Pacer Is a Mediator of mTORC1 and GSK3-TIP60 Signaling in Regulation of Autophagosome Maturation and Lipid Metabolism.

mTORC1 and GSK3 play critical roles in early stages of (macro)autophagy, but how they regulate late steps of autophagy remains poorly understood. Here we show that mTORC1 and GSK3-TIP60 signaling converge to modulate autophagosome maturation through Pacer, an autophagy regulator that was identified in our recent study. Hepatocyte-specific Pacer knockout in mice results in impaired autophagy flux, glycogen and lipid accumulation, and liver fibrosis. Under nutrient-rich conditions, mTORC1 phosphorylates Pacer at serine157 to disrupt the association of Pacer with Stx17 and the HOPS complex and thus abolishes Pacer-mediated autophagosome maturation. Importantly, dephosphorylation of Pacer under nutrient-deprived conditions promotes TIP60-mediated Pacer acetylation, which facilitates HOPS complex recruitment and is required for autophagosome maturation and lipid droplet clearance. This work not only identifies Pacer as a regulator in hepatic autophagy and liver homeostasis in vivo but also reveals a signal integration mechanism involved in late stages of autophagy and lipid metabolism.

GSK3 phosphorylates and regulates the Green Revolution protein Rht-B1b to reduce plant height in wheat.

The utilization of stabilized DELLA proteins Rht-B1b and Rht-D1b was crucial for increasing wheat (Triticum aestivum) productivity during the Green Revolution. However, the underlying mechanisms remain to be clarified. Here, we cloned a gain-of-function allele of the GSK3/SHAGGY-like kinase-encoding gene GSK3 by characterizing a dwarf wheat mutant. Furthermore, we determined that GSK3 interacts with and phosphorylates the Green Revolution protein Rht-B1b to promote it to reduce plant height in wheat. Specifically, phosphorylation by GSK3 may enhance the activity and stability of Rht-B1b, allowing it to inhibit the activities of its target transcription factors. Taken together, we reveal a positive regulatory mechanism for the Green Revolution protein Rht-B1b by GSK3, which might have contributed to the Green Revolution in wheat.

mTORC1 Promotes Metabolic Reprogramming by the Suppression of GSK3-Dependent Foxk1 Phosphorylation.

The mammalian Target of Rapamycin Complex 1 (mTORC1)-signaling system plays a critical role in the maintenance of cellular homeostasis by sensing and integrating multiple extracellular and intracellular cues. Therefore, uncovering the effectors of mTORC1 signaling is pivotal to understanding its pathophysiological effects. Here we report that the transcription factor forkhead/winged helix family k1 (Foxk1) is a mediator of mTORC1-regulated gene expression. Surprisingly, Foxk1 phosphorylation is increased upon mTORC1 suppression, which elicits a 14-3-3 interaction, a reduction of DNA binding, and nuclear exclusion. Mechanistically, this occurs by mTORC1-dependent suppression of nuclear signaling by the Foxk1 kinase, Gsk3. This pathway then regulates the expression of multiple genes associated with glycolysis and downstream anabolic pathways directly modulated by Foxk1 and/or by Foxk1-regulated expression of Hif-1α. Thus, Foxk1 mediates mTORC1-driven metabolic rewiring, and it is likely to be critical for metabolic diseases where improper mTORC1 signaling plays an important role.

Comparative analysis of BZR1/BES1 family transcription factors in Arabidopsis.

Brassinazole Resistant 1 (BZR1) and bri1 EMS Suppressor 1 (BES1) are key transcription factors that mediate brassinosteroid (BR)-responsive gene expression in Arabidopsis. The BZR1/BES1 family is composed of BZR1, BES1, and four BES1/BZR1 homologs (BEH1-BEH4). However, little is known about whether BEHs are regulated by BR signaling in the same way as BZR1 and BES1. We comparatively analyzed the functional characteristics of six BZR1/BES1 family members and their regulatory mechanisms in BR signaling using genetic and biochemical analyses. We also compared their subcellular localizations regulated by the phosphorylation status, interaction with GSK3-like kinases, and heterodimeric combination. We found that all BZR1/BES1 family members restored the phenotypic defects of bri1-5 by their overexpression. Unexpectedly, BEH2-overexpressing plants showed the most distinct phenotype with enhanced BR responses. RNA-Seq analysis indicated that overexpression of both BZR1 and BEH2 regulates BR-responsive gene expression, but BEH2 has a much greater proportion of BR-independent gene expression than BZR1. Unlike BZR1 and BES1, the BR-regulated subcellular translocation of the four BEHs was not tightly correlated with their phosphorylation status. Notably, BEH1 and BEH2 are predominantly localized in the nucleus, which induces the nuclear accumulation of other BZR1/BES1 family proteins through heterodimerization. Altogether, our comparative analyses suggest that BEH1 and BEH2 play an important role in the functional interaction between BZR1/BES1 family transcription factors.

Adenosine monophosphate deaminase modulates BIN2 activity through hydrogen peroxide-induced oligomerization.

The Arabidopsis thaliana GSK3-like kinase, BRASSINOSTEROID-INSENSITIVE2 (BIN2) is a key negative regulator of brassinosteroid (BR) signaling and a hub for crosstalk with other signaling pathways. However, the mechanisms controlling BIN2 activity are not well understood. Here we performed a forward genetic screen for resistance to the plant-specific GSK3 inhibitor bikinin and discovered that a mutation in the ADENOSINE MONOPHOSPHATE DEAMINASE (AMPD)/EMBRYONIC FACTOR1 (FAC1) gene reduces the sensitivity of Arabidopsis seedlings to both bikinin and BRs. Further analyses revealed that AMPD modulates BIN2 activity by regulating its oligomerization in a hydrogen peroxide (H2O2)-dependent manner. Exogenous H2O2 induced the formation of BIN2 oligomers with a decreased kinase activity and an increased sensitivity to bikinin. By contrast, AMPD activity inhibition reduced the cytosolic reactive oxygen species (ROS) levels and the amount of BIN2 oligomers, correlating with the decreased sensitivity of Arabidopsis plants to bikinin and BRs. Furthermore, we showed that BIN2 phosphorylates AMPD to possibly alter its function. Our results uncover the existence of an H2O2 homeostasis-mediated regulation loop between AMPD and BIN2 that fine-tunes the BIN2 kinase activity to control plant growth and development.

Glycogen Synthase Kinase 3 Inactivation Drives T-bet-Mediated Downregulation of Co-receptor PD-1 to Enhance CD8(+) Cytolytic T Cell Responses.

Despite the importance of the co-receptor PD-1 in T cell immunity, the upstream signaling pathway that regulates PD-1 expression has not been defined. Glycogen synthase kinase 3 (GSK-3, isoforms α and ß) is a serine-threonine kinase implicated in cellular processes. Here, we identified GSK-3 as a key upstream kinase that regulated PD-1 expression in CD8(+) T cells. GSK-3 siRNA downregulation, or inhibition by small molecules, blocked PD-1 expression, resulting in increased CD8(+) cytotoxic T lymphocyte (CTL) function. Mechanistically, GSK-3 inactivation increased Tbx21 transcription, promoting enhanced T-bet expression and subsequent suppression of Pdcd1 (encodes PD-1) transcription in CD8(+) CTLs. Injection of GSK-3 inhibitors in mice increased in vivo CD8(+) OT-I CTL function and the clearance of murine gamma-herpesvirus 68 and lymphocytic choriomeningitis clone 13 and reversed T cell exhaustion. Our findings identify GSK-3 as a regulator of PD-1 expression and demonstrate the applicability of GSK-3 inhibitors in the modulation of PD-1 in immunotherapy.

The F-box Protein KIB1 Mediates Brassinosteroid-Induced Inactivation and Degradation of GSK3-like Kinases in Arabidopsis.

The glycogen synthase kinase-3 (GSK3) family kinases are central cellular regulators highly conserved in all eukaryotes. In Arabidopsis, the GSK3-like kinase BIN2 phosphorylates a range of proteins to control broad developmental processes, and BIN2 is degraded through unknown mechanism upon receptor kinase-mediated brassinosteroid (BR) signaling. Here we identify KIB1 as an F-box E3 ubiquitin ligase that promotes the degradation of BIN2 while blocking its substrate access. Loss-of-function mutations of KIB1 and its homologs abolished BR-induced BIN2 degradation and caused severe BR-insensitive phenotypes. KIB1 directly interacted with BIN2 in a BR-dependent manner and promoted BIN2 ubiquitination in vitro. Expression of an F-box-truncated KIB1 caused BIN2 accumulation but dephosphorylation of its substrate BZR1 and activation of BR responses because KIB1 blocked BIN2 binding to BZR1. Our study demonstrates that KIB1 plays an essential role in BR signaling by inhibiting BIN2 through dual mechanisms of blocking substrate access and promoting degradation.

REDD1-dependent GSK3ß dephosphorylation promotes NF-κB activation and macrophage infiltration in the retina of diabetic mice.

Increasing evidence supports a role for inflammation in the early development and progression of retinal complications caused by diabetes. We recently demonstrated that the stress response protein regulated in development and DNA damage response 1 (REDD1) promotes diabetes-induced retinal inflammation by sustaining canonical activation of nuclear transcription factor, NF-κB. The studies here were designed to identify signaling events whereby REDD1 promotes NF-κB activation in the retina of diabetic mice. We observed increased REDD1 expression in the retina of mice after 16 weeks of streptozotocin (STZ)-induced diabetes and found that REDD1 was essential for diabetes to suppress inhibitory phosphorylation of glycogen synthase kinase 3ß (GSK3ß) at S9. In human retinal MIO-M1 Müller cell cultures, REDD1 deletion prevented dephosphorylation of GSK3ß and increased NF-κB activation in response to hyperglycemic conditions. Expression of a constitutively active GSK3ß variant restored NF-κB activation in cells deficient for REDD1. In cells exposed to hyperglycemic conditions, GSK3ß knockdown inhibited NF-κB activation and proinflammatory cytokine expression by preventing inhibitor of κB kinase complex autophosphorylation and inhibitor of κB degradation. In both the retina of STZ-diabetic mice and in Müller cells exposed to hyperglycemic conditions, GSK3 inhibition reduced NF-κB activity and prevented an increase in proinflammatory cytokine expression. In contrast with STZ-diabetic mice receiving a vehicle control, macrophage infiltration was not observed in the retina of STZ-diabetic mice treated with GSK3 inhibitor. Collectively, the findings support a model wherein diabetes enhances REDD1-dependent activation of GSK3ß to promote canonical NF-κB signaling and the development of retinal inflammation.

The novel potent GSK3 inhibitor AF3581 reverts fragile X syndrome phenotype.

Glycogen-synthase kinase 3 (GSK3) is a kinase mediating phosphorylation on serine and threonine amino acid residues of several target molecules. The enzyme is involved in the regulation of many cellular processes and aberrant activity of GSK3 has been linked to several disease conditions such as fragile X syndrome (FXS). Recent evidences demonstrating an increased activity of GSK3 in murine models of FXS, suggest that dysregulation/hyperactivation of the GSK3 path should contribute to FXS development. A likely possibility could be that in FXS there is a functional impairment of the upstream inhibitory input over GSK3 thus making overactive the kinase. Since GSK3 signaling is a central regulatory node for critical neurodevelopmental pathways, understanding the contribution of GSK3 dysregulation to FXS, may provide novel targets for therapeutic interventions for this disease. In this study we used AF3581, a potent GSK3 inhibitor that we recently discovered, in an in vivo FXS mouse model to elucidate the crucial role of GSK3 in specific behavioral patterns (locomotor activity, sensorimotor gating and social behavior) associated with this disease. All the behavioral alterations manifested by Fmr1 knockout mice were reverted after a chronic treatment with our GSK3 inhibitor, confirming the importance of this pathway as a therapeutic target.

Differential role of bovine serum albumin and HCO3- in the regulation of GSK3 alpha during mouse sperm capacitation.

To become fertile, mammalian sperm are required to undergo capacitation in the female tract or in vitro in defined media containing ions (e.g. HCO3 -, Ca2+, Na+, and Cl-), energy sources (e.g. glucose, pyruvate) and serum albumin (e.g. bovine serum albumin (BSA)). These different molecules initiate sequential and concomitant signaling pathways, leading to capacitation. Physiologically, capacitation induces changes in the sperm motility pattern (e.g. hyperactivation) and prepares sperm for the acrosomal reaction (AR), two events required for fertilization. Molecularly, HCO3 - activates the atypical adenylyl cyclase Adcy10 (aka sAC), increasing cAMP and downstream cAMP-dependent pathways. BSA, on the other hand, induces sperm cholesterol release as well as other signaling pathways. How these signaling events, occurring in different sperm compartments and with different kinetics, coordinate among themselves is not well established. Regarding the AR, recent work has proposed a role for glycogen synthase kinases (GSK3α and GSK3ß). GSK3α and GSK3ß are inactivated by phosphorylation of residues Ser21 and Ser9, respectively, in their N-terminal domain. Here, we present evidence that GSK3α (but not GSK3ß) is present in the anterior head and that it is regulated during capacitation. Interestingly, BSA and HCO3 - regulate GSK3α in opposite directions. While BSA induces a fast GSK3α Ser21 phosphorylation, HCO3 - and cAMP-dependent pathways dephosphorylate this residue. We also show that the HCO3--induced Ser21 dephosphorylation is mediated by hyperpolarization of the sperm plasma membrane potential (Em) and by intracellular pH alkalinization. Previous reports indicate that GSK3 kinases mediate the progesterone-induced AR. Here, we show that GSK3 inhibition also blocks the Ca2+ ionophore ionomycin-induced AR, suggesting a role for GSK3 kinases downstream of the increase in intracellular Ca2+ needed for this exocytotic event. Altogether, our data indicate a temporal and biphasic GSK3α regulation with opposite actions of BSA and HCO3 -. Our results also suggest that this regulation is needed to orchestrate the AR during sperm capacitation.

Positional cloning and characterization reveal the role of TaSRN-3D and TaBSR1 in the regulation of seminal root number in wheat.

Seminal roots play a critical role in water and nutrient absorption, particularly in the early developmental stages of wheat. However, the genes responsible for controlling SRN in wheat remain largely unknown. Genetic mapping and functional analyses identified a candidate gene (TraesCS3D01G137200, TaSRN-3D) encoding a Ser/Thr kinase glycogen synthase kinase 3 (STKc_GSK3) that regulated SRN in wheat. Additionally, experiments involving hormone treatment, nitrate absorption and protein interaction were conducted to explore the regulatory mechanism of TaSRN-3D. Results showed that the TaSRN-3D4332 allele inhibited seminal roots initiation and development, while loss-of-function mutants showed significantly higher seminal root number (SRN). Exogenous application of epi-brassinolide could increase the SRN in a HS2-allelic background. Furthermore, chlorate sensitivity and 15N uptake assays revealed that a higher number of seminal roots promoted nitrate accumulation. TaBSR1 (BIN2-related SRN Regulator 1, orthologous to OsGRF4/GL2 in rice) acts as an interactor of TaSRN-3D and promotes TaBSR1 degradation to reduce SRN. This study provides valuable insights into understanding the genetic basis and regulatory network of SRN in wheat, highlighting their roles as potential targets for root-based improvement in wheat breeding.

Plant-specific BLISTER interacts with kinase BIN2 and BRASSINAZOLE RESISTANT1 during skotomorphogenesis.

Brassinosteroids play an essential role in promoting skotomorphogenesis, yet the underlying mechanisms remain unknown. Here we report that a plant-specific BLISTER (BLI) protein functions as a positive regulator of both BR signaling and skotomorphogenesis in Arabidopsis (Arabidopsis thaliana). We found that the glycogen synthase kinase 3 (GSK3)-like kinase BRASSINOSTEROID INSENSITIVE2 interacts with and phosphorylates BLI at 4 phosphorylation sites (Ser70, Ser146, Thr256, and Ser267) for degradation; in turn, BR inhibits degradation of BLI. Specifically, BLI cooperates with the BRASSINAZOLE RESISTANT1 (BZR1) transcription factor to facilitate the transcriptional activation of BR-responsive genes. Genetic analyses indicated that BLI is essentially required for BZR1-mediated hypocotyl elongation in the dark. Intriguingly, we reveal that BLI and BZR1 orchestrate the transcriptional expression of gibberellin (GA) biosynthetic genes to promote the production of bioactive GAs. Our results demonstrate that BLI acts as an essential regulator of Arabidopsis skotomorphogenesis by promoting BR signaling and GA biosynthesis.

PP2A and GSK3 act as modifiers of FUS-ALS by modulating mitochondrial transport.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease which currently lacks effective treatments. Mutations in the RNA-binding protein FUS are a common cause of familial ALS, accounting for around 4% of the cases. Understanding the mechanisms by which mutant FUS becomes toxic to neurons can provide insight into the pathogenesis of both familial and sporadic ALS. We have previously observed that overexpression of wild-type or ALS-mutant FUS in Drosophila motor neurons is toxic, which allowed us to screen for novel genetic modifiers of the disease. Using a genome-wide screening approach, we identified Protein Phosphatase 2A (PP2A) and Glycogen Synthase Kinase 3 (GSK3) as novel modifiers of FUS-ALS. Loss of function or pharmacological inhibition of either protein rescued FUS-associated lethality in Drosophila. Consistent with a conserved role in disease pathogenesis, pharmacological inhibition of both proteins rescued disease-relevant phenotypes, including mitochondrial trafficking defects and neuromuscular junction failure, in patient iPSC-derived spinal motor neurons (iPSC-sMNs). In FUS-ALS flies, mice, and human iPSC-sMNs, we observed reduced GSK3 inhibitory phosphorylation, suggesting that FUS dysfunction results in GSK3 hyperactivity. Furthermore, we found that PP2A acts upstream of GSK3, affecting its inhibitory phosphorylation. GSK3 has previously been linked to kinesin-1 hyperphosphorylation. We observed this in both flies and iPSC-sMNs, and we rescued this hyperphosphorylation by inhibiting GSK3 or PP2A. Moreover, increasing the level of kinesin-1 expression in our Drosophila model strongly rescued toxicity, confirming the relevance of kinesin-1 hyperphosphorylation. Our data provide in vivo evidence that PP2A and GSK3 are disease modifiers, and reveal an unexplored mechanistic link between PP2A, GSK3, and kinesin-1, that may be central to the pathogenesis of FUS-ALS and sporadic forms of the disease.

A large-scale transgenic RNAi screen identifies transcription factors that modulate myofiber size in Drosophila.

Myofiber atrophy occurs with aging and in many diseases but the underlying mechanisms are incompletely understood. Here, we have used >1,100 muscle-targeted RNAi interventions to comprehensively assess the function of 447 transcription factors in the developmental growth of body wall skeletal muscles in Drosophila. This screen identifies new regulators of myofiber atrophy and hypertrophy, including the transcription factor Deaf1. Deaf1 RNAi increases myofiber size whereas Deaf1 overexpression induces atrophy. Consistent with its annotation as a Gsk3 phosphorylation substrate, Deaf1 and Gsk3 induce largely overlapping transcriptional changes that are opposed by Deaf1 RNAi. The top category of Deaf1-regulated genes consists of glycolytic enzymes, which are suppressed by Deaf1 and Gsk3 but are upregulated by Deaf1 RNAi. Similar to Deaf1 and Gsk3 overexpression, RNAi for glycolytic enzymes reduces myofiber growth. Altogether, this study defines the repertoire of transcription factors that regulate developmental myofiber growth and the role of Gsk3/Deaf1/glycolysis in this process.

HvGSK1.1 Controls Salt Tolerance and Yield through the Brassinosteroid Signaling Pathway in Barley.

Brassinosteroids (BRs) are a class of plant steroid hormones that are essential for plant growth and development. BRs control important agronomic traits and responses to abiotic stresses. Through the signaling pathway, BRs control the expression of thousands of genes, resulting in a variety of biological responses. The key effectors of the BR pathway are two transcription factors (TFs): BRASSINAZOLE RESISTANT 1 (BZR1) and BRI1-EMSSUPPRESSOR 1 (BES1). Both TFs are phosphorylated and inactivated by the Glycogen synthase kinase 3 BRASSINOSTEROID INSENSITIVE2 (BIN2), which acts as a negative regulator of the BR pathway. In our study, we describe the functional characteristics of HvGSK1.1, which is one of the GSK3/SHAGGY-like orthologs in barley. We generated mutant lines of HvGSK1.1 using CRISPR/Cas9 genome editing technology. Next Generation Sequencing (NGS) of the edited region of the HvGSK1.1 showed a wide variety of mutations. Most of the changes (frameshift, premature stop codon, and translation termination) resulted in the knock-out of the target gene. The molecular and phenotypic characteristics of the mutant lines showed that the knock-out mutation of HvGSK1.1 improved plant growth performance under salt stress conditions and increased the thousand kernel weight of the plants grown under normal conditions. The inactivation of HvGSK1.1 enhanced BR-dependent signaling, as indicated by the results of the leaf inclination assay in the edited lines. The plant traits under investigation are consistent with those known to be regulated by BRs. These results, together with studies of other GSK3 gene members in other plant species, suggest that targeted editing of these genes may be useful in creating plants with improved agricultural traits.

Proteomic Analysis of Prehypertensive and Hypertensive Patients: Exploring the Role of the Actin Cytoskeleton.

Hypertension is a pervasive and widespread health condition that poses a significant risk factor for cardiovascular disease, which includes conditions such as heart attack, stroke, and heart failure. Despite its widespread occurrence, the exact cause of hypertension remains unknown, and the mechanisms underlying the progression from prehypertension to hypertension require further investigation. Recent proteomic studies have shown promising results in uncovering potential biomarkers related to disease development. In this study, serum proteomic data collected from Qatar Biobank were analyzed to identify altered protein expression between individuals with normal blood pressure, prehypertension, and hypertension and to elucidate the biological pathways contributing to this disease. The results revealed a cluster of proteins, including the SRC family, CAMK2B, CAMK2D, TEC, GSK3, VAV, and RAC, which were markedly upregulated in patients with hypertension compared to those with prehypertension (fold change ≥ 1.6 or ≤-1.6, area under the curve ≥ 0.8, and q-value < 0.05). Pathway analysis showed that the majority of these proteins play a role in actin cytoskeleton remodeling. Actin cytoskeleton reorganization affects various biological processes that contribute to the maintenance of blood pressure, including vascular tone, endothelial function, cellular signaling, inflammation, fibrosis, and mechanosensing. Therefore, the findings of this study suggest a potential novel role of actin cytoskeleton-related proteins in the progression from prehypertension to hypertension. The present study sheds light on the underlying pathological mechanisms involved in hypertension and could pave the way for new diagnostic and therapeutic approaches for the treatment of this disease.

Functional inactivation of Plasmodium falciparum glycogen synthase kinase GSK3 modulates erythrocyte invasion and blocks gametocyte maturation.

Malaria is responsible for hundreds of thousands of deaths every year. The lack of an effective vaccine and the global spread of multidrug resistant parasites hampers the fight against the disease and underlines the need for new antimalarial drugs. Central to the pathogenesis of malaria is the proliferation of Plasmodium parasites within human erythrocytes. Parasites invade erythrocytes via a coordinated sequence of receptor-ligand interactions between the parasite and the host cell. Posttranslational modifications such as protein phosphorylation are known to be key regulators in this process and are mediated by protein kinases. For several parasite kinases, including the Plasmodium falciparum glycogen synthase kinase 3 (PfGSK3), inhibitors have been shown to block erythrocyte invasion. Here, we provide an assessment of PfGSK3 function by reverse genetics. Using targeted gene disruption, we show the active gene copy, PfGSK3ß, is not essential for asexual blood stage proliferation, although it modulates efficient erythrocyte invasion. We found functional inactivation leads to a 69% decreased growth rate and confirmed this growth defect by rescue experiments with wildtype and catalytically inactive mutants. Functional knockout of PfGSK3ß does not lead to transcriptional upregulation of the second copy of PfGSK3. We further analyze expression, localization, and function of PfGSK3ß during gametocytogenesis using a parasite line allowing conditional induction of sexual commitment. We demonstrate PfGSK3ß-deficient gametocytes show a strikingly malformed morphology leading to the death of parasites in later stages of gametocyte development. Taken together, these findings are important for our understanding and the development of PfGSK3 as an antimalarial target.

Placental growth factor stabilizes VEGF receptor-2 protein in retinal pigment epithelial cells by downregulating glycogen synthase kinase 3 activity.

Placental growth factor (PlGF) belongs to the vascular endothelial growth factor (VEGF) family of proteins that participate in angiogenesis and vasculogenesis. Anti-VEGF therapy has become the standard treatment for ocular angiogenic disorders in ophthalmological practice. However, there is emerging evidence that anti-VEGF treatment may increase the risk of atrophy of the retinal pigment epithelium (RPE), which is important for the homeostasis of retinal tissue. Whereas the cytoprotective role of VEGF family molecules, particularly that of VEGF A (VEGFA) through its receptor VEGF receptor-2 (VEGFR-2), has been recognized, the physiological role of PlGF in the retina is still unknown. In this study, we explored the role of PlGF in the RPE using PlGF-knockdown RPE cells generated by retrovirus-based PlGF-shRNA transduction. We show that VEGFA reduced apoptosis induced by serum starvation in RPE cells, whereas the antiapoptotic effect of VEGFA was abrogated by VEGFR-2 knockdown. Furthermore, PlGF knockdown increased serum starvation-induced cell apoptosis and unexpectedly reduced the protein level of VEGFR-2 in the RPE. The antiapoptotic effect of VEGFA was also diminished in PlGF-knockdown RPE cells. In addition, we found that glycogen synthase kinase 3 activity was involved in proteasomal degradation of VEGFR-2 in RPE cells and inactivated by PlGF via AKT phosphorylation. Overall, the present data demonstrate that PlGF is crucial for RPE cell viability and that PlGF supports VEGFA/VEGFR-2 signaling by stabilizing the VEGFR-2 protein levels through glycogen synthase kinase 3 inactivation.

Negative feedback by conserved kinases patterns the degradation of Caenorhabditiselegans Raf in vulval fate patterning.

Activation of a canonical EGFR-Ras-Raf-ERK cascade initiates patterning of multipotent vulval precursor cells (VPCs) of Caenorhabditis elegans We have previously shown that this pathway includes a negative-feedback component in which MPK-1/ERK activity targets the upstream kinase LIN-45/Raf for degradation by the SEL-10/FBXW7 E3 ubiquitin ligase. This regulation requires a Cdc4 phosphodegron (CPD) in LIN-45 that is conserved in BRAF. Here, we identify and characterize the minimal degron that encompasses the CPD and is sufficient for SEL-10-mediated, MPK-1-dependent protein degradation. A targeted screen of conserved protein kinase-encoding genes yielded gsk-3 (an ortholog of human GSK3B) and cdk-2 (a CDK2-related kinase) as required for LIN-45 degron-mediated turnover. Genetic analysis revealed that LIN-45 degradation is blocked at the second larval stage due to cell cycle quiescence, and that relief of this block during the third larval stage relies on activation of CDKs. Additionally, activation of MPK-1 provides spatial pattern to LIN-45 degradation but does not bypass the requirement for gsk-3 and cdk-2 This analysis supports a model whereby MPK-1/ERK, GSK-3/GSK3 and CDK-2/CDK2, along with SEL-10/FBXW7, constitute a regulatory network that exerts spatial and temporal control of LIN-45/Raf degradation during VPC patterning.

OsNAC016 regulates plant architecture and drought tolerance by interacting with the kinases GSK2 and SAPK8.

Ideal plant architecture and drought tolerance are important determinants of yield potential in rice (Oryza sativa). Here, we found that OsNAC016, a rice NAC (NAM, ATAF, and CUC) transcription factor, functions as a regulator in the crosslink between brassinosteroid (BR)-mediated plant architecture and abscisic acid (ABA)-regulated drought responses. The loss-of-function mutant osnac016 exhibited erect leaves and shortened internodes, but OsNAC016-overexpressing plants had opposite phenotypes. Further investigation revealed that OsNAC016 regulated the expression of the BR biosynthesis gene D2 by binding to its promoter. Moreover, OsNAC016 interacted with and was phosphorylated by GSK3/SHAGGY-LIKE KINASE2 (GSK2), a negative regulator in the BR pathway. Meanwhile, the mutant osnac016 had improved drought stress tolerance, supported by a decreased water loss rate and enhanced stomatal closure in response to exogenous ABA, but OsNAC016-overexpressing plants showed attenuated drought tolerance and reduced ABA sensitivity. Further, OSMOTIC STRESS/ABA-ACTIVATED PROTEIN KINASE8 (SAPK8) phosphorylated OsNAC016 and reduced its stability. The ubiquitin/26S proteasome system is an important degradation pathway of OsNAC016 via the interaction with PLANT U-BOX PROTEIN43 (OsPUB43) that mediates the ubiquitination of OsNAC016. Notably, RNA-sequencing analysis revealed global roles of OsNAC016 in promoting BR-mediated gene expression and repressing ABA-dependent drought-responsive gene expression, which was confirmed by chromatin immunoprecipitation quantitative PCR analysis. Our findings establish that OsNAC016 is positively involved in BR-regulated rice architecture, negatively modulates ABA-mediated drought tolerance, and is regulated by GSK2, SAPK8, and OsPUB43 through posttranslational modification. Our data provide insights into how plants balance growth and survival by coordinately regulating the growth-promoting signaling pathway and response under abiotic stresses.