Melatonin Inhibits the Migration of Human Lung Adenocarcinoma A549 Cell Lines Involving JNK/MAPK Pathway

Objective

Melatonin, an indolamine produced and secreted predominately by the pineal gland, exhibits a variety of physiological functions, possesses antioxidant and antitumor properties. But, the mechanisms for the anti-cancer effects are unknown. The present study explored the effects of melatonin on the migration of human lung adenocarcinoma A549 cells and its mechanism.

Methods

MTT assay was employed to measure the viability of A549 cells treated with different concentrations of melatonin. The effect of melatonin on the migration of A549 cells was analyzed by wound healing assay. Occludin location was observed by immunofluorescence. The expression of occludin, osteopontin (OPN), myosin light chain kinase (MLCK) and phosphorylation of myosin light chain (MLC), JNK were detected by western blots.

Results

After A549 cells were treated with melatonin, the viability and migration of the cells were inhibited significantly. The relative migration rate of A549 cells treated with melatonin was only about 20% at 24 h. The expression level of OPN, MLCK and phosphorylation of MLC of A549 cells were reduced, while the expression of occludin was conversely elevated, and occludin located on the cell surface was obviously increased. The phosphorylation status of JNK in A549 cells was also reduced when cells were treated by melatonin.

Conclusions

Melatonin significantly inhibits the migration of A549 cells, and this may be associated with the down-regulation of the expression of OPN, MLCK, phosphorylation of MLC, and up-regulation of the expression of occludin involving JNK/MAPK pathway.     

bFGF Regulates PI3-Kinase-Rac1-JNK Pathway and Promotes Fibroblast Migration in Wound Healing

Fibroblast proliferation and migration play important roles in wound healing. bFGF is known to promote both fibroblast proliferation and migration during the process of wound healing. However, the signal transduction of bFGF-induced fibroblast migration is still unclear, because bFGF can affect both proliferation and migration. Herein, we investigated the effect of bFGF on fibroblast migration regardless of its effect on fibroblast proliferation. We noticed involvement of the small GTPases of the Rho family, PI3-kinase, and JNK. bFGF activated RhoA, Rac1, PI3-kinase, and JNK in cultured fibroblasts. Inhibition of RhoA did not block bFGF-induced fibroblast migration, whereas inhibition of Rac1, PI3-kinase, or JNK blocked the fibroblast migration significantly. PI3-kinase-inhibited cells down-regulated the activities of Rac1 and JNK, and Rac1-inhibited cells down-regulated JNK activity, suggesting that PI3-kinase is upstream of Rac1 and that JNK is downstream of Rac1. Thus, we concluded that PI3-kinase, Rac1, and JNK were essential for bFGF-induced fibroblast migration, which is a novel pathway of bFGF-induced cell migration.     

Induction of Specific MicroRNAs Inhibits Cutaneous Wound Healing

Chronic nonhealing wounds, such as venous ulcers (VUs), are a widespread and serious medical problem with high morbidity and mortality. The molecular pathology of VUs remains poorly understood, impeding the development of effective treatment strategies. Using mRNA expression profiling of VUs biopsies and computational analysis, we identified a candidate set of microRNAs with lowered target gene expression. Among these candidates, miR-16, -20a, -21, -106a -130a, and -203 were confirmed to be aberrantly overexpressed in a cohort study of 10 VU patients by quantitative PCR and in situ hybridizations. These microRNAs were predicted to target multiple genes important for wound healing, including early growth response factor 3, vinculin, and leptin receptor (LepR). Overexpression of the top up-regulated miRNAs, miR-21 and miR-130a, in primary human keratinocytes down-regulated expression of the endogenous LepR and early growth response factor 3. The luciferase reporter assay verified LepR as a direct target for miR-21 and miR-130a. Both miR-21 and miR-130a delayed epithelialization in an acute human skin wound model. Furthermore, in vivo overexpression of miR-21 inhibited epithelialization and granulation tissue formation in a rat wound model. Our results identify a novel mechanism in which overexpression of specific set of microRNAs inhibits wound healing, resulting in new potential molecular markers and targets for therapeutic intervention.     

Zebrafish Keratocyte Explants to Study Collective Cell Migration and Reepithelialization in Cutaneous Wound Healing

Due to their unique motile properties, fish keratocytes dissociated from explant cultures have long been used to study the mechanisms of single cell migration. However, when explants are established, these cells also move collectively, maintaining many of the features which make individual keratocytes an attractive model to study migration: rapid rates of motility, extensive actin-rich lamellae with a perpendicular actin cable, and relatively constant speed and direction of migration. In early explants, the rapid interconversion of cells migrating individually with those migrating collectively allows the study of the role of cell-cell adhesions in determining the mode of migration, and emphasizes the molecular links between the two modes of migration. Cells in later explants lose their ability to migrate rapidly and collectively as an epithelial to mesenchymal transition occurs and genes associated with wound healing and inflammation are differentially expressed. Thus, keratocyte explants can serve as an in vitro model for the reepithelialization that occurs during cutaneous wound healing and can represent a unique system to study mechanisms of collective cell migration in the context of a defined program of gene expression changes. A variety of mutant and transgenic zebrafish lines are available, which allows explants to be established from fish with different genetic backgrounds. This allows the role of different proteins within these processes to be uniquely addressed. The protocols outlined here describe an easy and effective method for establishing these explant cultures for use in a variety of assays related to collective cell migration.     

Continual Cell Deformation Induced via Attachment to Oriented Fibers Enhances Fibroblast Cell Migration

Fibroblast migration is critical to the wound healing process. In vivo, migration occurs on fibrillar substrates, and previous observations have shown that a significant time lag exists before the onset of granulation tissue. We therefore conducted a series of experiments to understand the impact of both fibrillar morphology and migration time. Substrate topography was first shown to have a profound influence. Fibroblasts preferentially attach to fibrillar surfaces, and orient their cytoplasm for maximal contact with the fiber edge. In the case of en-mass cell migration out of an agarose droplet, fibroblasts on flat surfaces emerged with an enhanced velocity, v = 52μm/h, that decreases to the single cell value, v = 28μm/h within 24 hours and remained constant for at least four days. Fibroblasts emerging on fibrillar surfaces emerged with the single cell velocity, which remained constant for the first 24 hours and then increased reaching a plateau with more than twice the initial velocity within the next three days. The focal adhesions were distributed uniformly in cells on flat surfaces, while on the fibrillar surface they were clustered along the cell periphery. Furthermore, the number of focal adhesions for the cells on the flat surfaces remained constant, while it decreased on the fibrillar surface during the next three days. The deformation of the cell nuclei was found to be 50% larger on the fiber surfaces for the first 24 hours. While the mean deformation remained constant on the flat surface, it increased for the next three days by 24% in cells on fibers. On the fourth day, large actin/myosin fibers formed in cells on fibrillar surfaces only and coincided with a change from the standard migration mechanism involving extension of lamellipodia, and retraction of the rear, to one involving strong contractions oriented along the fibers and centered about the nucleus.     

Wnt-5a-induced Phosphorylation of DARPP-32 Inhibits Breast Cancer Cell Migration in a CREB-dependent Manner

Tumor cell migration plays a central role in the process of cancer metastasis. We recently identified dopamine and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) as an antimigratory phosphoprotein in breast cancer cells. Here we link this effect of DARPP-32 to Wnt-5a signaling by demonstrating that recombinant Wnt-5a triggers cAMP elevation at the plasma membrane and Thr34-DARPP-32 phosphorylation in MCF-7 cells. In agreement, both protein kinase A (PKA) inhibitors and siRNA-mediated knockdown of Frizzled-3 receptor or Gα_s expression abolished Wnt-5a-induced phosphorylation of DARPP-32. Furthermore, Wnt-5a induced DARPP-32-dependent inhibition of MCF-7 cell migration. Phospho-Thr-34-DARPP-32 interacted with protein phosphatase-1 (PP1) and potentiated the Wnt-5a-mediated phosphorylation of CREB, a well-known PP1 substrate, but had no effect on CREB phosphorylation by itself. Moreover, inhibition of the Wnt-5a/DARPP-32/CREB pathway, by expression of dominant negative CREB (DN-CREB), diminished the antimigratory effect of Wnt-5a-induced phospho-Thr-34-DARPP-32. Phalloidin-staining revealed that that the presence of phospho-Thr-34-DARPP-32 in MCF-7 cells results in reduced filopodia formation. In accordance, the activity of the Rho GTPase Cdc42, known to be crucial for filopodia formation, was reduced in MCF-7 cells expressing phospho-Thr-34-DARPP-32. The effects of DARPP-32 on cell migration and filopodia formation could be reversed in T47D breast cancer cells that were depleted of their endogenous DARPP-32 by siRNA targeting. Consequently, Wnt-5a activates a Frizzled-3/Gα_s/cAMP/PKA signaling pathway that triggers a DARPP-32- and CREB-dependent antimigratory response in breast cancer cells, representing a novel mechanism whereby Wnt-5a can inhibit breast cancer cell migration.Breast cancer is the most common form of cancer in women. Whereas the prognosis for breast cancer patients without local or distal dissemination is relatively favorable, the prognosis is considerably worse once distal metastasis has been established. It is therefore imperative to identify molecular targets and develop novel antimetastatic therapies that will stop, reduce, or delay the dissemination and growth of breast cancer metastasis.We recently isolated the dopamine and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32),² from a human breast expression library, as a DDR1-binding partner (1). Introduction of DARPP-32 in breast cancer cells lacking endogenous expression of this protein inhibited cell migration in a phospho-Thr-34-DARPP-32-dependent manner (1). DARPP-32 was originally identified 25 years ago as a dopamine and cAMP target enriched in dopaminoceptive neurones (2). Since then, a large body of work has shown that DARPP-32 is crucial for signal transmission by a wide array of neurotransmitters and drugs of abuse. DARPP-32 can act as either a phosphatase inhibitor or a kinase inhibitor depending on its phosphorylation state. Phosphorylation of Thr-34 by protein kinase A (PKA) converts DARPP-32 into a potent inhibitor of protein phosphatase-1 (PP1) (3), whereas phosphorylation at Thr-75 by Cdk5 turns DARPP-32 into an inhibitor of PKA (4). Downstream of DARPP-32 it has been shown that the activity of CREB and c-fos are strongly attenuated in DARPP-32 knockout mice (5). Despite the substantial amount of work done on DARPP-32 over the past 25 years, the role of this phosphoprotein outside the neuronal system has only recently started to be explored.Regarding the role of DARPP-32 in cancer, overexpression of DARPP-32 has been reported in gastric, colon, and prostate cancer (6, 7). In contrast, patients with esophageal squamous cell carcinoma that lacks DARPP-32 expression have reduced survival when compared with patients with DARPP-32-expressing esophageal squamous cell carcinomas (8, 9). Furthermore, DARPP-32 is needed to get a fully differentiated thyroid cell phenotype, and transformation of thyroid cells by constitutively activated Ras resulted in a loss of DARPP-32 expression (10). Thus, the role of DARPP-32 in cancer is somewhat uncertain, and little is known about the cell signaling mechanisms behind a possible suppressor or promotor role of DARPP-32 in cancer.Wnt-5a is a non-canonical member of the Wnt family of secreted glycoproteins that acts through the family of Frizzled G-protein-coupled receptor, Ror2, and co-receptors such as, LRP5/6, to mediate important events during development and cancer (11–15). In the breast, the non-transforming Wnt-5a has been shown to inhibit epithelial cell migration (16), and in breast cancer, expression of Wnt-5a has been shown to be a predictor of longer disease-free survival (17). Wnt-5a expression has been shown to increase activation of the receptor tyrosine kinase DDR1 in breast epithelial cells upon plating on collagen (18). As DDR1 is a collagen-binding adhesion receptor important for cell migration (19), and its binding partner DARPP-32 is a phospho-dependent antimigratory molecule (1), we wanted to test whether the functional overlaps between DARPP-32 and Wnt-5a, could be a result of Wnt-5a acting upstream in the signaling process leading to DARPP-32 phosphorylation.Here we demonstrate that Wnt-5a can trigger a Frizzled-3/Gαs/cAMP signal that results in PKA-dependent phosphorylation of DARPP-32. Furthermore, we show that phospho-DARPP-32 potentiates Wnt-5a-mediated CREB activity and suppresses filopodia formation.     

Fucoidan from Seaweed Fucus vesiculosus Inhibits Migration and Invasion of Human Lung Cancer Cell via PI3K-Akt-mTOR Pathways

Background

Recently there has been an increased interest in the pharmacologically active natural products associated with remedies of various kinds of diseases, including cancer. Fucoidan is a polysaccharide derived from brown seaweeds and has long been used as an ingredient in some dietary supplement products. Although fucoidan has been known to have anti-cancer activity, the anti-metastatic effects and its detailed mechanism of actions have been poorly understood. Therefore, the aims of this study were to demonstrate the anti-metastatic functions of fucoidan and its mechanism of action using A549, a highly metastatic human lung cancer cell line.

Methods and Principal Findings

Fucoidan inhibits the growth of A549 cells at the concentration of 400 µg/ml. Fucoidan treatment of non-toxic dose (0–200 µg/ml) exhibits a concentration-dependent inhibitory effect on the invasion and migration of the cancer cell via decreasing its MMP-2 activity. To know the mechanism of these inhibitory effects, Western blotting was performed. Fucoidan treatment down-regulates extracellular signal-related kinase 1 and 2 (ERK1/2) and phosphoinositide 3-kinase (PI3K)–Akt–mammalian target of rapamycin (PI3K-Akt-mTOR) pathways. Furthermore, fucoidan decreases the cytosolic and nuclear levels of Nuclear Factor-kappa B (p65).

Conclusions/Significance

The present study suggests that fucoidan exhibits anti-metastatic effect on A549 lung cancer cells via the down-regulation of ERK1/2 and Akt-mTOR as well as NF-kB signaling pathways. Hence, fucoidan can be considered as a potential therapeutic reagent against the metastasis of invasive human lung cancer cells.     

Bindarit Inhibits Human Coronary Artery Smooth Muscle Cell Proliferation,Migration and Phenotypic Switching

Bindarit, a selective inhibitor of monocyte chemotactic proteins (MCPs) synthesis, reduces neointimal formation in animal models of vascular injury and recently has been shown to inhibit in-stent late loss in a placebo-controlled phase II clinical trial. However, the mechanisms underlying the efficacy of bindarit in controlling neointimal formation/restenosis have not been fully elucidated. Therefore, we investigated the effect of bindarit on human coronary smooth muscle cells activation, drawing attention to the phenotypic modulation process, focusing on contractile proteins expression as well as proliferation and migration. The expression of contractile proteins was evaluated by western blot analysis on cultured human coronary smooth muscle cells stimulated with TNF-α (30 ng/mL) or fetal bovine serum (5%). Bindarit (100–300 µM) reduced the embryonic form of smooth muscle myosin heavy chain while increased smooth muscle α-actin and calponin in both TNF-α- and fetal bovine serum-stimulated cells. These effects were associated with the inhibition of human coronary smooth muscle cell proliferation/migration and both MCP-1 and MCP-3 production. The effect of bindarit on smooth muscle cells phenotypic switching was confirmed in vivo in the rat balloon angioplasty model. Bindarit (200 mg/Kg/day) significantly reduced the expression of the embryonic form of smooth muscle myosin heavy chain, and increased smooth muscle α-actin and calponin in the rat carodid arteries subjected to endothelial denudation. Our results demonstrate that bindarit induces the differentiated state of human coronary smooth muscle cells, suggesting a novel underlying mechanisms by which this drug inhibits neointimal formation.     

Circ_0084443 Inhibits Wound Healing Via Repressing Keratinocyte Migration Through Targeting the miR-17-3p/FOXO4 Axis

Biochemical Genetics - Keratinocyte migration is a crucial process during skin wound healing, and circular RNAs are associated with keratinocyte migration. The purpose of our study was to clarify...     

The Four-Herb Chinese Medicine ANBP Enhances Wound Healing and Inhibits Scar Formation via Bidirectional Regulation of Transformation Growth Factor Pathway

The four-herb Chinese medicine ANBP is a pulverized mixture of four herbs including Agrimonia Eupatoria (A), Nelumbo Nucifera Gaertn (N), Boswellia Carteri (B) and Pollen Typhae Angustifoliae (P). The combination of the four herbs was first described in Chinese canonical medicine about 2000 years ago for treatment of various trauma disorders, such as hemostasis, antiinflammatory, analgesia, and wound healing, etc. However, the precise mechanisms of ANBP are still unclear. In our study, using rabbit ear hypertrophic scar models of full-thickness skin defect, we showed that local ANBP treatment not only significantly enhanced wound healing by relieving inflammation, increasing formation of granulation tissue and accelerating re-epithelialization, but also reduced scar formation by decreasing collagen production, protuberant height and volume of scars, and increasing collagen maturity. We demonstrated that these effects of ANBP are associated with transforming growth factor (TGF)-β1-mediated signalling pathways through Smad-dependent pathways. ANBP treatment significantly increased expression of TGF-β1 and Smad2/3 mRNA at the early stage of wound healing, and led to markedly decrease expression of TGF-β1 and Smad2/3 compared with the control group after 14 days post-wounding. Taken together, our results defined a bidirectional regulation role of ANBP for TGF-β1/Smad pathway in promoting wound healing and alleviating scar formation, which may be an effective therapy for human wounds at the earliest stage.     

Knockdown of FRAT1 Expression by RNA Interference Inhibits Human Glioblastoma Cell Growth,Migration and Invasion

Background

FRAT1 positively regulates the Wnt/β-catenin signaling pathway by inhibiting GSK-3-mediated phosphorylation of β-catenin. It was originally characterized as a protein frequently rearranged in advanced T cell lymphoma, but has recently also been identified as a proto-oncogene involved in tumorigenesis. Our previous studies showed that FRAT1 was dramatically overexpressed in gliomas and its expression level was significantly increased along with clinicopathological grades.

Methods

In the current study, we used RT-PCR and Western blotting to assess the mRNA and protein levels of FRAT1 in three glioma cell lines. In addition, to evaluate its functional role in gliomas, we examined the effects of FRAT1 knockdown on proliferation, migration and invasion in vitro and tumor growth in vivo using glioblastoma U251 cells and RNAi.

Results

FRAT1 was highly expressed in all three glioma cell lines. RNAi-mediated down-regulation of endogenous FRAT1 in human glioblastoma U251 cells resulted in suppression of cell proliferation, arrest of cell cycle, inhibition of cell migration and invasion in vitro. Moreover, FRAT1 depletion significantly impaired tumor xenograft growth in nude mice.

Conclusions

Our results highlight the potential role of FRAT1 in tumorigenesis and progression of glioblastoma. These findings provide a biological basis for FRAT1 as a potential molecular marker for improved pathological grading and as a novel candidate therapeutic target for glioblastoma management.     

Cinnamtannin B-1 Promotes Migration of Mesenchymal Stem Cells and Accelerates Wound Healing in Mice

Substances that enhance the migration of mesenchymal stem cells to damaged sites have the potential to improve the effectiveness of tissue repair. We previously found that ethanol extracts of Mallotus philippinensis bark promoted migration of mesenchymal stem cells and improved wound healing in a mouse model. We also demonstrated that bark extracts contain cinnamtannin B-1, a flavonoid with in vitro migratory activity against mesenchymal stem cells. However, the in vivo effects of cinnamtannin B-1 on the migration of mesenchymal stem cells and underlying mechanism of this action remain unknown. Therefore, we examined the effects of cinnamtannin B-1 on in vivo migration of mesenchymal stem cells and wound healing in mice. In addition, we characterized cinnamtannin B-1-induced migration of mesenchymal stem cells pharmacologically and structurally. The mobilization of endogenous mesenchymal stem cells into the blood circulation was enhanced in cinnamtannin B-1-treated mice as shown by flow cytometric analysis of peripheral blood cells. Whole animal imaging analysis using luciferase-expressing mesenchymal stem cells as a tracer revealed that cinnamtannin B-1 increased the homing of mesenchymal stem cells to wounds and accelerated healing in a diabetic mouse model. Additionally, the cinnamtannin B-1-induced migration of mesenchymal stem cells was pharmacologically susceptible to inhibitors of phosphatidylinositol 3-kinase, phospholipase C, lipoxygenase, and purines. Furthermore, biflavonoids with similar structural features to cinnamtannin B-1 also augmented the migration of mesenchymal stem cells by similar pharmacological mechanisms. These results demonstrate that cinnamtannin B-1 promoted mesenchymal stem cell migration in vivo and improved wound healing in mice. Furthermore, the results reveal that cinnamtannin B-1-induced migration of mesenchymal stem cells may be mediated by specific signaling pathways, and the flavonoid skeleton may be relevant to its effects on mesenchymal stem cell migration.     

PTEN对SMMC—7721人肝癌细胞迁移增殖及粘着斑激酶磷酸化水平的影响

抑癌基因PTEN编码产物具有双专一性磷酸酶活性 ,并与细胞骨架张力蛋白同源。PTEN可参与粘着斑的形成和解聚而影响细胞迁移。现用PTEN表达质粒转染SMMC 772 1肝癌细胞 ,研究SMMC 772 1细胞运动能力的变化及PTEN与粘着斑激酶 (FAK)酪氨酸磷酸化水平之间的关系。PTEN过表达能够显著抑制细胞在Fn基质上的活动 :细胞在Fn基质上的迁移下降了 35 % ;在 30min和 6 0min两个时间点 ,Fn基质上细胞铺展分别降低了 2 9%和 2 6 % ;而在多聚赖氨酸基质上细胞铺展并没有变化。运用免疫沉淀和Western印迹方法 ,分析FAK及其酪氨酸磷酸化水平 ,发现PTEN过表达不影响FAK表达 ,但显著降低Fn诱导的FAK酪氨酸磷酸化水平 ,两者水平呈负相关。流式细胞仪分析细胞周期结果表明 ,PTEN抑制细胞 ,S期细胞下降了 16 %。上述结果提示 ,PTEN抑制肝癌细胞迁移铺展和增殖 ;PTEN对细胞运动的影响可能通过调节FAK酪氨酸磷酸化水平而实现。     

A Human Model of Small Fiber Neuropathy to Study Wound Healing

The aim of this study was to develop a human model of acute wound healing that isolated the effects of small fiber neuropathy on the healing process. Twenty-five healthy subjects had the transient receptor vanilloid 1 agonist capsaicin and placebo creams topically applied to contralateral areas on the skin of the thigh for 48 hours. Subjects had shallow (1.2 millimeter) and deep (>3 millimeter) punch skin biopsies from each thigh on days 1 and 14. Biopsy wound healing was monitored photographically until closure. Intra-epidermal and sweat-gland nerve fiber densities were measured for each biopsy. Shallow wounds in capsaicin-treated sites healed more slowly than in placebo treated skin with biopsies taken on day 1 (P<0.001) and day 14 (P<0.001). Deep biopsies in the capsaicin and placebo areas healed at similar rates at both time points. Nerve fiber densities were reduced only in capsaicin treated regions (P<0.01). In conclusion, topical application of capsaicin causes a small fiber neuropathy and is associated with a delay in healing of shallow, but not deep wounds. This novel human model may prove valuable in the study of wound healing in patients with neuropathy.     

Down-Regulation of Gab1 Inhibits Cell Proliferation and Migration in Hilar Cholangiocarcinoma

Hilar cholangiocarcinoma is a highly aggressive malignancy originating from the hilar biliary duct epithelium. Due to few effective comprehensive treatments, the prognosis of hilar cholangiocarcinoma is poor. In this study, immunohistochemistry was first used to detect and analyze the expression of Gab1, VEGFR-2, and MMP-9 in hilar cholangiocarcinoma solid tumors and the relationships to the clinical pathological features. Furthermore, Gab1 and VEGFR-2 siRNA were used to interfere the hilar cholangiocarcinoma cell line ICBD-1 and then detect the PI3K/Akt signaling pathway, MMP-9 levels and malignant biological behaviors of tumor cells. The data showed that 1. Gab1, VEGFR-2, and MMP-9 were highly expressed and positively correlated with each other in hilar cholangiocarcinoma tissues, which were related to lymph node metastasis and differentiation. 2. After Gab1 or VEGFR-2 siRNA interference, PI3K/Akt pathway activity and MMP-9 levels were decreased in ICBD-1 cells. At the same time, cell proliferation decreased, cell cycle arrested in G1 phase, apoptosis increased and invasion decreased. These results suggest that the expression of Gab1, VEGFR-2, and MMP-9 are significantly related to the malignant biological behavior of hilar cholangiocarcinoma. Gab1 regulates growth, apoptosis and invasion through the VEGFR-2/Gab1/PI3K/Akt signaling pathway in hilar cholangiocarcinoma cells and influences the invasion of tumor cells via MMP-9.     

Phosphorylation of Phytochrome B Inhibits Light-Induced Signaling via Accelerated Dark Reversion in Arabidopsis

The photoreceptor phytochrome B (phyB) interconverts between the biologically active Pfr (λ_max = 730 nm) and inactive Pr (λ_max = 660 nm) forms in a red/far-red–dependent fashion and regulates, as molecular switch, many aspects of light-dependent development in Arabidopsis thaliana. phyB signaling is launched by the biologically active Pfr conformer and mediated by specific protein–protein interactions between phyB Pfr and its downstream regulatory partners, whereas conversion of Pfr to Pr terminates signaling. Here, we provide evidence that phyB is phosphorylated in planta at Ser-86 located in the N-terminal domain of the photoreceptor. Analysis of phyB-9 transgenic plants expressing phospho-mimic and nonphosphorylatable phyB–yellow fluorescent protein (YFP) fusions demonstrated that phosphorylation of Ser-86 negatively regulates all physiological responses tested. The Ser86Asp and Ser86Ala substitutions do not affect stability, photoconversion, and spectral properties of the photoreceptor, but light-independent relaxation of the phyB^Ser86Asp Pfr into Pr, also termed dark reversion, is strongly enhanced both in vivo and in vitro. Faster dark reversion attenuates red light–induced nuclear import and interaction of phyB^Ser86Asp-YFP Pfr with the negative regulator PHYTOCHROME INTERACTING FACTOR3 compared with phyB–green fluorescent protein. These data suggest that accelerated inactivation of the photoreceptor phyB via phosphorylation of Ser-86 represents a new paradigm for modulating phytochrome-controlled signaling.