PPM1A as a key target of the application of Jiawei­Maxing­Shigan decoction for the attenuation of radiation­induced epithelial­mesenchymal transition in type II alveolar epithelial cells.

Radiation­induced lung tissue injury is an important reason for the limited application of radiotherapy on thoracic malignancies. Previously, we reported that administration of Jiawei­Maxing­Shigan decoction (JMSD) attenuated the radiation­induced epithelial­mesenchymal transition (EMT) in alveolar epithelial cells (AECs) via TGF­ß/Smad signaling. The present study aimed to examine the role of protein phosphatase Mg2+/Mn2+­dependent 1A (PPM1A) in the anti­EMT activity of JMSD on AECs. The components in the aqueous extract of JMSD were identified by high­performance liquid chromatography coupled with electrospray mass spectrometry. Primary rat type II AECs were treated with radiation (60Co γ­ray at 8 Gy) and JMSD­medicated serum. PPM1A was overexpressed and knocked down in the AECs via lentivirus transduction and the effects of JMSD administration on the key proteins related to TGF­ß1/Smad signaling were measured by western blotting. It was found that radiation decreased the PPM1A expression in the AECs and JMSD­medicated serum upregulated the PPM1A expressions in the radiation­induced AECs. PPM1A overexpression increased the E­cadherin level but decreased the phosphorylated (p­)Smad2/3, vimentin and α­smooth muscle actin (α­SMA) levels in the AECs. By contrast, the PPM1A knockdown decreased the E­cadherin level and increased the p­Smad2/3, vimentin and α­SMA levels in the AECs and these effects could be blocked by SB431542 (TGF­ß1/Smad signaling inhibitor). JMSD administration increased the E­cadherin level and decreased the p­Smad2/3, vimentin and α­SMA levels in the AECs; however, these effects could be blocked by siPPM1A­2. In conclusion, PPM1A is a key target of JMSD administration for the attenuation of the radiation­induced EMT in primary type II AECs via the TGF­ß1/Smad pathway.

Genome-wide analysis of PYL-PP2C-SnRK2s family in Camellia sinensis.

Abscisic acid (ABA) signaling regulates plant growth and development and participates in response to abiotic stressors. However, details about the PYL-PP2C-SnRK2 gene family, which is the core component of ABA signaling in Camellia sinensis, are unknown. In this work, we identified 14 pyrabactin resistance-likes (PYLs), 84 type 2C protein phosphatase (PP2Cs), and 8 SNF1-related protein kinase 2s (SnRK2s) from C. sinensis. The transcriptomic analysis indicated that PYL-PP2C-SnRK2s were associated with changes of leaf color and the response of C. sinensis to drought and salt stressors. Changes of the expression of Snrk2s were not significant in the process of leaf color change or drought and salt stress response, suggesting that PYLs and PP2Cs may not interact with SnRK2s in C. sinensis during these processes. Finally, Gene Regulatory Network (GRN) construction and interaction networks analysis demonstrated that PYLs and PP2Cs were associated with multiple metabolic pathways during the changes of leaf color.

A trapped human PPM1A-phosphopeptide complex reveals structural features critical for regulation of PPM protein phosphatase activity.

Metal-dependent protein phosphatases (PPM) are evolutionarily unrelated to other serine/threonine protein phosphatases and are characterized by their requirement for supplementation with millimolar concentrations of Mg2+ or Mn2+ ions for activity in vitro The crystal structure of human PPM1A (also known as PP2Cα), the first PPM structure determined, displays two tightly bound Mn2+ ions in the active site and a small subdomain, termed the Flap, located adjacent to the active site. Some recent crystal structures of bacterial or plant PPM phosphatases have disclosed two tightly bound metal ions and an additional third metal ion in the active site. Here, the crystal structure of the catalytic domain of human PPM1A, PPM1Acat, complexed with a cyclic phosphopeptide, c(MpSIpYVA), a cyclized variant of the activation loop of p38 MAPK (a physiological substrate of PPM1A), revealed three metal ions in the active site. The PPM1Acat D146E-c(MpSIpYVA) complex confirmed the presence of the anticipated third metal ion in the active site of metazoan PPM phosphatases. Biophysical and computational methods suggested that complex formation results in a slightly more compact solution conformation through reduced conformational flexibility of the Flap subdomain. We also observed that the position of the substrate in the active site allows solvent access to the labile third metal-binding site. Enzyme kinetics of PPM1Acat toward a phosphopeptide substrate supported a random-order, bi-substrate mechanism, with substantial interaction between the bound substrate and the labile metal ion. This work illuminates the structural and thermodynamic basis of an innate mechanism regulating the activity of PPM phosphatases.

Functional analysis of a type 2C protein phosphatase gene from Ammopiptanthus mongolicus.

In Arabidopsis and certain other plant species, the type 2C protein phosphatases (PP2Cs) of the clade A class have been demonstrated to act as negative regulators in ABA-induced stress responses, such as stomatal closure. The present study reports the identification of a PP2C ortholog from the ancient desert shrub Ammopiptanthus mongolicus (Maxim.) Cheng f. (AmPP2C), which is functionally conserved over its counterparts reported from other plant species. AmPP2C was primarily expressed in leaves, with strong transcriptional accumulation being observed in the guard cells. The expression of AmPP2C was induced in response to PEG or ABA treatments, implying the potential involvement in ABA-induced stress responses. The GFP-tagging observation revealed that AmPP2C was predominantly localized to the nuclei and partly to the cytoplasm. Furthermore, BiFC assays demonstrated an interaction between AmPP2C and the typical protein kinase SnRK2.6 (AmOST1). Overexpression of AmPP2C in Arabidopsis significantly overcame the inhibition of seed germination by ABA. The transgenic Arabidopsis lines exhibited larger stomatal apertures and significantly reduced sensitivity to ABA-induced stomatal closure, which subsequently led to greater water loss and decreased biomass under PEG-simulated drought stress treatments. Under limited nitrogen or potassium supplements, plants overexpressing AmPP2C obtained a superior capability of nitrogen (N) and potassium (K) acquisition in the green parts. Therefore, the impairment of ABA-induced stomatal closure rendered by the function of PP2C helped to identify a potential survival strategy in plants suffering persistent drought stress via the maintenance of the necessary mineral nutrient acquisition driven by transpirational solute flow.

Functions and dysfunctions of Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP/PPM1F) and CaMKP-N/PPM1E.

Intracellular signal transduction is built on the basis of the subtle balance between phosphorylation and dephosphorylation. Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP/PPM1F/POPX2) and CaMKP-N (PPM1E/POPX1) are Ser/Thr phosphatases that belong to the PPM (protein phosphatase, Mg2+/Mn2+-dependent) family. The former was discovered in rat brain as a novel protein phosphatase regulating Ca2+/calmodulin-dependent protein kinases (CaMKs), whereas the latter was first identified in human cDNA databases using the rat CaMKP sequence. Subsequent studies have revealed that they are involved in various cellular functions through regulation of not only CaMKs but also other protein kinases such as AMP-activated protein kinase. Furthermore, accumulating evidence shows possible involvement of CaMKP and CaMKP-N in the pathogenesis of various diseases including cancer. Therefore, the biochemistry of CaMKP and CaMKP-N largely contributes to molecular medicine targeting these phosphatases. In this review, we summarized recent progress in the enzymology and biology of CaMKP and CaMKP-N. We also focused on etiology studies in which CaMKP and CaMKP-N are involved. Based on the emerging evidence, future perspectives of studies on these phosphatases and related issues to be elucidated are discussed.