploited in the close future. Among the many yet unexplored targets of PPs, modulation of plasma membrane ion channel function and participation in mechanotransduction pathways are emerging novel aspects of signalling during chondrogenesis that should be further elucidated. Besides the regulation of cellular ion homeostasis, other potentially significant novel roles for PPs during the regulation of in vitro chondrogenesis are discussed.