Styxl2 regulates de novo sarcomere assembly by binding to non-muscle myosin IIs and promoting their degradation.

Styxl2, a poorly characterized pseudophosphatase, was identified as a transcriptional target of the Jak1-Stat1 pathway during myoblast differentiation in culture. Styxl2 is specifically expressed in vertebrate striated muscles. By gene knockdown in zebrafish or genetic knockout in mice, we found that Styxl2 plays an essential role in maintaining sarcomere integrity in developing muscles. To further reveal the functions of Styxl2 in adult muscles, we generated two inducible knockout mouse models: one with Styxl2 being deleted in mature myofibers to assess its role in sarcomere maintenance, and the other in adult muscle satellite cells (MuSCs) to assess its role in de novo sarcomere assembly. We find that Styxl2 is not required for sarcomere maintenance but functions in de novo sarcomere assembly during injury-induced muscle regeneration. Mechanistically, Styxl2 interacts with non-muscle myosin IIs, enhances their ubiquitination, and targets them for autophagy-dependent degradation. Without Styxl2, the degradation of non-muscle myosin IIs is delayed, which leads to defective sarcomere assembly and force generation. Thus, Styxl2 promotes de novo sarcomere assembly by interacting with non-muscle myosin IIs and facilitating their autophagic degradation.

PJA1-mediated suppression of pyroptosis as a driver of docetaxel resistance in nasopharyngeal carcinoma.

Chemoresistance is a main reason for treatment failure in patients with nasopharyngeal carcinoma, but the exact regulatory mechanism underlying chemoresistance in nasopharyngeal carcinoma remains to be elucidated. Here, we identify PJA1 as a key E3 ubiquitin ligase involved in nasopharyngeal carcinoma chemoresistance that is highly expressed in nasopharyngeal carcinoma patients with nonresponse to docetaxel-cisplatin-5-fluorouracil induction chemotherapy. We find that PJA1 facilitates docetaxel resistance by inhibiting GSDME-mediated pyroptosis in nasopharyngeal carcinoma cells. Mechanistically, PJA1 promotes the degradation of the mitochondrial protein PGAM5 by increasing its K48-linked ubiquitination at K88, which further facilitates DRP1 phosphorylation at S637 and reduced mitochondrial reactive oxygen species production, resulting in suppression of GSDME-mediated pyroptosis and the antitumour immune response. PGAM5 knockdown fully restores the docetaxel sensitization effect of PJA1 knockdown. Moreover, pharmacological targeting of PJA1 with the small molecule inhibitor RTA402 enhances the docetaxel sensitivity of nasopharyngeal carcinoma in vitro and in vivo. Clinically, high PJA1 expression indicates inferior survival and poor clinical efficacy of TPF IC in nasopharyngeal carcinoma patients. Our study emphasizes the essential role of E3 ligases in regulating chemoresistance and provides therapeutic strategies for nasopharyngeal carcinoma based on targeting the ubiquitin-proteasome system.

A CPF-like phosphatase module links transcription termination to chromatin silencing.

The interconnections between co-transcriptional regulation, chromatin environment, and transcriptional output remain poorly understood. Here, we investigate the mechanism underlying RNA 3' processing-mediated Polycomb silencing of Arabidopsis FLOWERING LOCUS C (FLC). We show a requirement for ANTHESIS PROMOTING FACTOR 1 (APRF1), a homolog of yeast Swd2 and human WDR82, known to regulate RNA polymerase II (RNA Pol II) during transcription termination. APRF1 interacts with TYPE ONE SERINE/THREONINE PROTEIN PHOSPHATASE 4 (TOPP4) (yeast Glc7/human PP1) and LUMINIDEPENDENS (LD), the latter showing structural features found in Ref2/PNUTS, all components of the yeast and human phosphatase module of the CPF 3' end-processing machinery. LD has been shown to co-associate in vivo with the histone H3 K4 demethylase FLOWERING LOCUS D (FLD). This work shows how the APRF1/LD-mediated polyadenylation/termination process influences subsequent rounds of transcription by changing the local chromatin environment at FLC.

Rapeseed PP2C37 Interacts with PYR/PYL Abscisic Acid Receptors and Negatively Regulates Drought Tolerance.

Global water deficit is a severe abiotic stress threatening the yielding and quality of crops. Abscisic acid (ABA) is a phytohormone that mediates drought tolerance. Protein kinases and phosphatases function as molecular switches in eukaryotes. Protein phosphatases type 2C (PP2Cs) are a major family that play essential roles in ABA signaling and stress responses. However, the role and underlying mechanism of PP2C in rapeseed (Brassica napus L.) mediating drought response has not been reported yet. Here, we characterized a PP2C family member, BnaPP2C37, and its expression level was highly induced by ABA and dehydration treatments. It negatively regulates drought tolerance in rapeseed. We further identified that BnaPP2C37 interacted with multiple PYR/PYL receptors and a drought regulator BnaCPK5 (calcium-dependent protein kinase 5) through yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays. Specifically, BnaPYL1 and BnaPYL9 repress BnaPP2C37 phosphatase activity. Moreover, the pull-down assay and phosphatase assays show BnaPP2C37 interacts with BnaCPK5 to dephosphorylate BnaCPK5 and its downstream BnaABF3. Furthermore, a dual-luciferase assay revealed BnaPP2C37 transcript level was enhanced by BnaABF3 and BnaABF4, forming a negative feedback regulation to ABA response. In summary, we identified that BnaPP2C37 functions negatively in drought tolerance of rapeseed, and its phosphatase activity is repressed by BnaPYL1/9 whereas its transcriptional level is upregulated by BnaABF3/4.

Structure and mechanism of the human CTDNEP1-NEP1R1 membrane protein phosphatase complex necessary to maintain ER membrane morphology.

C-terminal Domain Nuclear Envelope Phosphatase 1 (CTDNEP1) is a noncanonical protein serine/threonine phosphatase that has a conserved role in regulating ER membrane biogenesis. Inactivating mutations in CTDNEP1 correlate with the development of medulloblastoma, an aggressive childhood cancer. The transmembrane protein Nuclear Envelope Phosphatase 1 Regulatory Subunit 1 (NEP1R1) binds CTDNEP1, but the molecular details by which NEP1R1 regulates CTDNEP1 function are unclear. Here, we find that knockdown of NEP1R1 generates identical phenotypes to reported loss of CTDNEP1 in mammalian cells, establishing CTDNEP1-NEP1R1 as an evolutionarily conserved membrane protein phosphatase complex that restricts ER expansion. Mechanistically, NEP1R1 acts as an activating regulatory subunit that directly binds and increases the phosphatase activity of CTDNEP1. By defining a minimal NEP1R1 domain sufficient to activate CTDNEP1, we determine high-resolution crystal structures of the CTDNEP1-NEP1R1 complex bound to a peptide sequence acting as a pseudosubstrate. Structurally, NEP1R1 engages CTDNEP1 at a site distant from the active site to stabilize and allosterically activate CTDNEP1. Substrate recognition is facilitated by a conserved Arg residue in CTDNEP1 that binds and orients the substrate peptide in the active site. Together, this reveals mechanisms for how NEP1R1 regulates CTDNEP1 and explains how cancer-associated mutations inactivate CTDNEP1.

Mechanisms involved in the regulation of mitochondrial quality control by PGAM5 in heart failure.

Heart failure (HF) refers to a group of clinical syndromes in which various heart diseases lead to the inability of cardiac output to meet the metabolic needs of the body's tissues. Cardiac metabolism requires enormous amounts of energy; thus, impaired myocardial energy metabolism is considered a key factor in the occurrence and development of HF. Mitochondria serve as the primary energy source for cardiomyocytes, and their regular functionality underpins healthy cardiac function. The mitochondrial quality control system is a crucial mechanism for regulating the functionality of cardiomyocytes, and any abnormality in this system can potentially impact the morphology and structure of mitochondria, as well as the energy metabolism of cardiomyocytes. Phosphoglycerate mutase 5 (PGAM5), a multifunctional protein, plays a key role in the regulation of mitochondrial quality control through multiple pathways. Therefore, abnormal PGAM5 function is closely related to mitochondrial damage. This article reviews the mechanism of PGAM5's involvement in the regulation of the mitochondrial quality control system in the occurrence and development of HF, thereby providing a theoretical basis for future in-depth research.

Ctdnep1 phosphatase is required for negative regulation of RANKL-induced osteoclast differentiation in RAW264.7 cells.

Osteoclasts are multinucleated cells with bone resorption activity. Excessive osteoclast activity has been implicated in osteoporosis, rheumatoid arthritis, and bone destruction due to bone metastases from cancer, making osteoclasts essential target cells in bone and joint diseases. C-terminal domain nuclear envelope phosphatase 1 (Ctdnep1, formerly Dullard) is a negative regulator of transforming growth factor (TGF)-ß superfamily signaling and regulates endochondral ossification in mesenchymal cells during skeletal development. In this study, we investigated the role of Ctdnep1 in the Receptor activator of nuclear factor-kappa B ligand (RANKL)-induced RAW264.7 osteoclast differentiation. Expression of Ctdnep1 did not change during osteoclast differentiation; Ctdnep1 protein localized to the cytoplasm before and after osteoclast differentiation. Small interfering RNA-mediated knockdown of Ctdnep1 increased tartrate-resistant acid phosphatase-positive multinucleated osteoclasts and the expression of osteoclast marker genes, including Acp5, Ctsk, and Nfatc1. Interestingly, the knockdown of Ctdnep1 increased the protein level of Nfatc1 in cells unstimulated with RANKL. Knockdown of Ctdnep1 also enhanced calcium-resorbing activity. Mechanistically, the knockdown of Ctdnep1 increased the phosphorylation of RANKL signaling components. These results suggest that Ctdnep1 negatively regulates osteoclast differentiation by suppressing the RANKL signaling pathway.

Pgam5 aggravates hyperglycemia-induced myocardial dysfunction through disrupting Phb2-dependent mitochondrial dynamics.

This study aims to elucidate the roles of Phosphoglycerate Mutase Family Member 5 (Pgam5) and Prohibitin 2 (Phb2) in the context of hyperglycemia-induced myocardial dysfunction, a critical aspect of diabetic cardiomyopathy. The research employed primary cardiomyocytes, which were then subjected to hyperglycemia treatment to mimic diabetic conditions. We used siRNA transfection to knock down Pgam5 and overexpressed Phb2 using adenovirus transfection to assess their individual and combined effects on cardiomyocyte health. Mitochondrial function was evaluated through measurements of mitochondrial membrane potential using the JC-1 probe, and levels of mitochondrial reactive oxygen species (ROS) were assessed. Additionally, the study involved qPCR analysis to quantify the transcriptional changes in genes related to mitochondrial fission and mitophagy. Our findings indicate that hyperglycemia significantly reduces cardiomyocyte viability and impairs mitochondrial function, as evidenced by decreased mitochondrial membrane potential and increased ROS levels. Pgam5 knockdown was observed to mitigate these adverse effects, preserving mitochondrial function and cardiomyocyte viability. On the molecular level, Pgam5 was found to regulate genes associated with mitochondrial fission (such as Drp1, Mff, and Fis1) and mitophagy (including Parkin, Bnip3, and Fundc1). Furthermore, overexpression of Phb2 countered the hyperglycemia-induced mitochondrial dysfunction and normalized the levels of key mitochondrial antioxidant enzymes. The combined data suggest a protective role for both Pgam5 knockdown and Phb2 overexpression against hyperglycemia-induced cellular and mitochondrial damage. The study elucidates the critical roles of Pgam5 and Phb2 in regulating mitochondrial dynamics in the setting of hyperglycemia-induced myocardial dysfunction. By modulating mitochondrial fission and mitophagy, Pgam5 and Phb2 emerge as key players in preserving mitochondrial integrity and cardiomyocyte health under diabetic conditions. These findings contribute significantly to our understanding of the molecular mechanisms underlying diabetic cardiomyopathy and suggest potential therapeutic targets for mitigating myocardial dysfunction in diabetes.

The protein phosphatase PP6 promotes RIPK1-dependent PANoptosis.

BACKGROUND: The innate immune system serves as the first line of host defense. Transforming growth factor-ß-activated kinase 1 (TAK1) is a key regulator of innate immunity, cell survival, and cellular homeostasis. Because of its importance in immunity, several pathogens have evolved to carry TAK1 inhibitors. In response, hosts have evolved to sense TAK1 inhibition and induce robust lytic cell death, PANoptosis, mediated by the RIPK1-PANoptosome. PANoptosis is a unique innate immune inflammatory lytic cell death pathway initiated by an innate immune sensor and driven by caspases and RIPKs. While PANoptosis can be beneficial to clear pathogens, excess activation is linked to pathology. Therefore, understanding the molecular mechanisms regulating TAK1 inhibitor (TAK1i)-induced PANoptosis is central to our understanding of RIPK1 in health and disease. RESULTS: In this study, by analyzing results from a cell death-based CRISPR screen, we identified protein phosphatase 6 (PP6) holoenzyme components as regulators of TAK1i-induced PANoptosis. Loss of the PP6 enzymatic component, PPP6C, significantly reduced TAK1i-induced PANoptosis. Additionally, the PP6 regulatory subunits PPP6R1, PPP6R2, and PPP6R3 had redundant roles in regulating TAK1i-induced PANoptosis, and their combined depletion was required to block TAK1i-induced cell death. Mechanistically, PPP6C and its regulatory subunits promoted the pro-death S166 auto-phosphorylation of RIPK1 and led to a reduction in the pro-survival S321 phosphorylation. CONCLUSIONS: Overall, our findings demonstrate a key requirement for the phosphatase PP6 complex in the activation of TAK1i-induced, RIPK1-dependent PANoptosis, suggesting this complex could be therapeutically targeted in inflammatory conditions.

Protein phosphatases regulate the formation of Müller glia-derived progenitor cells in the chick retina.

Different kinase-dependent cell signaling pathways are known to play important roles in glia-mediated neuroprotection and reprogramming of Müller glia (MG) into Müller glia-derived progenitor cells (MGPCs) in the retina. However, very little is known about the phosphatases that regulate kinase-dependent signaling in MG. Using single-cell RNA-sequencing (scRNA-seq) databases, we investigated patterns of expression of Dual Specificity Phosphatases (DUSP1/6) and other protein phosphatases in normal and damaged chick retinas. We found that DUSP1, DUSP6, PPP3CB, PPP3R1 and PPPM1A/B/D/E/G are widely expressed by many types of retinal neurons and are dynamically expressed by MG and MGPCs in retinas during the process of reprogramming. We find that inhibition of DUSP1/6 and PP2C phosphatases enhances the formation of proliferating MGPCs in damaged retinas and in retinas treated with insulin and FGF2 in the absence of damage. By contrast, inhibition of PP2B phosphatases suppressed the formation of proliferating MGPCs, but increased numbers of proliferating MGPCs in undamaged retinas treated with insulin and FGF2. In damaged retinas, inhibition of DUSP1/6 increased levels of pERK1/2 and cFos in MG whereas inhibition of PP2B's decreased levels of pStat3 and pS6 in MG. Analyses of scRNA-seq libraries identified numerous differentially activated gene modules in MG in damaged retinas versus MG in retinas treated with insulin+FGF2 suggesting significant differences in kinase-dependent signaling pathways that converge on the formation of MGPCs. Inhibition of phosphatases had no significant effects upon numbers of dying cells in damaged retinas. We conclude that the activity of different protein phosphatases acting through retinal neurons and MG "fine-tune" the cell signaling responses of MG in damaged retinas and during the reprogramming of MG into MGPCs.

Tracking Inhibition of Human Small C-Terminal Domain Phosphatase 1 Using 193 nm Ultraviolet Photodissociation Mass Spectrometry.

Working in tandem with kinases via a dynamic interplay of phosphorylation and dephosphorylation of proteins, phosphatases regulate many cellular processes and thus represent compelling therapeutic targets. Here we leverage ultraviolet photodissociation to shed light on the binding characteristics of two covalent phosphatase inhibitors, T65 and rabeprazole, and their respective interactions with the human small C-terminal domain phosphatase 1 (SCP1) and its single-point mutant C181A, in which a nonreactive alanine replaces one key reactive cysteine. Top-down MS/MS analysis is used to localize the binding of T65 and rabeprazole on the two proteins and estimate the relative reactivities of each cysteine residue.

High PPP4C expression predicts poor prognosis in diffuse large B-cell lymphoma.

The significance of Protein phosphatase 4 catalytic subunit (PPP4C) in diffuse large B-cell lymphoma (DLBCL) prognosis is not well understood. This work aimed to investigate the expression of PPP4C in DLBCL, investigate the correlation between PPP4C expression and clinicopathological parameters, and assess the prognostic significance of PPP4C. The mRNA expression of PPP4C was investigated using data from TCGA and GEO. To further analyze PPP4C expression, immunohistochemistry was performed on tissue microarray samples. Correlation analysis between clinicopathological parameters and PPP4C expression was conducted using Pearson's chi-square test or Fisher's exact test. Univariate and multivariate Cox hazard models were utilized to determine the prognostic significance of clinicopathological features and PPP4C expression. Additionally, survival analysis was performed using Kaplan-Meier survival curves. In both TCGA and GEO datasets, we identified higher mRNA levels of PPP4C in tumor tissues compared to normal tissues. Upon analysis of various clinicopathological features of DLBCL, we observed a correlation between high PPP4C expression and ECOG score (P = 0.003). Furthermore, according to a Kaplan-Meier survival analysis, patients with DLBCL who exhibit high levels of PPP4C had worse overall survival (P = 0.001) and progression-free survival (P = 0.002). PPP4C was shown to be an independent predictive factor for OS and PFS in DLBCL by univariate and multivariate analysis (P = 0.011 and P = 0.040). This study's findings indicate that high expression of PPP4C is linked to a poor prognosis for DLBCL and may function as an independent prognostic factors.

C-TERMINAL DOMAIN PHOSPHATASE-LIKE 3 contributes to GA-mediated growth and flowering by interaction with DELLA proteins.

Gibberellic acid (GA) plays a central role in many plant developmental processes and is crucial for crop improvement. DELLA proteins, the core suppressors in the GA signaling pathway, are degraded by GA via the 26S proteasomal pathway to release the GA response. However, little is known about the phosphorylation-mediated regulation of DELLA proteins. In this study, we combined GA response assays with protein-protein interaction analysis to infer the connection between Arabidopsis thaliana DELLAs and the C-TERMINAL DOMAIN PHOSPHATASE-LIKE 3 (CPL3), a phosphatase involved in the dephosphorylation of RNA polymerase II. We show that CPL3 directly interacts with DELLA proteins and promotes DELLA protein stability by inhibiting its degradation by the 26S proteasome. Consequently, CPL3 negatively modulates multiple GA-mediated processes of plant development, including hypocotyl elongation, flowering time, and anthocyanin accumulation. Taken together, our findings demonstrate that CPL3 serves as a novel regulator that could improve DELLA stability and thereby participate in GA signaling transduction.

The CaMK Family Differentially Promotes Necroptosis and Mouse Cardiac Graft Injury and Rejection.

Organ transplantation is associated with various forms of programmed cell death which can accelerate transplant injury and rejection. Targeting cell death in donor organs may represent a novel strategy for preventing allograft injury. We have previously demonstrated that necroptosis plays a key role in promoting transplant injury. Recently, we have found that mitochondria function is linked to necroptosis. However, it remains unknown how necroptosis signaling pathways regulate mitochondrial function during necroptosis. In this study, we investigated the receptor-interacting protein kinase 3 (RIPK3) mediated mitochondrial dysfunction and necroptosis. We demonstrate that the calmodulin-dependent protein kinase (CaMK) family members CaMK1, 2, and 4 form a complex with RIPK3 in mouse cardiac endothelial cells, to promote trans-phosphorylation during necroptosis. CaMK1 and 4 directly activated the dynamin-related protein-1 (Drp1), while CaMK2 indirectly activated Drp1 via the phosphoglycerate mutase 5 (PGAM5). The inhibition of CaMKs restored mitochondrial function and effectively prevented endothelial cell death. CaMKs inhibition inhibited activation of CaMKs and Drp1, and cell death and heart tissue injury (n = 6/group, p < 0.01) in a murine model of cardiac transplantation. Importantly, the inhibition of CaMKs greatly prolonged heart graft survival (n = 8/group, p < 0.01). In conclusion, CaMK family members orchestrate cell death in two different pathways and may be potential therapeutic targets in preventing cell death and transplant injury.

SMEK1 ablation promotes glucose uptake and improves obesity-related metabolic dysfunction via AMPK signaling pathway.

Obesity has become a major risk of global public health. SMEK1 is also known as a regulatory subunit of protein phosphatase 4 (PP4). Both PP4 and SMEK1 have been clarified in many metabolic functions, including the regulation of hepatic gluconeogenesis and glucose transporter gene expression in yeast. Whether SMEK1 participates in obesity and the broader metabolic role in mammals is unknown. Thus, we investigated the function of SMEK1 in white adipose tissue and glucose uptake. GWAS/GEPIA/GEO database was used to analyze the correlation between SMEK1 and metabolic phenotypes/lipid metabolism-related genes/obesity. Smek1 KO mice were generated to identify the role of SMEK1 in obesity and glucose homeostasis. Cell culture and differentiation of stromal-vascular fractions (SVFs) and 3T3-L1 were used to determine the mechanism. 2-NBDG was used to measure the glucose uptake. Compound C was used to confirm the role of AMPK. We elucidated that SMEK1 was correlated with obesity and adipogenesis. Smek1 deletion enhanced adipogenesis in both SVFs and 3T3-L1. Smek1 KO protected mice from obesity and had protective effects on metabolic disorders, including insulin resistance and inflammation. Smek1 KO mice had lower levels of fasting serum glucose. We found that SMEK1 ablation promoted glucose uptake by increasing p-AMPKα(T172) and the transcription of Glut4 when the effect on AMPK-regulated glucose uptake was due to the PP4 catalytic subunits (PPP4C). Our findings reveal a novel role of SMEK1 in obesity and glucose homeostasis, providing a potential new therapeutic target for obesity and metabolic dysfunction.NEW & NOTEWORTHY Our study clarified the relationship between SMEK1 and obesity for the first time and validated the conclusion in multiple ways by combining available data from public databases, human samples, and animal models. In addition, we clarified the role of SMEK1 in glucose uptake, providing an in-depth interpretation for the study of its function in glucose metabolism.

Pleckstrin Homology Domain Leucine-rich Repeat Protein Phosphatase Acts as a Tumor Suppressor in Oral Squamous Cell Carcinoma.

The pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP) family has been found to have both tumor-suppressor and oncogenic properties across various types and locations of cancer. Given that PHLPP has not been previously studied in oral squamous cell carcinoma (SCC), we conducted an assessment of the expression of both its isoforms in oral SCC tissues and cell lines and compared these findings to their corresponding normal counterparts. In addition, we assessed the relationship between PHLPP and clinicopathological factors and patient survival. Quantitative real-time polymerase chain reaction was used to detect the mRNA levels of PHLPP1 and PHLPP2 in cancerous and normal cell lines in addition to 124 oral SCC and noncancerous adjacent epithelia (N = 62, each). Correlations between their expression rate and clinicopathological parameters were further evaluated in 57 patients. Data were statistically analyzed with t test and paired t test, analysis of variance, Mann-Whitney U , and Cox Regression tests ( P < 0.05). We found significantly lower levels of both PHLPP isoforms in oral SCC tissues compared with noncancerous epithelia ( P < 0.001, for both). However, in the cell lines, this difference was significant only for PHLPP1 ( P = 0.027). The correlation between the two isoforms was significant only in cancerous tissues ( P < 0.001). None of the clinicopathologic factors showed significant associations with either of the isoforms and there was no correlation with survival. We showed for the first time that PHLPP1 and PHLPP2 act as tumor suppressors in oral SCC at the mRNA level. The regulation of their mRNA appears to be different between normal and cancerous tissues.

Identification of Inhibitors of the Disease-Associated Protein Phosphatase Scp1 Using Antibody Mimetic Molecules.

Protein phosphorylation is a prevalent translational modification, and its dysregulation has been implicated in various diseases, including cancer. Despite its significance, there is a lack of specific inhibitors of the FCP/SCP-type Ser/Thr protein phosphatase Scp1, characterized by high specificity and affinity. In this study, we focused on adnectin, an antibody-mimetic protein, aiming to identify Scp1-specific binding molecules with a broad binding surface that target the substrate-recognition site of Scp1. Biopanning of Scp1 was performed using an adnectin-presenting phage library with a randomized FG loop. We succeeded in identifying FG-1Adn, which showed high affinity and specificity for Scp1. Ala scanning analysis of the Scp1-binding sequence in relation to the FG-1 peptide revealed that hydrophobic residues, including aromatic amino acids, play important roles in Scp1 recognition. Furthermore, FG-1Adn was found to co-localize with Scp1 in cells, especially on the plasma membrane. In addition, Western blotting analysis showed that FG-1Adn increased the phosphorylation level of the target protein of Scp1 in cells, indicating that FG-1Adn can inhibit the function of Scp1. These results suggest that FG-1Adn can be used as a specific inhibitor of Scp1.

Microcystin-LR, a cyanotoxin, modulates division of higher plant chloroplasts through protein phosphatase inhibition and affects cyanobacterial division.

Microcystin-LR (MC-LR) is a harmful cyanotoxin that inhibits 1 and 2A serine-threonine protein phosphatases. This study examines the influence of MC-LR on chloroplast division and the underlying mechanisms and consequences in Arabidopsis. MC-LR increased the frequency of dividing chloroplasts in hypocotyls in a time range of 1-96 h. At short-term exposures to MC-LR, small-sized chloroplasts (longitudinal diameters ≤6 µm) were more sensitive to these stimulatory effects, while both small and large chloroplasts showed stimulations at long-term exposure. After 48 h, the cyanotoxin increased the frequency of small-sized chloroplasts, indicating the stimulation of division. MC-LR inhibited protein phosphatases in whole hypocotyls and isolated chloroplasts, while it did not induce oxidative stress. We show for the first time that total cellular phosphatases play important roles in chloroplast division and that particular chloroplast phosphatases may be involved in these processes. Interestingly, MC-LR has a protective effect on cyanobacterial division during methyl-viologen (MV) treatments in Synechococcus PCC6301. MC-LR production has harmful effects on ecosystems and it may have an ancient cell division regulatory role in stressed cyanobacterial cells, the evolutionary ancestors of chloroplasts. We propose that cytoplasmic (eukaryotic) factors also contribute to the relevant effects of MC-LR in plants.

Functional screening of the Arabidopsis 2C protein phosphatases family identifies PP2C15 as a negative regulator of plant immunity by targeting BRI1-associated receptor kinase 1.

Genetic engineering using negative regulators of plant immunity has the potential to provide a huge impetus in agricultural biotechnology to achieve a higher degree of disease resistance without reducing yield. Type 2C protein phosphatases (PP2Cs) represent the largest group of protein phosphatases in plants, with a high potential for negative regulatory functions by blocking the transmission of defence signals through dephosphorylation. Here, we established a PP2C functional protoplast screen using pFRK1::luciferase as a reporter and found that 14 of 56 PP2Cs significantly inhibited the immune response induced by flg22. To verify the reliability of the system, a previously reported MAPK3/4/6-interacting protein phosphatase, PP2C5, was used; it was confirmed to be a negative regulator of PAMP-triggered immunity (PTI). We further identified PP2C15 as an interacting partner of BRI1-associated receptor kinase 1 (BAK1), which is the most well-known co-receptor of plasma membrane-localized pattern recognition receptors (PRRs), and a central component of PTI. PP2C15 dephosphorylates BAK1 and negatively regulates BAK1-mediated PTI responses such as MAPK3/4/6 activation, defence gene expression, reactive oxygen species bursts, stomatal immunity, callose deposition, and pathogen resistance. Although plant growth and 1000-seed weight of pp2c15 mutants were reduced compared to those of wild-type plants, pp2c5 mutants did not show any adverse effects. Thus, our findings strengthen the understanding of the mechanism by which PP2C family members negatively regulate plant immunity at multiple levels and indicate a possible approach to enhance plant resistance by eliminating specific PP2Cs without affecting plant growth and yield.