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Fibroblast growth factor receptor 3 (FGFR3) is a member of receptor tyrosine kinases, which is involved in skeletal cell growth, differentiation, and migration. FGFR3 transduces biochemical signals from the extracellular ligand-binding domain to the intracellular kinase domain through the conformational changes of the transmembrane (TM) helix dimer. Here, we apply generalized replica exchange with solute tempering method to wild type (WT) and G380R mutant (G380R) of FGFR3. The dimer interface in G380R is different from WT and the simulation results are in good agreement with the solid-state nuclear magnetic resonance (NMR) spectroscopy. TM helices in G380R are extended more than WT, and thereby, G375 in G380R contacts near the N-termini of the TM helix dimer. Considering that both G380R and G375C show the constitutive activation, the formation of the N-terminal contacts of the TM helices can be generally important for the activation mechanism. © 2019 Wiley Periodicals, Inc.

Triacylglycerols (TAGs) are the major component of plant storage lipids. Acyl-CoA:diacylglycerol acyltransferase (DGAT) catalyzes the final step of the Kennedy pathway, and responsible for plant oil accumulation. We previously found DGAT activity of Vernonia galamensis DGAT1 was distinctively higher than that of Arabidopsis thaliana DGAT1 and soybean DGAT1 in a yeast microsome assay. In this study, the DGAT1 cDNAs of Arabidopsis, Vernonia, soybean, and castor were introduced into Arabidopsis (ecotype Col-0). All Vernonia DGAT1 expressing lines showed a significantly higher oil content (average 49% relative increase compared to the wild type) followed by soybean, and castor. Most Arabidopsis DGAT1 over-expressing lines did not show a significant increase. In addition to these four DGAT1s, sunflower, Jatropha and sesame DGAT1 genes were introduced into the TAG biosynthesis defective yeast mutant (H1246). In the yeast expression culture, DGAT1s from Arabidopsis, castor, and soybean only slightly increased TAG content, however, DGAT1s from Vernonia, sunflower, Jatropha, and sesame remarkably increased TAG content more than 10 times higher than the former three DGAT1s. Three amino acid residues were characteristically common in the latter four DGAT1s. Using soybean DGAT1, these amino acid substitutions by site-directed mutagenesis was performed and analyzed. These substitutions substantially increased the TAG content.HighlightDGAT1s from several plant species were tested their TAG accumulation promotion in Arabidopsis and yeast. They were divided into high and low function and single amino acid substitution enhanced function

Background Accurate evaluation of kidney function before and after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is important for both informed decision making and detection of chronic kidney disease. However, to the best of our knowledge, no report has evaluated the glomerular filtration rate (GFR) in pediatric patients who underwent HSCT using the gold standard GFR measurement, as well as inulin-based GFR (iGFR). Methods We assessed iGFR before and after allo-HSCT to evaluate the impact of allo-HSCT on GFR in a prospective cohort study of 17 pediatric patients. We also assessed the accuracy and bias of the values of estimated GFR (eGFR) calculated using serum creatinine (Cr), cystatin C (CysC), beta-2 microglobulin (β2 MG), 24-h creatinine clearance (24hCcr), and the full chronic kidney disease in children (CKiD) index that combines Cr, CysC, and blood urea nitrogen-based equations with iGFR as a reference to identify the most reliable equation for GFR. Results There was no significant difference between the values before and after allo-HSCT. CKiD CysC-, 24hCcr-, and full CKiD-based values showed good within 30% (P30) accuracy (80.6%, 79.3%, and 80.6%, respectively), but only 24hCcr and full CKiD had good mean bias (8.5% and 8.9%, respectively) and narrow 95% limits of agreement (-32.2 to 52.7 mL/min/1.73 m2 and -29.3 to 47.4 mL/min/1.73 m2 , respectively) compared with the corresponding iGFR. Conclusion There was no significant impact of allo-HSCT on GFR in our cohort. The most reliable equations for pediatric patients with allo-HSCT were eGFR-24hCcr and eGFR-full CKiD.

To understand the genetics of steroid-sensitive nephrotic syndrome (SSNS), we conducted a genome-wide association study in 987 childhood SSNS patients and 3,206 healthy controls with Japanese ancestry. Beyond known associations in the HLA-DR/DQ region, common variants in NPHS1-KIRREL2 (rs56117924, P=4.94E-20, odds ratio (OR) =1.90) and TNFSF15 (rs6478109, P=2.54E-8, OR=0.72) regions achieved genome-wide significance and were replicated in Korean, South Asian and African populations. Trans-ethnic meta-analyses including Japanese, Korean, South Asian, African, European, Hispanic and Maghrebian populations confirmed the significant associations of variants in NPHS1-KIRREL2 (P(meta)=6.71E-28, OR=1.88) and TNFSF15 (P(meta)=5.40E-11, OR=1.33) loci. Analysis of the NPHS1 risk alleles with glomerular NPHS1 mRNA expression from the same person revealed allele specific expression with significantly lower expression of the transcript derived from the risk haplotype (Wilcox test p=9.3E-4). Because rare pathogenic variants in NPHS1 cause congenital nephrotic syndrome of the Finnish type (CNSF), the present study provides further evidence that variation along the allele frequency spectrum in the same gene can cause or contribute to both a rare monogenic disease (CNSF) and a more complex, polygenic disease (SSNS).

Abscisic acid (ABA) plays an important role in plant growth, development, and stress responses. ABA regulates many aspects of plant growth and development, including seed maturation, dormancy, germination, the transition from vegetative to reproductive growth, leaf senescence and responses to environmental stresses, such as drought, high salinity and cold. It is also known that mitogen-activated protein kinase (MAPK) cascades function in ABA signaling. Recently, we and another group have identified the ABA-inducible MAP3Ks MAP3K17 and MAP3K18 as the upstream MAP3Ks of MKK3, implicating the MAP3K17/18-MKK3-MPK1/2/7/14 cascade in ABA signaling. It has also been reported that overexpression of MAP3K18 in Arabidopsis causes an early leaf senescence phenotype, ABA hypersensitive stomata closing, and drought tolerance. In this study, we generated transgenic plants overexpressing MAP3K17 (35S:MAP3K17) and its kinase-inactive form (35S:MAP3K17KN). The bolting of 35S:MAP3K17 was earlier than WT, and the fresh weights of the seedlings were smaller, whereas 35S:MAP3K17KN showed the opposite phenotype. These results indicate that the transition from vegetative to reproductive growth can be regulated by overexpression of MAP3K17 and its kinase-inactive form. Moreover, 35S:MAP3K17 showed lower sensitivity to ABA during post-germinated growth, whereas 35S:MAP3K17 KN showed the opposite phenotype, suggesting the negative roles of MAP3K17 in the response to ABA. Our work provides the possibility to regulate plant growth and development by the genetic manipulation of ABA-induced MAPK cascades, leading to improved crop growth and productivity.

Abscisic acid (ABA) is a phytohormone that regulates many physiological functions, such as plant growth, development and stress responses. The MAPK cascade plays an important role in ABA signal transduction. Several MAPK and MAPKK molecules are reported to function in ABA signaling; however, there have been few studies related to the identification of MAPKKK upstream of MAPKK in ABA signaling. In this study, we show that an Arabidopsis MAPKKK, MAPKKK18 functions in ABA signaling. The expression of MAPKKK18 was induced by ABA treatment. Yeast two-hybrid analysis revealed that MAPKKKK18 interacted with MKK3, which interacted with C-group MAPK, MPK1/2/7. Immunoprecipitated kinase assay showed that the 3xFlag-tagged MAPKKK18, expressed in Arabidopsis plants, was activated when treated with ABA. These results indicate the possibility that the MAPK cascade is composed of MAPKKK18, MKK3 and MPK1/2/7 in ABA signaling. The transgenic plants overexpressing MAPKKK18 (35S:MAPKKK18) and its kinase negative mutant (35S:MAPKKK18 KN) were generated, and their growth was monitored. Compared with the WT plant, 35S:MAPKKK18 and 35S:MAPKKK18 KN showed smaller and bigger phenotypes, respectively. Senescence of the rosette leaves was promoted in 35S:MAPKKK18, but suppressed in 35S:MAPKKK18 KN. Furthermore, ABA-induced leaf senescence was accelerated in 35S:MAPKKK18. These results suggest that MAPKKK18 controls the plant growth by adjusting the timing of senescence via its protein kinase activity in ABA dependent manners.

Mitogen-activated protein kinase (MAPK) cascades play important roles in plant responses to various environmental stimuli, including high salt or drought levels. Arabidopsis MAP3Kδ4 with PAS (period circadian protein, arylhydrocarbon receptor nuclear translocator protein and single-minded protein) domain is one of the Raftype MAPKKKs whose function has not been identified to date. Previous studies have shown that the MAP3Kδ4 overexpressing transformant exhibits vigorous growth. In this study, RT-PCR analysis showed that MAP3Kδ4 transcripts are increased through stress treatments, such as high salt, osmosis, drought and cold and the plant hormone, abscisic acid (ABA). The precipitation of MAP3Kδ4 using its specific antibody showed that ABA treatment markedly induces the activity of this enzyme. Furthermore, the ABA-mediated inhibition of seed germination was relieved in transgenic Arabidopsis over-expressing MAP3Kδ4. These results suggested that MAP3Kδ4 plays an important role in ABA signalling. Transgenic Arabidopsis over-expressing MAP3Kδ4 also exhibited enhanced tolerance to salt stress. The results obtained in this study demonstrated that MAP3Kδ4 was active during ABA-related responses and is involved in both stress tolerance and increased biomass. Therefore, MAP3Kδ4 and its counterpart genes are important with respect to agricultural developments.

MAP3Kδ4 (At4g23050) is a Raf-like mitogen-activated protein kinase kinase kinase (MAP3K) in Arabidopsis thaliana, classified as subgroup B2. The B2 subgroup MAP3Ks have a kinase domain in the C-terminus and a PAS domain in the N-terminal region. PAS domains are found in a variety of proteins and are reported to mediate protein–protein interactions and to sense environmental stimuli. However, the function of MAP3Kδ4 has not yet been determined. We generated transgenic plants constitutively expressing MAP3Kδ4 or its kinase-negative mutant (MAP3Kδ4KN) and characterized their physiological traits. The transgenic plants overexpressing MAP3Kδ4 showed earlier bolting than wild-type plants. They also showed more vigorous growth by both fresh weight and stem length. In contrast, the transgenic plants overexpressing MAP3Kδ4KN showed a highly branched phenotype; MAP3Kδ4 overexpression had no effect on branch number. These results indicated that MAP3Kδ4 is crucial to regulating both plant growth and shoot branching. In addition, MAP3Kδ4 transcripts were found in all Arabidopsis tissues examined and upregulated by auxin treatment in seedlings, suggesting that MAP3Kδ4 functions in an auxin-dependent manner.

Alternative splicing (AS) generates multiple forms of proteins. A role for AS in the plant mitogen-activated protein kinase (MAPK) cascade has not been clarified. In this study, we analyzed expression of 20 Arabidopsis MAPK genes by RT-PCR and found five that generate splice variants. The MPK13 gene, with six exons and five introns, generates at least three splice variants, one in which complete splicing of five introns occurs (MPK13 Full), and ones in which the 4th and 5th introns are retained (MPK13 I4 and I5). Translation products of the splice variants MPK13 Full and I4, were found in Arabidopsis tissues by Western blot. Yeast two-hybrid analysis and protein kinase assays of recombinant proteins showed that neither I4 nor I5 interacted with upstream MAPKKs, and neither had protein kinase activity. However, MPK13 I4 protein enhanced the MKK6-dependent activation of MPK13 Full, indicating the possibility of an additional mechanism to regulate the MAPK cascade by AS.

Object: For ectothermic insects, thermal tolerance is one of the most essential properties for survival in ambient environments. However, the molecular mechanisms involved in thermal tolerance in the silkworm, Bombyx mori L. (Lepidoptera: Bombycidae) have not been fully elucidated. The present study investigated mechanisms that provide heat tolerance in embryos of Bombyx mori . Materials and Methods: Eggs of the bivoltine silkworm strain p50 were exposed to different temperatures, to determine the lethal threshold temperature and to assess the effects of mild and low temperature incubation on tolerance to heat shock and on embryonic protein profiles. Protein levels were measured by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Results: When eggs were exposed to transient heat shock for one hour, the lethal threshold temperature was between 47.0°C and 48.0°C. However, exposure to 40.0°C for four hours, substantially elevated tolerance to the threshold heat shock and simultaneously increased levels of 70 and 27 kDa proteins in eggs, whereas exposure to 35.0°C did not. Exposure to 10.0°C for four hours lowered heat tolerance and did not alter the expression of 70 and 27 kDa proteins. Conclusion: The present findings indicated that hardening silkworm eggs at mild temperatures increases heat tolerance in embryos. To our knowledge this is the first observation of heat hardening in silkworm embryos. These putative heat-shock proteins of 70 and 27 kDa might be involved in the effect of mild temperature hardening on heat tolerance. Cold stress might deprive embryos of energy reserves available for protection against heat damage. Protective mechanisms against heat and cold stress are probably different in this species. Further studies of the molecular mechanisms of heat tolerance should provide insight into the development of novel, high-yield silkworm strains in tropical environments.

Phototropins (phot) are blue light receptors in plants which are involved in phototropism, stomatal opening, and chloroplast movements. Phototropin has two LOV domains (LOV1 and LOV2), and the LOV2 domain is responsible for activation of Ser/Thr kinase. There is an alpha-helix at the C-terminal side of the LOV2 domain, which is called the Jalpha helix. The functional importance of the Jalpha helix has been established for Arabidopsis phot1, where light-induced structural perturbation takes place in the Jalpha helix during the photocycle of LOV2 domains. However, the present FTIR study reports a different role of the Jalpha helix in light-induced signal transduction of LOV2 domains. Here we construct LOV2 domains with (LOV-Jalpha) and without (LOV-core) the Jalpha helix for Arabidopsis phot1 and phot2 and Adiantum neochrome 1 and compare their light-induced difference FTIR spectra. Light-induced protein structural changes differ significantly between LOV-Jalpha and LOV-core for Arabidopsis phot1 [Yamamoto, A., Iwata, T., Sato, Y., Matsuoka, D., Tokutomi, S., and Kandori, H. (2009) Biophys. J. 96, 2771-2778]. In contrast, the difference spectra are identical between LOV-Jalpha and LOV-core for Adiantum neochrome 1. In Arabidopsis phot2, the protein structural changes are intermediate between Arabidopsis phot1 and Adiantum neochrome 1. These results suggest that the conformational changes of the Jalpha helix and the interaction between the LOV-core and the Jalpha helix are different among phototropins. The role of the Jalpha helix for signal transduction in phototropins is discussed.

In the plant blue-light sensor phototropin, illumination of the chromophoric LOV domains causes activation of the serine/threonine kinase domain. Flavin mononucleotide (FMN) is a chromophore molecule in the two LOV domains (LOV1 and LOV2), but only LOV2 is responsible for kinase activation. Previous studies reported an important role of an additional helix connected to the C-terminal of LOV2 (Jalpha helix) for the function of phototropin; however, it remains unclear how the Jalpha helix affects light-induced structural changes in LOV2. In this study we compared light-induced protein structural changes of the LOV2 domain of Arabidopsis phot1 in the absence (LOV2-core) and presence (LOV2-Jalpha) of the Jalpha helix by Fourier-transform infrared spectroscopy. Prominent peaks were observed only in the amide-I region (1650 (-)/1625 (+) cm(-1)) of LOV2-Jalpha at physiological temperatures (>/=260 K), corresponding to structural perturbation of the alpha-helix. The peaks were diminished by point mutation of functionally important amino acids such as Phe-556 between FMN and the beta-sheet, Gln-575 being hydrogen-bonded with FMN, and Ile-608 on the Jalpha helix. We thus conclude that a light signal is relayed from FMN through these amino acids and eventually changes the interaction between LOV2-core and the Jalpha helix in Arabidopsis phot1.

Phototropins are plant blue-light photoreceptors containing two light-, oxygen-, or voltage-sensitive (LOV) domains and a C-terminal kinase domain. The two LOV domains bind noncovalently flavin mononucleotide as a chromophore. We investigated the photocycle of fast-recovery mutant LOV2-I403V from Arabidopsis phototropin 2 by step-scan Fourier transform infrared spectroscopy. The reaction of the triplet excited state of flavin with cysteine takes place with a time constant of 3 micros to yield the covalent adduct. Our data provide evidence that the flavin is unprotonated in the productive triplet state, disfavoring an ionic mechanism of bond formation. An intermediate adduct species was evident that displayed changes in secondary structure in the helix or loop region, and relaxed with a time constant of 120 micros. In milliseconds, the final adduct state is formed by further alterations of secondary structure, including beta-sheets. A comparison with wild-type adduct spectra shows that the mutation does not interfere with the functionality of the domain. All signals originate from within the LOV domain, because the construct does not comprise the adjacent Jalpha helix required for signal transduction. The contribution of early and late adduct intermediates to signal transfer to the Jalpha helix outside of the domain is discussed.

Phototropin is a blue-light receptor protein in plants that is responsible for phototropic responses, stomata opening and photo-induced relocation of chloroplasts. Higher plants such as Arabidopsis thaliana have two isoforms of phototropin: phototropin 1 and phototropin 2. Both isoforms comprise a tandem pair of blue-light-absorbing light–oxygen–voltage domains named LOV1 and LOV2 in the N-terminal half and a serine/threonine kinase domain in the C-­terminal half. The LOV1 domain is thought to function as a dimerization site. In the present study, recombinant LOV1 domains of A. thaliana phototropin 1 and phototropin 2 were crystallized. The crystal of the LOV1 domain of phototropin 1 belonged to the orthorhombic space group P212121, with unit-cell parameters a = 61.2, b = 64.9, c = 70.8 A, and diffracted X-rays to a resolution of 2.1 A. The crystal of the LOV1 domain of phototropin 2 belonged to space group P21, with unit-cell parameters a = 32.5, b = 66.5, c = 56.7 A, β = 92.4°, and diffracted X-rays to beyond 2.0 A resolution. In both crystals, two LOV1 domains occupied the crystallographic asymmetric unit.

Phototropin (phot) is a blue light photoreceptor in plants that mediates phototropism, chloroplast movement, stomata opening and leaf expansion. The phot molecule has two photoreceptive domains, LOV 1 and 2, in the N-terminal half and the C-terminal half forms Ser/Thr kinase. Phot acts as a blue light-regulated protein kinase. Each LOV domain binds a FMN and undergoes a unique cyclic reaction upon blue light absorption that induces conformational changes in the protein moiety and leads to regulation of the kinase activity, in which LOV2 plays a predominant role in the switching and LOV1 acts to attenuate the light sensitivity. Phot kinase is classified into the AGC kinase group since the consensus amino acid residues and the motifs are well conserved except for the lack of the hydrophobic motif and the presence of additional amino acid sequence in the activation loop. Secondary structure prediction and 3D structure simulation show a alpha/beta fold of the phot kinase similar to that of the catalytic subunit of PKA. The additional sequence forms an extra helix and loops. Docking simulation of the LOV2 domain with phot kinase provided useful information regarding the molecular mechanism underlying the photoregulation of phot kinase.

本研究ではマイタケ子実体の生体電位信号をモニタリングし，信号中の概周期的な生体リズム成分より，光照射のタイミングを制御する方法を考案した．すなわち，子実体をバイオセンサとして用いることで，生体電位の自発性リズム変動に連動させた光照射を用いる新たな栽培技術を提案した．約1時間分析区間長毎の生体電位の上昇や下降の変動特性に応じて，光源装置の点灯あるいは消灯タイミングを試行的に設定した．その結果，12L12D(12時間Light，12時間Dark)の間欠照射あるいは連続照射に比べて，生体電位のリズム変動に連動させた光源制御の方が菌傘の展開が発達したり，新鮮重が増加する傾向となり，茸工場内での生産性向上が見込まれる結果を得た．このように，本研究では特定の個体をバイオセンサとして生体電位信号のリズム変動を計測して，その信号に連動させて茸工場全体の光源を制御するシステムの基盤が構築できたと考えている．なお，本研究成果の一部は文部科学省科学研究費(基盤研究(C)：課題番号16560420)の助成による．

Plants photoreceptors are divided into two groups, well-known phytochromes and blue-light receptors, such as cryptochromes and phototropins. Regarding one of the later, we proved the role of the LOV domains in the kinase photoactivation. In addition to the plant phytochromes, many bacteriophytochromes have been found in both photosynthetic and nonphotosynthetic prokaryotic microorganisms. One of them has been found to undergo not red/far-red but blue/green photoreversible transformaion. Furthermore, new members of blue-light receptors, FKF1 families and BULF photoreceptors, have been found. Their structures and functions are reviewed.

本論文では，子実体の形態形成に影響を及ぼす環境因子の科学的解明を目的とし，ナメコを供試体として光強度が生体電位および形態形成に与える影響について調べた．その結果，以下の結論を得た．生体電位反応は光強度2 W m-2と比較して10 W m-2で大となる傾向を得た．この生体電位応答特性を踏まえ，2 W m-2と10 W m-2における形態形成を比較した結果，2 W m-2と比較して10 W m-2では菌傘が大きく発達し，色が濃くなる傾向を得た．過去の実験においても，生体電位反応が大となる環境条件下では菌傘が発達してその色は濃くなる傾向にあり，生体電位と形態形成との関係が示唆された．今後は，生体電位が具体的にどのような生長に関する現象の指標となるのかを系統的に実験計画を立てて解明し，生体電位を指標とした生長制御を試みたい．また，今回はストロボ光によるミリ秒オーダの光刺激が生体電位と形態形成に与える影響についても試行的に調べた．その結果，約10 msec幅のストロボ光に対して電位反応が数秒間継続することを突き止め，子実体がミリ秒オーダの刺激にも反応することを明らかにした．このストロボ光による子実体内部での生体電位の誘発は，生長促進に効果的であることも示唆され，新たな光照射手法による生産コスト削減の可能性も見出した．

Flavin-binding, Kelch repeat, F-box (FKF1) protein is a photoreceptor to regulate flowering of Arabidopsis. The protein has a light, oxygen and voltage (LOV)-sensing domain binding a flavin mononucleotide. The photo-activation of the domain is an indispensable step to initiate the cellular signaling for flowering. In the present study, a LOV-containing polypeptide of FKF1 was prepared by an overexpression system, and the quaternary structure of it was studied by size exclusion chromatography and small-angle X-ray scattering. The apparent molecular weight from chromatography suggested a globular trimeric or an anisotropic-shaped dimeric association of the polypeptide in solution. The scattering experiment demonstrated a dimeric association of the polypeptides with an elongated molecular shape displaying the radius of gyration of 27Å and the maximum dimension of 94Å. The molecular shape simulated from scattering profiles suggests an antiparallel association of the LOV domains in the dimer. Though the absorption spectrum of blue-light irradiated polypeptide was stable in the photoactivated state for a long period, the scattering profiles showed very small changes between the dark and light conditions. Based on the homologies in the amino-acid sequences and the scattering profiles, these results are discussed in connection with the structures and function of LOV domains of phototropin.

本論文では, 茸の至適生育環境条件を工学の視点から科学的に解明することを目的に, 光の波長がエリンギの生体電位および形態形成に与える影響について調べた.その結果, エリンギの生体電位反応は青色域の波長の光で大となり, 逆に赤色域の波長の光では小となる傾向を得た. この生体電位応答特性を踏まえて, 各種光源における形態形成を比較したところ, 生体電位反応が大となった青色光下では菌傘の生長が促進され菌柄の伸長が抑制されるといった, 元来自生している場合の形態形成を得た. これより, 生体電位を指標とすることで, 茸に適した環境条件を解明することができると結論した.

The mitogen-activated protein kinase (MAPK) cascade, consisting of MAPK, MAPK kinase (MAPKK) and MAPK kinase kinase (MAPKKK), is the signaling system that relays various external signals, including mitogens and stresses in eukaryotes. MAPKK is activated by phosphorylation in the consensus motif, SXXXS/T, in animals, but the regulation mechanism for the plant MAPKK by phosphorylation, having the putative phosphorylation motif of S/TXXXXXS/T, is not yet fully clarified. Here we constructed a series of mutants of AtMEK1, an Arabidopsis MAPKK, having the sequence T218-X-S220-X-X-X-S224 that fits both of the plant- and animal-type motifs. We show that the two double-mutant proteins replacing Thr-218/Ser-224 and Ser-220/Ser-224 by Glu expressed in Escherichia coli show a constitutive activity to phosphorylate the Thr and Tyr residues of the kinase-negative mutant of an Arabidopsis MAPK, named ATMPK4, in vitro. The mutation analysis of AtMEK1 replacing Thr-218 and Ser-220 to Ala suggested that Thr-218 is autophosphorylated by the enzyme. The wild-type ATMPK4 was also phosphorylated by the active mutants of AtMEK1 and showed a high protein kinase activity toward myelin basic proteins. In contrast, ATMPK3, another Arabidopsis MAPK, was a poor substrate of this plant MAPKK, indicating that AtMEK1 has a substrate specificity preferring ATMPK4 to ATMPK3, at least in vitro. Furthermore, AtMEK1 immunoprecipitated from Arabidopsis seedlings stimulated with wounding, cold, drought, and high salt showed an elevated protein kinase activity toward the kinase-negative ATMPK4, while the amounts of the AtMEK1 protein did not change significantly. These data indicate that the AtMEK1 becomes an active form through phosphorylation and activates its downstream target ATMPK4 in stress response in Arabidopsis.

The MEKK1-MKK2-MPK4 cascade is activated during cold acclimation. However, little is known regarding the perception of low temperature. In this study, we demonstrate that treatment of Arabidopsis with a membrane rigidifier, DMSO, caused MPK4 activation concomitantly with MEKK1 and MKK2 phosphorylation, as well as the cold-inducible gene COR15a expression. These processes are similar to the effects of cold treatment, whereas benzyl alcohol (BA), a membrane fluidizer, prevented such cold-induced events. Moreover, the DMSO-treated seedlings acquired freezing tolerance without cold acclimation. In contrast, the BA-pretreated seedlings did not show freezing tolerance. These results suggest that membrane rigidification activates this MAPK cascade and contributes to the acquisition of freezing tolerance.

A gene named epk2 that encodes the amino acid sequence of a protein kinase was identified from the photosynthetic flagellate, Euglena gracilis Z. Homology search and phylogenetic analysis revealed that the deduced amino acid sequence of epk2 is most similar to that of the catalytic subunit of cAMP-dependent protein kinase (PKA). Northern blot analysis showed that Euglena cells express a 1.4-kb transcript of this gene. When the EPK2 protein was coexpressed with the rat regulatory subunit of PKA in cultured mammalian cells, these two proteins were coimmunoprecipitated. The association of EPK2 and the rat regulatory subunit of PKA was not detected in the cell lysate incubated with cAMP. EPK2 immunoprecipitated from the transfected cells phosphorylated Kemptide, a synthetic peptide substrate for PKA, and the phosphorylation was inhibited by PKI, a PKA-selective protein kinase inhibitor. These results indicate that EPK2 is a PKA homologue in the photosynthetic flagellate, and this is the first evidence for the occurrence of the PKA catalytic subunit in photosynthetic organisms.

Dent disease is an inherited tubulopathy caused by a mutation in the CLCN5 chloride channel gene. In cases of Dent disease in Japan (Japanese Dent, J-Dent), renal function is generally preserved and rarely progresses to advanced kidney dysfunction. However, the long-term prognosis of J-Dent remains unknown. We report the case of a 32-year-old man with J-Dent who developed advanced kidney dysfunction. Since the age of 3 years, he persistently exhibited proteinuria, and examination of a kidney biopsy specimen indicated focal segmental glomerulosclerosis (FSGS)-like lesions. Repeated corticosteroid treatments were found to be ineffective. After the age of 18 years, the patient was lost to follow-up and treatment was discontinued. The patient presented to our hospital again at the age of 32 years with advanced kidney dysfunction with low-molecular-weight proteinuria (LMWP), along with proximal tubular dysfunction and nephrocalcinosis. The patient’s 5-year-old nephew was also found to have LMWP from the age of 7 months. Therefore, Dent disease was suspected and genetic testing in the patient and his nephew revealed a CLCN5 mutation. Our case report suggests that J-Dent may cause advanced kidney dysfunction in adulthood, and, therefore, close collaboration between pediatricians and nephrologists is essential for the early identification of this complication. When male patients exhibit chronic kidney disease (CKD) of unknown etiology along with proximal tubular dysfunction and nephrocalcinosis, Dent disease should be considered. Investigations of undiagnosed adult J-Dent cases and further research on the natural history of J-Dent will help us better understand its clinical characteristics, prognosis, and effective treatment options.

The Arabidopsis mitogen activated protein kinase kinase kinase (MEKK1) plays an important role in stress signaling. However, little is known about the upstream pathways of MEKK1. This report describes the regulation of MEKK1 activity during cold signaling. Immunoprecipitated MEKK1 from cold-treated Arabidopsis seedlings showed elevated kinase activity towards mitogen activated protein kinase kinase2 (MKK2), one of the candidate MEKK1 substrates. To clarify how MEKK1 becomes active in response to cold stress signaling, MEKK1 phosphorylation was monitored by an enzyme extracted from the seedlings grown under cold stress with or without EGTA. MEKK1 was phosphorylated after cold stress, but EGTA inhibited the phosphorylation. MKK2 was also phosphorylated by the same extract, but only when EGTA was absent. These results suggested that Ca(2+) signaling occurred upstream of the MEKK1-MKK2 pathway. Full-length MEKK1 showed almost no activity but MEKK1 without the N-terminal region (MEKK1 KD) that retained the kinase domain had a strong ability to phosphorylate MKK2, demonstrating the inhibitory role of the N-terminal region of MEKK1. In addition, MEKK1 was phosphorylated by calcium/calmodulin-regulated receptor-like kinase (CRLK1), which suggested that CRLK1 is one of candidates located upstream of MEKK1.

Flavin-binding Kelch repeat F-box (FKF1) protein plays important roles in the photoregulation of flowering in Arabidopsis. FKF1 has a light, oxygen, and voltage (LOV) sensing domain binding a flavin mononucleotide (FMN) as a chromophore noncovalently. Photoreaction of the FKF1-LOV polypeptide was studied by low-temperature absorption spectroscopy. Upon blue light irradiation, a ground state, D(450), is converted to S(390) known as a cysteinyl-flavin adduct intermediate in the photoreaction of phototropin. Below 150 K, bleaching of D(450) was much reduced and a new photoproduct, Z(370), appeared as well as S(390) formation. The calculated absorption spectrum for Z(370) is very similar to those of flavoproteins in an anion radical state. On the basis of the results that S(390) formation proceeds to Z(370) formation and that Z(370) formed at low temperatures reverts to D(450) upon temperature increase, Z(370) is concluded to be not an intermediate from D(450) to S(390). Z(370) is suggested to be formed from the biradical triplet-excited state after relaxing to the ground state with the FMN anion radical trapped at the low temperature, in which the SH of the cysteine is in the wrong position that is able to produce a radical pair but unable to form the cysteinyl-flavin adduct. The counter SH in the cationic radical state may revert to the ground state by extracting an electron from the unidentified amino acid residue. Interestingly, S(390) that has been thought to be irreversible to D(450) was revealed to revert to D(450) very slowly with a half-life time of 62.5 h in solution at 298 K. The photoreaction mechanism is discussed in reference to the calculated activation energy of the reaction processes.

Phototropin is a blue light photoreceptor for tropic responses, relocation of chloroplasts, and stomata opening in plants. Phototropin has two chromophoric domains named light-oxygen-voltage-sensing (LOV) 1 and 2 in the N-terminal half, and a serine/threonine (Ser/Thr) protein kinase motif in the C-terminal half. Concerning the kinase activity of phototropin, only autophosphorylation has been detected so far. However, we found that phototropin can phosphorylate a protein other than phototropin itself. Bacterially expressed Arabidopsis phototropin 2 kinase domain (KD) with GST-tag showed a constitutive kinase activity on casein, a common in vitro substrate of Ser/Thr protein kinase. By using this in vitro assay system, the roles of each LOV domain were studied. Addition of LOV2 to KD (GST-L2-KD) inhibits the kinase activity that is canceled by light. This light activation of kinase disappeared on introduction of a mutation blocking photochemical reaction in the LOV2 domain. Accordingly, LOV2 domain acts as a major light-regulated molecular switch of casein phosphorylation. Interestingly, isolated LOV2 from the KD still binds to the KD in a light-dependent manner and functions in similar ways, indicating the role of LOV2 domain as an inhibitor of the kinase activity in the substrate phosphorylation. LOV1, in contrast, contributes little to the photoactivation in GST-L1-L2-KD; however, it acts as an attenuator of the light activation of the kinase by LOV2.

We have constructed a series of deletion mutants of Arabidopsis MAPK kinase kinase (AtMEKK1) and obtained a constitutively active mutant, AtMEKK1Delta166, which lacks in self-inhibitory sequence of N-terminal 166 amino acids but still has substrate specificity. AtMEKK1Delta166 predominantly phosphorylates AtMEK1, an Arabidopsis MAPKK, but not its double mutant (AtMEK1T218A/S224E), suggesting that Thr-218 and Ser-224 are the phosphorylation sites. In wounded seedlings, AtMEKK1 was activated and phosphorylated its downstream AtMEK1. Furthermore, analysis using anti-AtMEKK1 and anti-AtMEK1 antibodies revealed that the interaction between the two proteins was signal dependent. These results suggest the presence of AtMEKK1-AtMEK1 pathway induced by wounding.

Phototropin is a blue light receptor in plants and is thought to be a light-regulated protein kinase. Previously, we defined the role of the photoreceptive domains, LOV1 and 2, in the light activation of the kinase in Arabidopsis phototropin2 (phot2) [1] . In this study, photoregulation of the kinase in phototropin1 (phot1) was studied using LOV2-linker-kinase polypeptide. We designed a new substrate consisting of the N-terminal part of the phot1 with autophosphorylation sites. The LOV2-linker-kinase had the same spectroscopic properties as those of the LOV2 core and phosphorylated the substrate in a light-dependent manner. Amino acid substitution experiments proved that the phosphorylation comes from the activation of the kinase via photoreaction of LOV2. Structured summary of protein interactions: AtPhot1-LOV2 phosphorylates AtPhot1-Nt by protein kinase assay (View Interaction: 1 , 2 , 3 )