Phosphorylated Amino Acid Research Articles

Insulin-induced de novo lipid synthesis occurs mainly via mTOR-dependent regulation of proteostasis of SREBP-1c.

Insulin stimulates de novo lipid synthesis in the liver and in cultured hepatocytes via its ability to activate sterol regulatory element-binding protein 1c (SREBP-1c). Although PI3K-AKT-mTORC1-p70S6K-signaling kinases are known to drive feed-forward expression of SREBP-1c, the identity of the phosphorylated amino acid residue(s) putatively involved in insulin-stimulated de novo lipogenesis remains elusive. We obtained in silico and mass spectrometry evidence, that was combined with siRNA strategies, to discover that insulin-induced phosphorylation of serine 418, serine 419, and serine 422 in rat SREBP-1c was most likely mediated by p70S6 kinase. Here, for the first time, we show that insulin-induced phosphorylation of these 3 serine residues mainly impinged on the mechanisms of proteostasis of both full-length and mature SREBP-1c in the McArdle-RH7777 hepatoma cells. Consistent with this conclusion, nascent SREBP-1c, substituted with phosphomimetic aspartic acid residues at these 3 sites, was resistant to proteasomal degradation. As a consequence, endoplasmic reticulum to Golgi migration and proteolytic maturation of pSREBP-1c was significantly enhanced which led to increased accumulation of mature nSREBP-1c, even in the absence of insulin. Remarkably, aspartic acid substitutions at S418, S419 and S422 also protected the nascent SREBP-1c from ubiquitin-mediated proteasome degradation thus increasing its steady-state levels and transactivation potential in the nucleus. These complementary effects of p70S6K-mediated phosphorylation on proteostasis of pSREBP-1c were necessary and sufficient to account for insulin's ability to enhance transcription of genes controlling de novo lipogenesis in hepatocytes.

Biomimetic Composite Scaffold With Phosphoserine Signaling for Bone Tissue Engineering Application.

In guided bone tissue engineering, successful ingrowth of MSCs depends primarily on the nature of the scaffold. It is well-known that only seconds after implantation, biomaterials are coated by a layer of adsorbed proteins/peptides which modulates the subsequent cell/scaffold interactions, especially at early times after implantation. In this work, nanohydroxyapatite and collagen based composite materials (Coll/nanoHA) were modified with phosphorylated amino acid (O-phospho-L-serine–OPS) to mimic bone tissue, and induce cell differentiation. The choice for this phosphorylated amino acid is due to the fact that osteopontin is a serine-rich glycol-phosphoprotein and has been associated to the early stages of bone formation, and regeneration. Several concentrations of OPS were added to the Coll/nanoHA scaffold and physico-chemical, mechanical, and in vitro cell behavior were evaluated. Afterwards, the composite scaffold with stronger mechanical and best cellular behavior was tested in vivo, with or without previous in vitro culture of human MSC's (bone tissue engineering). The OPS signaling of the biocomposite scaffolds showed similar cellular adhesion and proliferation, but higher ALP enzyme activity (HBMSC). In vivo bone ectopic formation studies allowed for a thorough evaluation of the materials for MSC's osteogenic differentiation. The OPS-scaffolds results showed that the material could modulated mesenchymal cells behavior in favor of osteogenic differentiation into late osteoblasts that gave raised to their ECM with human bone proteins (osteopontin) and calcium deposits. Finally, OPS-modified scaffolds enhanced cell survival, engraftment, migration, and spatial distribution within the 3D matrix that could be used as a cell-loaded scaffold for tissue engineering applications and accelerate bone regeneration processes.

Tocotrienol-Rich Fraction Modulates Genes Expression in Oxidative Stress-induced Caenorhabditis e legans

Objective: The study was done to determine the effect of Tocotrienol rich fraction (TRF) on the expression of RNAs in C. elegans under oxidative stress.
 Methods: The nematodes were divided into 4 groups and treated accordingly: control; TRF; hydrogen peroxide (H202); TRF treatment before and after H202-induction (TRF+H202+TRF). Expressions of RNAs were analyzed with Affymetrix Genechip C. elegans Genome Array and Genespring GX11 software where differentially expressed genes were further analyzed using gene ontology (GO). Selected genes (unc-15, cit-1.2, ftn-1, rsks-1, unc-4 and daf-12) were analyzed with RT-qPCR to validate the results.
 Results: TRF modulated the expression of 314 genes involved in determination of adult lifespan, regulation of growth and lipid modification. A total of 440 genes involved in RNA metabolic processes, transcription, growth and differentiation of muscle and nerve cells were differently expressed following H202 induction. TRF treatment before and after H202- induction resulted in 438 differentially expressed genes involved in RNA metabolic processes, transcription, response to xenobiotic stimulus and protein amino acid phosphorylation.
 Conclusion: TRF modulates the expression of genes involved in the regulation of lifespan in C. elegans.
 Bangladesh Journal of Medical Science Vol.18(4) 2019 p.711-721

Analysis of the influence of cyclo (L-phenylalanine-L-proline) on the proteome of Staphylococcus aureus using iTRAQ

Cyclo (L-phenylalanine-L-proline) (cFP) is an extracellular quorum sensing (QS) signal molecule that coordinates communication between Gram-negative bacteria. Some studies have also reported QS in Gram-positive bacteria. However, the effect of cFP on Gram-positive bacteria remains unknown. Therefore, an isobaric tags for relative and absolute quantitation (iTRAQ) proteomic experiment were designed to elucidate whether cFP influences protein expression in Staphylococcus aureus (S. aureus). The iTRAQ proteomics method was used to analyze untreated (control) and S. aureus treated with cFP for 12 h. Samples were then processed by liquid-phase tandem mass spectrometry (LC-MS/MS) and analyzed using bioinformatics tools. The results identified 1296 proteins from the S. aureus CGMCC 1.1861 proteome. Twenty-two proteins, including some two-component regulatory systems (TCRS), were associated with signal transduction. Differential expression analysis revealed that only 43 proteins were up-regulated and 41 proteins were down-regulated by cFP. The most significantly different pathways were amino acid metabolism, fatty acid degradation, and metabolism of cofactors and vitamins. Results showed that cFP down-regulated virulence factors, up-regulated lipid and amino acid metabolism, promoted acetylation and phosphorylation, and decreased alcohol dehydrogenase expression. A total of 12 significantly differentially expressed proteins (DEPs) were related to signal transduction. Among them, Rot (Q9RFJ6) and SarR (Q9F0R1), which can inhibit transcription of the Agr system, were up-regulated, whereas virulence factors such as ESAT-6 protein A (Q2G189), phenol soluble modulin (Psm, Q2FZA4), and a peptide ABC transporter permease (Q2G168) were down-regulated. AgrA (Q2FWM4) was down-regulated by cFP in S. aureus. cFP reduced AgrA and the expression of some exotoxins but increased Rot and SarR expression.

Phosphoproteomics reveals conserved exercise-stimulated signaling and AMPK regulation of store-operated calcium entry.

Exercise stimulates cellular and physiological adaptations that are associated with widespread health benefits. To uncover conserved protein phosphorylation events underlying this adaptive response, we performed mass spectrometry-based phosphoproteomic analyses of skeletal muscle from two widely used rodent models: treadmill running in mice and insitu muscle contraction in rats. We overlaid these phosphoproteomic signatures with cycling in humans to identify common cross-species phosphosite responses, as well as unique model-specific regulation. We identified >22,000 phosphosites, revealing orthologous protein phosphorylation and overlapping signaling pathways regulated by exercise. This included two conserved phosphosites on stromal interaction molecule 1 (STIM1), which we validate as AMPK substrates. Furthermore, we demonstrate that AMPK-mediated phosphorylation of STIM1 negatively regulates store-operated calcium entry, and this is beneficial for exercise in Drosophila. This integrated cross-species resource of exercise-regulated signaling in human, mouse, and rat skeletal muscle has uncovered conserved networks and unraveled crosstalk between AMPK and intracellular calcium flux.

Phosphoproteomic identification of vasopressin V2 receptor-dependent signaling in the renal collecting duct.

Vasopressin controls water balance largely through PKA-dependent effects to regulate the collecting duct water channel aquaporin-2 (AQP2). Although considerable information has accrued regarding the regulation of water and solute transport in collecting duct cells, information is sparse regarding the signaling connections between PKA and transport responses. Here, we exploited recent advancements in protein mass spectrometry to perform a comprehensive, multiple-replicate analysis of changes in the phosphoproteome of native rat inner medullary collecting duct cells in response to the vasopressin V2 receptor-selective agonist 1-desamino-8D-arginine vasopressin. Of the 10,738 phosphopeptides quantified, only 156 phosphopeptides were significantly increased in abundance, and only 63 phosphopeptides were decreased, indicative of a highly selective response to vasopressin. The list of upregulated phosphosites showed several general characteristics: 1) a preponderance of sites with basic (positively charged) amino acids arginine (R) and lysine (K) in position -2 and -3 relative to the phosphorylated amino acid, consistent with phosphorylation by PKA and/or other basophilic kinases; 2) a greater-than-random likelihood of sites previously demonstrated to be phosphorylated by PKA; 3) a preponderance of sites in membrane proteins, consistent with regulation by membrane association; and 4) a greater-than-random likelihood of sites in proteins with class I COOH-terminal PDZ ligand motifs. The list of downregulated phosphosites showed a preponderance of those with proline in position +1 relative to the phosphorylated amino acid, consistent with either downregulation of proline-directed kinases (e.g., MAPKs or cyclin-dependent kinases) or upregulation of one or more protein phosphatases that selectively dephosphorylate such sites (e.g., protein phosphatase 2A). The phosphoproteomic data were used to create a web resource for the investigation of G protein-coupled receptor signaling and regulation of AQP2-mediated water transport.

Selective Enrichment of Phosphorylated Peptides by Monolithic Polymers Surface Imprinted with bis-Imidazolium Moieties by UV-Initiated Cryopolymerization.

Reversible protein phosphorylation on serine, threonine, and tyrosine residues is essential for fast, specific, and accurate signal transduction in cells. Up to now, the identification and quantification of phosphorylated amino acids, peptides, and proteins continue to be one of the significant challenges in contemporary bioanalytical research. In this paper, a series of surface grafted monoliths in the capillary format targeting phosphorylated serine has been prepared by first synthesizing a monolithic core substrate material based on trimethylolpropane trimethacrylate, onto which a thin surface-imprinted layer was established by oriented photografting of a variety of mono- and bis-imidazolium host monomers at subzero temperature, using six different continuous or pulsed UV light sources. The imprinted monolith capillaries were evaluated in a capillary liquid chromatographic system connected to a mass spectrometer in order to test the specific retention of phosphorylated peptides. Site-specific recognition selectivity and specificity for phosphorylated serine was demonstrated when separating amino acids and peptides, proving that the optimized materials could be used as novel trapping media in affinity-based phosphoproteomic analysis.

Proteomic characterization of the hepatopancreas in the Pacific white shrimp Litopenaeus vannamei under cold stress: Revealing the organism homeostasis mechanism

To understand the homeostasis mechanism of crustacean hepatopancreas to cold stress, iTRAQ proteomics based on the genome database of Litopenaeus vannamei (L. vannamei) was applied to investigate proteins changes and variety of the hepatopancreas during cold stress stage in this study. A total of 4062 distinct proteins were identified, 137 differentially expressed proteins (DEPs) including 62 differentially up-regulated proteins (DUPs) and 75 differentially down-regulated proteins (DDPs) were identified in G1 (18 °C) compared with CK (28 °C), 359 DEPs including 131 DUPs and 228 DDPs were identified in G2 (13 °C for 24 h) compared with CK. Based on bioinformatics analysis, the cold tolerance of L. vannamei might be related to energy metabolism such as amino acid, carbohydrate, lipid, and oxidative phosphorylation. Moreover, shrimp immunity was declined during cold stress stage. However, L. vannamei could cope with cold stress by enhancing the production of ATP and UFA. Notably, arginine kinase, heat shock proteins, and histones may act as positive regulators in L. vannamei under cold stress. Ten randomly selected proteins were used for validation using qRT-PCR and the expressions on the transcription level for most of the genes were similar to the results of iTRAQ. These results indicated that L. vannamei can maintain the organism homeostasis by a series of orderly regulatory process during cold stress. Furthermore, the results can provide guidance for shrimp farming.

Integrated miRNA profiling and bioinformatics analyses reveal upregulated miRNAs in gastric cancer.

Gastric cancer is one of the most common malignancies in China and exhibits a poor prognosis. The most significant challenge for gastric cancer treatment is the absence of early diagnostic biomarkers. MicroRNAs (miRNAs) are small non-coding RNAs, which possess clinical value in a number of different types of cancer. The current study identified 13 miRNAs (hsa-miR-22, hsa-miR-545, hsa-let-7i, hsa-miR-15b, hsa-miR-221, hsa-miR-196a, hsa-miR-20a, hsa-miR-196b, hsa-miR-93, hsa-miR-19a, hsa-miR-503, hsa-miR-106b and hsa-miR-18a) that were significantly overexpressed in GC, by analyzing 1,000 GC samples included in four public datasets, including GSE23739, GSE78091, GSE30070 and The Cancer Genome Atlas. Furthermore, it was revealed that the expression levels of these 13 miRNAs were significantly higher in gastric cancer tissues of grades I, II and III compared with normal controls. Gene ontology analysis and Kyoto Encyclopedia of Genes and Genomes analysis demonstrated that the differentially expressed miRNAs were involved in regulating transcription, protein amino acid phosphorylation, signal transduction, protein binding, zinc ion binding, the mitogen-activated protein kinase signaling pathway and focal adhesion. In summary, the present study may provide potential new therapeutic and prognostic targets for gastric cancer.

Comparison of the mutation profile between pancreatic head/neck and body/tail cancers.

e15752 Background: Carcinomas in the pancreatic head/neck and body/tail represent different clinicopathological characteristics, but little is known about the underlying genetic mechanisms. The present study aims to explore the differences in mutation profile between pancreatic head and body/tail cancers. Methods: Fifty-four patients were retrospectively enrolled in the present study. DNA was purified from qualified formalin-fixed, paraffin-embedded tissue blocks of the tumor and corresponding adjacent normal tissue and subjected to target-capture next generation sequencing (TGS). Somatic mutations were identified, which further underwent gene ontology (GO) and KEGG pathway analysis. Results: The most frequently mutated genes in carcinomas of the pancreatic head were KRAS (61.3%), TP53(38.7%), and CDKN2A (16.1%), while that of the pancreatic body/tail were KRAS (95.7%), TP53(87%), CDKN2A (26.1%), and ARID1A (26.1%). The prevalence of TP53 and KRAS mutations in pancreatic head and body/tail cancers were significantly different from each other (p = 0.0006 and p = 0.0037, respectively). Pancreatic head/neck cancers also preserved a broader KRAS mutation spectrum than their counterparts of the body/tail. Pathway analyses revealed that mutations in cancer of pancreatic head/neck were enriched for genes involved in the protein amino acid phosphorylation, regulation of cell proliferation, transmembrane receptor protein tyrosine kinase signaling pathway, and phosphorylation pathways, while cancers of the pancreatic body/tail were enriched for genes involved in colorectal cancers. Conclusions: Cancer of the pancreatic head/neck and body/tail have distinct mutation profiles.

Profiling of Histidine Phosphoproteome in Danio rerio by TiO2 Enrichment.

Histidine phosphorylation is a reversible post-translational modification that is known to regulate signal transduction in prokaryotes. However, functional studies in eukaryotes have been largely neglected due to the labile nature of N-linked phosphorylated amino acids. In an effort to help elucidate the heretofore hidden vertebrate phosphoproteome, this report presents a global phosphorylation analysis of Danio rerio (zebrafish) larvae. Phosphopeptide enrichment is performed using a TiO2 affinity technique. A total of 68 unique phosphohistidine sites are detected on 63 proteins among 1076 unique phosphosites on 708 proteins. Data are available via ProteomeXchange with identifier PXD012735. This report provides the first phosphohistidine dataset obtained from zebrafish.

Determination of protein phosphorylation by polyacrylamide gel electrophoresis.

Phosphorylation is the most important modification for protein regulation; it controls many signal transduction pathways in all organisms. While several tools to detect phosphorylated proteins have been developed to study a variety of basic cellular processes involving protein phosphorylation, these methods have several limitations. Many proteins exhibit a phosphorylation-dependent electrophoretic mobility shift (PDEMS) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the molecular mechanism responsible for this phenomenon has been elucidated recently. The method for detecting phosphorylated proteins can be simplified by the application of the PDEMS. Herein, we present a novel simple method to detect protein phosphorylation, which is based on the construction of a variant protein displaying a PDEMS. The PDEMS of proteins is caused by the distribution of negatively charged amino acids around the phosphorylation site, i.e. an electrophoretic mobility shift (EMS)-related motif (ΘX1-3ΘX1-3Θ, where Θ corresponds to an acidic or phosphorylated amino acid and X represents any amino acid). The EMS-related motif can be constructed by the introduction of a negative charge by phosphorylation; it results in the decreased binding of SDS to the proteins, consequently inducing the retardation of the mobility of the protein during SDS-PAGE. Based on these molecular analyses of the PDEMS, a protein with the EMSrelated motif is designed and used to determine the in vivo phosphorylation state of the protein. This method may be used as a general strategy to easily measure the ratio of protein phosphorylation in cells.

Detection of Phytochrome Phosphorylation in Plants.

Posttranslational modification (PTM) of proteins occurs during or after translation and in most cases means covalent binding of a functional group to certain amino acid side chains. Among PTMs, phosphorylation is extensively studied for decades. During phosphorylation, a phosphate group is added to the target residue that is dominantly serine, threonine, and tyrosine in eukaryotes. The phosphate group attachment is catalyzed by kinases, whereas the removal of phosphate (dephosphorylation) is performed by phosphatases. Phosphorylation of phytochrome photoreceptors alters light signaling in multiple ways, thus the examination of this PTM is an expanding aspect of light signaling research. Although this chapter presents methods for detecting phosphorylated phytochrome B molecules, it can be applied on other phytochrome species. The first presented protocol of this chapter shows how the phosphorylation state of phytochrome photoreceptors can be monitored in a modified polyacrylamide gel electrophoresis system. The second protocol describes in detail how phosphorylated amino acids of a target molecule can be identified using mass spectrometry analysis.

Expression of authentic post-translationally modified proteins in organisms with expanded genetic codes.

Cellular signaling and regulatory cascades often rely on post-translational modification of proteins, particularly phosphorylation, to quickly and effectively relay signals from a variety of inputs. Numerous kinases, the effectors of phosphorylation, and kinase networks have been implicated in human diseases. Until recently, an inability to produce high yields of physiologically phosphorylated proteins has proven to be a substantial barrier toward our understanding of many enzymatic processes. Orthogonal translation systems provide the means to overcome many of these limitations by enabling site-specific incorporation of phosphorylated amino acids into recombinantly expressed proteins. Site-by-site, combinatorial assessment of phosphorylation site function is unique to orthogonal translation system based approaches and offers unmatched precision in the study of PTM-enzymology, extending well beyond the scope of kinase biology.

Integrated network analysis to explore the key mRNAs and lncRNAs in acute myocardial infarction.

Acute myocardial infarction (AMI) is the most severe cardiovascular event in the world. However, the molecular mechanisms underlying AMI remained largely unclear. Recently, long non-coding RNAs (lncRNAs) were reported to play important roles in human diseases. In the present work, we analyzed a public dataset GSE48060 to confirm key lncRNAs and mRNAs in AMI. We observed 4835 mRNAs and 442 lncRNAs were significantly differently expressed in AMI. Then, we for the first time constructed PPI networks and lncRNA co-expression networks in AMI. The protein-protein interaction (PPI) networks revealed several mRNAs such as RHOA, GNB1, GNG, RAC1, FBXO32, DET1, MEX3C and HECTD1 functioned as key regulators in AMI. LncRNA co-expression network analysis showed 8 lncRNAs (CA5BP1, LOC101927608, BZRAP1-AS1, EBLN3, FGD5-AS1, HNRNPU-AS1, LINC00342, and LOC101927204) played key roles in AMI. Gene ontology (GO) analysis demonstrated these differently expressed lncRNAs were associated with more signaling pathways, such as regulating transcription, protein amino acid phosphorylation, signal transduction, development. Taken together, our research unveiled a series of key lncRNAs and mRNAs in AMI. Several lncRNAs, including CA5BP1, LOC101927608, BZRAP1-AS1, EBLN3, FGD5-AS1, HNRNPU-AS1, LINC00342, and LOC101927204 were identified as key lncRNAs. PPI networks were constructed to reveal key mRNAs in AMI. These results provided useful information for exploring novel molecular target therapy for AMI.

Bioinformatics analysis of four proteins of Leishmania donovani to guide epitopes vaccine design and drug targets selection

Visceral leishmaniasis (VL) is a serious and widespread parasitic disease caused by Leishmania donovani complex. The threat of this fatal disease continues due to the lack of ideal drugs or vaccines. In this study, we selected Amastin, CaNA2, Kmp-11 and PDI proteins of Leishmania donovani for study, which are VL vaccine candidates or possible drug targets. Eleven bioinformatics tools were used to analyze different aspects of these proteins, including amino acid composition, topology, signal peptide, secondary structure, surface properties, phosphorylation sites and kinases, protein binding sites, 3D homology modeling, B cell epitopes, MHC class Ⅰ and Ⅱ epitopes and protein-protein interactions. Finally, the functionally related amino acid sites and dominant epitopes of these proteins were founded. Some possible relationships between protein structure, phosphorylation sites, protein binding sites and epitopes were also discovered. High flexibility and random coils regions of protein have a tendency to be phosphorylated, bind proteins and present epitopes. Since some phosphorylation sites and their kinases are involved in Leishmania invasion and survival in host cells, they may be potential drug targets. Bioinformatics analysis helps us better understand protein function and find dominant epitopes to guide drug design and vaccine development.

Serine arginine protein kinase 1 (SRPK1): a moonlighting protein with theranostic ability in cancer prevention.

Serine/arginine protein kinase 1 (SRPK1); a versatile functional moonlighting protein involved in varied cellular activities comprised of cell cycle progression, innate immune response, chromatin reorganization, negative and positive regulation of viral genome replication, protein amino acid phosphorylation, regulation of numerous mRNA-processing pathways, germ cell development as well as inflammation due to acquaintances with many transcription factors and signaling pathways. Several diseases including cancer have been associated with dysregulation of SRPK1. The function of SRPK1 in cancer is contradictory and inexplicable because it acts as both tumor suppressor and promoter based on the type of cell and locale. Over expression of SRPK1 including its role has been recently narrated and associated with several cancers, which includes, lung, glioma, prostate and breast via dysregulated signals from the Akt/eIF4E/HIF-1/VEGF, Erk or MAPK, PI3K/AKT/mTOR, TGF-β, and Wnt/β-catenin signaling pathways. Therefore, SRPK1 has occurred as a promising and possible curative target in cancer. In recent years, few natural and synthetic SRPK1 inhibitors have been discovered. This review emphasizes and highlights the complicated connections between SRPK1 and oncogenic signaling circuits together with the possibility of aiming SRPK1 in the treatment of cancer.

A Novel Class of Injectable Bioceramics that Glue Tissues and Biomaterials.

Calcium phosphate cements (CPCs) are clinically effective void fillers that are capable of bridging calcified tissue defects and facilitating regeneration. However, CPCs are completely synthetic/inorganic, unlike the calcium phosphate that is found in calcified tissues, and they lack an architectural organization, controlled assembly mechanisms, and have moderate biomechanical strength, which limits their clinical effectiveness. Herein, we describe a new class of bioinspired CPCs that can glue tissues together and bond tissues to metallic and polymeric biomaterials. Surprisingly, alpha tricalcium phosphate cements that are modified with simple phosphorylated amino acid monomers of phosphoserine (PM-CPCs) bond tissues up to 40-fold stronger (2.5–4 MPa) than commercial cyanoacrylates (0.1 MPa), and 100-fold stronger than surgical fibrin glue (0.04 MPa), when cured in wet-field conditions. In addition to adhesion, phosphoserine creates other novel properties in bioceramics, including a nanoscale organic/inorganic composite microstructure, and templating of nanoscale amorphous calcium phosphate nucleation. PM-CPCs are made of the biocompatible precursors calcium, phosphate, and amino acid, and these represent the first amorphous nano-ceramic composites that are stable in liquids.

Role of G-proteins and phosphorylation in the distribution of AGS3 to cell puncta

Activator of G-protein signaling 3 (AGS3, also known as GPSM1) exhibits broad functional diversity and oscillates among different subcellular compartments in a regulated manner. AGS3 consists of a tetratricopeptide repeat (TPR) domain and a G-protein regulatory (GPR) domain. Here, we tested the hypothesis that phosphorylation of the AGS3 GPR domain regulates its subcellular distribution and functionality. In contrast to the cortical and/or diffuse non-homogeneous distribution of wild-type (WT) AGS3, an AGS3 construct lacking all 24 potential phosphorylation sites in the GPR domain localized to cytosolic puncta. This change in localization was revealed to be dependent upon phosphorylation of a single threonine amino acid (T602). The punctate distribution of AGS3-T602A was rescued by co-expression of Gαi and Gαo but not Gαs or Gαq Following treatment with alkaline phosphatase, both AGS3-T602A and WT AGS3 exhibited a gel shift in SDS-PAGE as compared to untreated WT AGS3, consistent with a loss of protein phosphorylation. The punctate distribution of AGS3-T602A was lost in an AGS3-A602T conversion mutant, but was still present upon T602 mutation to glutamate or aspartate. These results implicate dynamic phosphorylation as a discrete mechanism to regulate the subcellular distribution of AGS3 and associated functionality.

Potential Root Foraging Strategy of Wheat (Triticum aestivum L.) for Potassium Heterogeneity.

Potassium (K) distribution is horizontally heterogeneous under the conservation agriculture approach of no-till with strip fertilization. The root foraging strategy of wheat for K heterogeneity is poorly understood. In this study, WinRHIZO, microarray, Non-invasive Micro-test Technology (NMT) and a split-root system were performed to investigate root morphology, gene expression profiling and fluxes of K+ and O2 under K heterogeneity and homogeneity conditions. The split-root system was performed as follows: C. LK (both compartments had low K), C. NK (both compartments had normal K), Sp. LK (one compartment had low K) and Sp. NK (the other compartment had normal K). The ratio of total root length and root tips in Sp. NK was significantly higher than that in C. NK, while no significant differences were found between Sp. LK and C. LK. Differential expression genes in C. LK vs. C. NK had opposite responses in Sp. LK vs. C. LK and similar responses in Sp. NK vs. C. NK. Low-K responsive genes, such as peroxidases, mitochondrion, transcription factor activity, calcium ion binding, glutathione transferase and cellular respiration genes were found to be up-regulated in Sp. NK. However, methyltransferase activity, protein amino acid phosphorylation, potassium ion transport, and protein kinase activity genes were found to be down-regulated in Sp. LK. The up-regulated gene with function in respiration tended to increase K+ uptake through improving O2 influx on the root surface in Sp. NK, while the down-regulated genes with functions of K+ and O2 transport tended to reduce K+ uptake on the root surface in Sp. LK. To summarize, wheat roots tended to perform active-foraging strategies in Sp. NK and dormant-foraging strategies in Sp. LK through the following patterns: (1) root development in Sp. NK but not in Sp. LK; (2) low-K responsive genes, such as peroxidases, mitochondrion, transcription factor activity, calcium ion binding and respiration, were up-regulated in Sp. NK but not in Sp. LK; and (3) root K+ and O2 influxes increased in Sp. NK but not in Sp. LK. Our findings may better explain the optimal root foraging strategy for wheat grown with heterogeneous K distribution in the root zone.