NDPK2 as a signal transducer in the phytochrome-mediated light signaling

Nucleoside-diphosphate kinase (NDPK) 2 in Arabidopsis has been identified as a phytochrome-interacting protein by using the C-terminal domain of phytochrome A (PhyA) as the bait in yeast two-hybrid screening. The far-red light-absorbing form of phytochrome (Pfr) A stimulates NDPK2 gamma-phosphate exchange activity in vitro. To better understand the multiple functions of NDPK and its role in phytochrome-mediated signaling, we characterized the interaction between phytochrome and NDPK2. Domain studies revealed that PER-ARNT-SIM domain A in the C-terminal domain of phytochrome is the binding site for NDPK2. Additionally, phytochrome recognizes both the NDPK2 C-terminal fragment and the NDPK2 hexameric structure to fulfill its binding. To illustrate the mechanism of how the Pfr form of phytochrome stimulates NDPK2, His-197-surrounding residue mutants were made and tested. Results suggested that the H-bonding with His-197 inside the nucleotide-binding pocket is critical for NDPK2 functioning. The pH dependence profiles of NDPK2 indicated that mutants with different activities from the wild type have different pK(a) values of His-197 and that NDPK2 hyperactive mutants possess lower pK(a) values. Because a lower pK(a) value of His-197 accelerates NDPK2 autophosphorylation and the phospho-transfer between the phosphorylated NDPK2 and its kinase substrate, we concluded that the Pfr form of phytochrome stimulates NDPK2 by lowering the pK(a) value of His-197.     

On the phylogeny, expression and targeting of plant nucleoside diphosphate kinases

The plant nucleoside diphosphate kinase (NDPK, EC 2.7.4.6) gene family consists of three groups whose gene products are found in different subcellular locations. In this study we discuss the evolutionary history, localization and expression of the NDPK genes, addressing the question of functional specialization of the different NDPKs. A phylogenetic analysis revealed that the three NDPK isoforms were present already in the last common ancestor of vascular plants and mosses. Our data also imply that the NDPK3 genes possess a higher degree of conservation than the NDPK1 and NDPK2 genes. The expression levels of the different NDPKs in Arabidopsis thaliana inflorescences, leaves and roots were evaluated using quantitative PCR as well as in silico methods. This analysis showed that NDPK1 is the most highly expressed NDPK gene in all the studied tissues. NDPK3a has the second highest NDPK expression, while NDPK3b is expressed to a very low extent. However, expression of NDPK3b is elevated in inflorescence tissue. In situ hybridization experiments performed on inflorescences showed NDPK3a expression in actively dividing cells. NDPK3b expression was observed during later stages of flower development, specifically in the tapetum, ovules and petals. Additionally, we show that an NDPK3 protein is able to direct the green fluorescent protein to both mitochondria and chloroplasts using transient expression in leaf protoplasts. The dual localisation of NDPK3 was confirmed by Western blot, which also demonstrated that the majority of the NDPK3 protein is found in the mitochondria.     

The protein phosphatase 7 regulates phytochrome signaling in Arabidopsis

The psi2 mutant of Arabidopsis displays amplification of the responses controlled by the red/far red light photoreceptors phytochrome A (phyA) and phytochrome B (phyB) but no apparent defect in blue light perception. We found that loss-of-function alleles of the protein phosphatase 7 (AtPP7) are responsible for the light hypersensitivity in psi2 demonstrating that AtPP7 controls the levels of phytochrome signaling. Plants expressing reduced levels of AtPP7 mRNA display reduced blue-light induced cryptochrome signaling but no noticeable deficiency in phytochrome signaling. Our genetic analysis suggests that phytochrome signaling is enhanced in the AtPP7 loss of function alleles, including in blue light, which masks the reduced cryptochrome signaling. AtPP7 has been found to interact both in yeast and in planta assays with nucleotide-diphosphate kinase 2 (NDPK2), a positive regulator of phytochrome signals. Analysis of ndpk2-psi2 double mutants suggests that NDPK2 plays a critical role in the AtPP7 regulation of the phytochrome pathway and identifies NDPK2 as an upstream element involved in the modulation of the salicylic acid (SA)-dependent defense pathway by light. Thus, cryptochrome- and phytochrome-specific light signals synchronously control their relative contribution to the regulation of plant development. Interestingly, PP7 and NDPK are also components of animal light signaling systems.     

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.     

The Signaling Mechanism of Arabidopsis CRY1 Involves Direct Interaction with COP1

Dark-grown transgenic Arabidopsis seedlings expressing the C-terminal domains (CCT) of the cryptochrome (CRY) blue light photoreceptors exhibit features that are normally associated only with light-grown seedlings, indicating that the signaling mechanism of Arabidopsis CRY is mediated through CCT. The phenotypic properties mediated by CCT are remarkably similar to those of the constitutive photomorphogenic1 (cop1) mutants. Here we show that Arabidopsis cryptochrome 1 (CRY1) and its C-terminal domain (CCT1) interacted strongly with the COP1 protein. Coimmunoprecipitation studies showed that CRY1 was bound to COP1 in extracts from both dark- and light-grown Arabidopsis. An interaction also was observed between the C-terminal domain of Arabidopsis phytochrome B and COP1, suggesting that phytochrome signaling also proceeds, at least in part, through direct interaction with COP1. These findings give new insight into the initial step in light signaling in Arabidopsis, providing a molecular link between the blue light receptor, CRY1, and COP1, a negative regulator of photomorphogenesis.     

Arabidopsis nucleoside diphosphate kinase-2 as a plant GTPase activating protein

Nucleoside diphosphate kinase (NDPK) is involved in multiple signaling pathways in mammalian systems, including G-protein signaling. Arabidopsis NDPK2, like its mammalian counterparts, is multifunctional despite its initial discovery phytochrome-interacting protein. This similarity raises the possibility that NDPK2 may play a role in G-protein signaling in plants. In the present study, we explore the potential relationship between NDPK2 and the small G proteins, Pra2 and Pra3, as well as the heterotrimeric G protein, GPA1. We report a physical interaction between NDPK2 and these small G proteins, and demonstrate that NDPK2 can stimulate their GTPase activities. Our results suggest that NDPK2 acts as a GTPase-activating protein for small G proteins in plants. We propose that NDPK2 might be a missing link between the phytochromemediated light signaling and G protein-mediated signaling.     

Missense mutation in the PAS2 domain of phytochrome A impairs subnuclear localization and a subset of responses

Phytochrome A signaling shows two photobiologically discrete outputs: so-called very-low-fluence responses (VLFR) and high-irradiance responses (HIR). By modifying previous screening protocols, we isolated two Arabidopsis mutants retaining VLFR and lacking HIR. Phytochrome A negatively or positively regulates phytochrome B signaling, depending on light conditions. These mutants retained the negative but lacked the positive regulation. Both mutants carry the novel phyA-302 allele, in which Glu-777 (a residue conserved in angiosperm phytochromes) changed to Lys in the PAS2 motif of the C-terminal domain. The phyA-302 mutants showed a 50% reduction in phytochrome A levels in darkness, but this difference was compensated for by greater stability under continuous far-red light. phyA-302:green fluorescent protein fusion proteins showed normal translocation from the cytosol to the nucleus under continuous far-red light but failed to produce nuclear spots, suggesting that nuclear speckles could be involved in HIR signaling and phytochrome A degradation. We propose that the PAS2 domain of phytochrome A is necessary to initiate signaling in HIR but not in VLFR, likely via interaction with a specific partner.     

Trypanosoma cruzi: multiple nucleoside diphosphate kinase isoforms in a single cell

Nucleoside diphosphate kinases (NDPKs) are multifunctional enzymes involved mainly in the conservation of nucleotides and deoxynucleotides at intracellular levels. Here we report the characterization of two NDPKs from the protozoan parasite Trypanosoma cruzi, the etiological agent of Chagas disease. TcNDPK1 and TcNDPK2 were biochemically characterized presenting different kinetic parameters and regulation mechanisms. NDPK activity was mainly detected in soluble fractions according to the digitonin extraction technique; however 20% of the activity remains insoluble at digitonin concentrations up to 5 mg ml⁻¹. TcNDPK1 is a short enzyme isoform, whereas TcNDPK2 is a long one containing a DM10 motif. In addition, two other putative NDPK genes (TcNPDK3 and TcNDPK4) were detected by data mining at the T. cruzi genome database. The large number and diversity of NDPK isoforms are in agreement with those previously observed for other T. cruzi phosphotransferases, such as adenylate kinases.     

Molecular cloning and characterization of nucleoside diphosphate (NDP) kinases from Chinese cabbage (Brassica campestris)

Nucleoside diphosphate kinases (NDPKs) are key metabolic enzymes that catalyze the synthesis of non-adenosine nucleoside triphosphates (NTP) by transfer of the terminal phosphate between NDP and NTP. Recently we isolated three NDPK cDNAs from Chinese cabbage cDNA library. BcNDK1 has 636 bp and encodes a putative 17.4 kDa protein, BcNDK2 has 854 bp and encodes a putative 25.5 kDa protein, and BcNDK3 is 986 bp long and encodes a putative 25.7 kDa protein. The precursor proteins of BcNDK2 and BcNDK3 have long N-terminal extensions containing putative chloroplast or mitochondrial targeting sequences. A phylogenic tree showed that the 3 BcNDKs are highly homologous to other plant NDPK genes, especially those of Arabidopsis. Expression of the BcNDK genes depended on the developmental stage and the conditions of seed germination. Most notably, expression of BcNDK2 increased dramatically in seedlings transferred to the light after germinating in the dark. In addition, BcNDK3 differed from BcNDK1 in being highly expressed in the hooks and cotyledons of seedlings. Although all BcNDKs were highly expressed in petals, BcNDK1 was also expressed in pistils. Expression of each of the BcNDKs increased as the flower bud matured. These results indicate that NDPKs are involved in physiological pathways activated by a variety of environmental conditions and at different developmental stages.     

Nucleoside Diphosphate Kinase Is a Possible Component of the Ethylene Signal Transduction Pathway

In etiolated seedlings of Pisum sativum and leaves of Arabidopsis thaliana, in vivo ethylene treatment resulted in an increase in in vitro phosphorylation of 17 kD (P. sativum) or 16 and 17 kD (A. thaliana) polypeptides. These polypeptides were identified as nucleoside diphosphate kinase (NDPK) based on both biochemical properties and interaction with antibodies against NDPK from P. sativum. Using the receptor-directed antagonist of ethylene action 2,5-norbornadiene and the ethylene-insensitive mutants of A. thaliana etr1-1 and eti5, ethylene specificity and receptor dependence of NDPK phosphorylation have been demonstrated. In pea epicotyls, ethylene treatment also led to increase in nucleoside transferase activity unlike in A. thaliana leaves. The increases in nucleoside transferase activity and NDPK phosphorylation were very rapid and transient. The results suggest a role for NDPK as a possible component of the ethylene signal transduction chain.     

Nucleoside diphosphate kinase activity in soluble transducin preparations biochemical properties and possible role of transducin-beta as phosphorylated enzyme intermediate.

Known nucleoside diphosphate kinases (NDPKs) are oligomers of 17-23-kDa subunits and catalyze the reaction N1TP + N2DP --> N1DP + N2TP via formation of a histidine-phosphorylated enzyme intermediate. NDPKs are involved in the activation of heterotrimeric GTP-binding proteins (G-proteins) by catalyzing the formation of GTP from GDP, but the properties of G-protein-associated NDPKs are still incompletely known. The aim of our present study was to characterize NDPK in soluble preparations of the retinal G-protein transducin. The NDPK is operationally referred to as transducin-NDPK. Like known NDPKs, transducin-NDPK utilizes NTPs and phosphorothioate analogs of NTPs as substrates. GDP was a more effective phosphoryl group acceptor at transducin-NDPK than ADP and CDP, and guanosine 5'-[gamma-thio]triphosphate (GTP[S]) was a more effective thiophosphoryl group donor than adenosine 5'-[gamma-thio]triphosphate (ATP[S]). In contrast with their action on known NDPKs, mastoparan and mastoparan 7 had no stimulatory effect on transducin-NDPK. Guanosine 5'-[beta, gamma-imido]triphosphate (p[NH]ppG) potentiated [3H]GTP[S] formation from [3H]GDP and ATP[S] but not [3H]GTP[S] formation from [3H]GDP and GTP[S]. Depending on the thiophosphoryl group acceptor and donor, [3H]NTP[S] formation was differentially regulated by Mg2+, Mn2+, Co2+, Ca2+ and Zn2+. [gamma-32P]ATP and [gamma-32P]GTP [32P]phosphorylated, and [35S]ATP[S] [35S]thiophosphorylated, a 36-kDa protein comigrating with transducin-beta. p[NH]ppG potentiated [35S]thiophosphorylation of the 36-kDa protein. 32P-labeling of the 36-kDa protein showed characteristics of histidine phosphorylation. There was no evidence for (thio)phosphorylation of 17-23-kDa proteins. Our data show the following: (a) soluble transducin preparations contain a GDP-prefering and guanine nucleotide-regulated NDPK; (b) transducin-beta may serve as a (thio)phosphorylated NDPK intermediate; (c) transducin-NDPK is distinct from known NDPKs and may consist of multiple kinases or a single kinase with multiple regulatory domains.     

Inter-domain crosstalk in the phytochrome molecules

Phytochromes are bifunctional photoreceptors with a two-domain structure, consisting of the N-terminal photosensory domain and the C-terminal regulatory domain. The photo-induced Pr <--> Pfr phototransformation accompanies subtle conformational changes, primarily triggered by the apoprotein-chromophore interactions in the N-terminal domain. The conformational signals are subsequently transmitted to the C-terminal domain through various inter-domain crosstalks, resulting in the interaction of the activated C-terminal domain with phytochrome interacting factors. Thus the inter-domain crosstalks play critical roles in the photoactivation of the phytochromes. Protein phosphorylation, such as that of Ser-598, is implicated in this process by inducing conformational changes and by modulating inter-domain signaling.     

Evidence for two distinct effector-binding sites in threonine deaminase by site-directed mutagenesis, kinetic, and binding experiments

A three-dimensional structure comparison between the dimeric regulatory serine-binding domain of Escherichia coli D-3-phosphoglycerate dehydrogenase [Schuller, D. J., Grant, G. A., and Banaszak, L. J. (1995) Nat. Struct. Biol. 2, 69-76] and the regulatory domain of E. coli threonine deaminase [Gallagher, D. T., Gilliland, G. L., Xiao, G., Zondlo, J., Fisher, K. E., Chinchilla, D. , and Eisenstein, E. (1998) Structure 6, 465-475] led us to make the hypothesis that threonine deaminase could have two binding sites per monomer. To test this hypothesis about the corresponding plant enzyme, site-directed mutagenesis was carried out on the recombinant Arabidopsis thaliana threonine deaminase. Kinetic and binding experiments demonstrated for the first time that each regulatory domain of the monomers of A. thaliana threonine deaminase possesses two different effector-binding sites constituted in part by Y449 and Y543. Our results demonstrate that Y449 belongs to a high-affinity binding site whose interaction with a first isoleucine induces conformational modifications yielding a conformer displaying a higher activity and with enhanced ability to bind a second isoleucine on a lower-affinity binding site containing Y543. Isoleucine interaction with this latter binding site is responsible for conformational modifications leading to final inhibition of the enzyme. Y449 interacts with both regulators, isoleucine and valine. However, interaction of valine with the high-affinity binding site induces different conformational modifications leading to reversal of isoleucine binding and reversal of inhibition.     

PIP kinases and their role in plant tip growing cells

Phosphatidylinositol (4,5) bisphosphate, [PtdIns(4,5)P2], is a signaling lipid involved in many important processes in animal cells such as cytoskeleton organization, intracellular vesicular trafficking, secretion, cell motility, regulation of ion channels, and nuclear signaling pathways. In the last years PtdIns(4,5)P2 and its synthesizing enzyme, phosphatidylinositol phosphate kinase (PIPK), has been intensively studied in plant cells, revealing a key role in the control of polar tip growth. Analysis of the PIPK members from Arabidopsis thaliana, Oryza sativa and Physcomitrella patens showed that they share some regulatory features with animal PIPKs but also exert plant-specific modes of regulation. This review aims at giving an overview on the PIPK family from Arabidopsis thaliana and Physcomitrella patens. Even though their basic structure, modes of activation and physiological role is evolutionary conserved, modules responsible for plasma membrane localization are distinct for different PIPKs, depending on differences in physiological and/or developmental status of cells, such as polarized and non-polarized.     

Degradation of Arabidopsis CRY2 is regulated by SPA proteins and phytochrome A

The UV-A/blue light photoreceptor crytochrome2 (cry2) plays a fundamental role in the transition from the vegetative to the reproductive phase in the facultative long-day plant Arabidopsis thaliana. The cry2 protein level strongly decreases when etiolated seedlings are exposed to blue light; cry2 is first phosphorylated, polyubiquitinated, and then degraded by the 26S proteasome. COP1 is involved in cry2 degradation, but several cop1 mutants show only reduced but not abolished cry2 degradation. SUPPRESSOR OF PHYA-105 (SPA) proteins are known to work in concert with COP1, and recently direct physical interaction between cry2 and SPA1 was demonstrated. Thus, we hypothesized that SPA proteins could also play a role in cry2 degradation. To this end, we analyzed cry2 protein levels in spa mutants. In all spa mutants analyzed, cry2 degradation under continuous blue light was alleviated in a fluence rate-dependent manner. Consistent with a role of SPA proteins in phytochrome A (phyA) signaling, a phyA mutant had enhanced cry2 levels, particularly under low fluence rate blue light. Fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy studies showed a robust physical interaction of cry2 with SPA1 in nuclei of living cells. Our results suggest that cry2 stability is controlled by SPA and phyA, thus providing more information on the molecular mechanisms of interaction between cryptochrome and phytochrome photoreceptors.     

The metastasis suppressor Nm23 as a modulator of Ras/ERK signaling

NM23-H1 (also known as NME1) was the first identified metastasis suppressor, which displays a nucleoside diphosphate kinase (NDPK) and histidine protein kinase activity. NDPKs are linked to many processes, such as cell migration, proliferation, differentiation, but the exact mechanism whereby NM23-H1 inhibits the metastatic potential of cancer cells remains elusive. However, some recent data suggest that NM23-H1 may exert its anti-metastatic effect by blocking Ras/ERK signaling. In mammalian cell lines NDPK-mediated attenuation of Ras/ERK signaling occurs through phosphorylation (thus inactivation) of KSR (kinase suppressor of Ras) scaffolds. In this review I summarize our knowledge about KSR’s function and its regulation in mammals and in C. elegans. Genetic studies in the nematode contributed substantially to our understanding of the function and regulation of the Ras pathway (i.e. KSR’s discovery is also linked to the nematode). Components of the RTK/Ras/ERK pathway seem to be highly conserved between mammals and worms. NDK-1, the worm homolog of NM23-H1 affects Ras/MAPK signaling at the level of KSRs, and a functional interaction between NDK-1/NDPK and KSRs was first demonstrated in the worm in vivo. However, NDK-1 is a factor, which is necessary for proper MAPK activation, thus it activates rather than suppresses Ras/MAPK signaling in the worm. The contradiction between results in mammalian cell lines and in the worm regarding NDPKs’ effect exerted on the outcome of Ras signaling might be resolved, if we better understand the function, structure and regulation of KSR scaffolds.