The PIN-FORMED (PIN) proteins are secondary transporters acting in the efflux of the plant signal molecule auxin from cells. They are asymmetrically localized within cells and their polarity determines the directionality of intercellular auxin flow. PIN genes are found exclusively in the genomes of multicellular plants and play an important role in regulating asymmetric auxin distribution in multiple developmental processes, including embryogenesis, organogenesis, tissue differentiation and tropic responses. All PIN proteins have a similar structure with amino- and carboxy-terminal hydrophobic, membrane-spanning domains separated by a central hydrophilic domain. The structure of the hydrophobic domains is well conserved. The hydrophilic domain is more divergent and it determines eight groups within the protein family. The activity of PIN proteins is regulated at multiple levels, including transcription, protein stability, subcellular localization and transport activity. Different endogenous and environmental signals can modulate PIN activity and thus modulate auxin-distribution-dependent development. A large group of PIN proteins, including the most ancient members known from mosses, localize to the endoplasmic reticulum and they regulate the subcellular compartmentalization of auxin and thus auxin metabolism. Further work is needed to establish the physiological importance of this unexpected mode of auxin homeostasis regulation. Furthermore, the evolution of PIN-based transport, PIN protein structure and more detailed biochemical characterization of the transport function are important topics for further studies.

• MeSH
• Arabidopsis genetics   MeSH
• Phylogeny MeSH
• Protein Conformation MeSH
• Indoleacetic Acids metabolism   MeSH
• Membrane Transport Proteins genetics   metabolism   MeSH
• Evolution, Molecular MeSH
• Models, Molecular MeSH
• Multigene Family genetics   MeSH
• Arabidopsis Proteins genetics   metabolism   MeSH
• Gene Expression Profiling MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH

Auxin is unique among plant hormones due to its directional transport that is mediated by the polarly distributed PIN auxin transporters at the plasma membrane. The canalization hypothesis proposes that the auxin feedback on its polar flow is a crucial, plant-specific mechanism mediating multiple self-organizing developmental processes. Here, we used the auxin effect on the PIN polar localization in Arabidopsis thaliana roots as a proxy for the auxin feedback on the PIN polarity during canalization. We performed microarray experiments to find regulators of this process that act downstream of auxin. We identified genes that were transcriptionally regulated by auxin in an AXR3/IAA17- and ARF7/ARF19-dependent manner. Besides the known components of the PIN polarity, such as PID and PIP5K kinases, a number of potential new regulators were detected, among which the WRKY23 transcription factor, which was characterized in more detail. Gain- and loss-of-function mutants confirmed a role for WRKY23 in mediating the auxin effect on the PIN polarity. Accordingly, processes requiring auxin-mediated PIN polarity rearrangements, such as vascular tissue development during leaf venation, showed a higher WRKY23 expression and required the WRKY23 activity. Our results provide initial insights into the auxin transcriptional network acting upstream of PIN polarization and, potentially, canalization-mediated plant development.

• MeSH
• Arabidopsis genetics   growth & development   MeSH
• Plants, Genetically Modified MeSH
• Gene Regulatory Networks * drug effects   MeSH
• Plant Roots drug effects   genetics   growth & development   metabolism   MeSH
• Indoleacetic Acids metabolism   pharmacology   MeSH
• Membrane Transport Proteins genetics   metabolism   MeSH
• Microarray Analysis MeSH
• Cell Polarity * genetics   MeSH
• Arabidopsis Proteins genetics   metabolism   physiology   MeSH
• Gene Expression Regulation, Plant drug effects   MeSH
• Gene Expression Profiling MeSH
• Transcription Factors physiology   MeSH
• Feedback, Physiological drug effects   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Directional transport of the phytohormone auxin is a versatile, plant-specific mechanism regulating many aspects of plant development. The recently identified plant hormones, strigolactones (SLs), are implicated in many plant traits; among others, they modify the phenotypic output of PIN-FORMED (PIN) auxin transporters for fine-tuning of growth and developmental responses. Here, we show in pea and Arabidopsis that SLs target processes dependent on the canalization of auxin flow, which involves auxin feedback on PIN subcellular distribution. D14 receptor- and MAX2 F-box-mediated SL signaling inhibits the formation of auxin-conducting channels after wounding or from artificial auxin sources, during vasculature de novo formation and regeneration. At the cellular level, SLs interfere with auxin effects on PIN polar targeting, constitutive PIN trafficking as well as clathrin-mediated endocytosis. Our results identify a non-transcriptional mechanism of SL action, uncoupling auxin feedback on PIN polarity and trafficking, thereby regulating vascular tissue formation and regeneration.

• MeSH
• Arabidopsis genetics   metabolism   MeSH
• Heterocyclic Compounds, 3-Ring metabolism   MeSH
• Pisum sativum genetics   metabolism   MeSH
• Indoleacetic Acids metabolism   MeSH
• Lactones metabolism   MeSH
• Arabidopsis Proteins genetics   metabolism   MeSH
• Gene Expression Regulation, Plant genetics   physiology   MeSH
• Plant Growth Regulators metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Auxin and cytokinin are key endogenous regulators of plant development. Although cytokinin-mediated modulation of auxin distribution is a developmentally crucial hormonal interaction, its molecular basis is largely unknown. Here we show a direct regulatory link between cytokinin signalling and the auxin transport machinery uncovering a mechanistic framework for cytokinin-auxin cross-talk. We show that the CYTOKININ RESPONSE FACTORS (CRFs), transcription factors downstream of cytokinin perception, transcriptionally control genes encoding PIN-FORMED (PIN) auxin transporters at a specific PIN CYTOKININ RESPONSE ELEMENT (PCRE) domain. Removal of this cis-regulatory element effectively uncouples PIN transcription from the CRF-mediated cytokinin regulation and attenuates plant cytokinin sensitivity. We propose that CRFs represent a missing cross-talk component that fine-tunes auxin transport capacity downstream of cytokinin signalling to control plant development.

• MeSH
• Arabidopsis MeSH
• Chromatin Immunoprecipitation MeSH
• Cytokinins metabolism   MeSH
• Plants, Genetically Modified MeSH
• Microscopy, Confocal MeSH
• Plant Roots metabolism   MeSH
• Real-Time Polymerase Chain Reaction MeSH
• Indoleacetic Acids metabolism   MeSH
• Membrane Transport Proteins genetics   metabolism   MeSH
• Arabidopsis Proteins genetics   metabolism   MeSH
• Gene Expression Regulation, Plant MeSH
• Response Elements MeSH
• Signal Transduction MeSH
• Transcription Factors genetics   metabolism   MeSH
• Green Fluorescent Proteins MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Shoot branching is one of the major determinants of plant architecture. Polar auxin transport in stems is necessary for the control of bud outgrowth by a dominant apex. Here, we show that following decapitation in pea (Pisum sativum L.), the axillary buds establish directional auxin export by subcellular polarization of PIN auxin transporters. Apical auxin application on the decapitated stem prevents this PIN polarization and canalization of laterally applied auxin. These results support a model in which the apical and lateral auxin sources compete for primary channels of auxin transport in the stem to control the outgrowth of axillary buds.

• MeSH
• Pisum sativum genetics   growth & development   metabolism   MeSH
• Indoleacetic Acids metabolism   MeSH
• Gene Expression Regulation, Plant MeSH
• Plant Growth Regulators metabolism   MeSH
• Plant Proteins genetics   metabolism   MeSH
• Plant Stems growth & development   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Class C G-protein coupled receptors form obligatory dimers. Metabotropic glutamate receptors (mGluRs) are found commonly as homodimers. Alternative splicing of mGluR1 gene results in vivo in the expression of a long variant mGluR1a and at least two short variants mGluR1b and d. The amino acid sequences diverge within their carboxyl-termini six amino acid residues following RRKK motif. This four basic residue sequence was shown to have pronounced impact on function and trafficking of the short variants, while for mGluR1a the long C-terminus reduces the effects caused by presence of the RRKK motif. Here we investigated consequences of interactions between long mGluR1a and short mGluR1b variants. Our results show that mGluR1a interferes with mGluR1b trafficking to the cell surface in HEK293 transfected cells. Expression of a mGlu1a mutant incapable of activating G-proteins with mGluR1b mutated in the glutamate binding site led to the formation of a functional heterodimer. Moreover, we show that swapping long mGluR1a and/or short mGluR1b C-termini with corresponding regions in chimerical GB1 and GB2 gamma-amino butyric acid b (GABAb) receptor subunits do not exclude heterodimerization. These data reveal that the C-terminal ends of mGluR1 do not control subunit association, such that mGluR1 dimers with two distinct C-termini can form and function properly.

• MeSH
• Alternative Splicing genetics   MeSH
• Gene Expression physiology   MeSH
• Phosphoric Monoester Hydrolases metabolism   MeSH
• Immunoprecipitation methods   MeSH
• Humans MeSH
• Mutagenesis physiology   MeSH
• Receptors, Metabotropic Glutamate genetics   classification   metabolism   MeSH
• Protein Structure, Tertiary physiology   MeSH
• Transfection methods   MeSH
• Cell Line, Transformed physiology   MeSH
• Protein Transport physiology   MeSH
• Calcium metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Research Support, Non-U.S. Gov't MeSH

Insect larvae develop in decaying organic matter and their defence against various microorganisms must therefore be highly efficient. In the present study, we explored the transcriptional kinetics and induction levels of eight genes in Sarcophaga bullata larvae after infection or aseptic injury. Using real-time PCR, we studied the time-dependent immune response of larvae of the fleshfly S. bullata. We compared the mRNA levels of eight selected genes in induced and non-induced larvae. The third-instar larvae of S. bullata were induced by injecting a bacterial suspension of Escherichia coli, Staphylococcus aureus or Pseudomonas aeruginosa, or by simple aseptic injury with an entomological pin. We used intact larvae as a control for basal mRNA expression. Total RNA was isolated from the whole body, fat body and haemocytes. We determined the mRNA levels of genes encoding sapecin, transferrin, prophenoloxidase 1 and 2, storage-binding protein, cathe psin L, sarcocystatin, and 26/29 kDa protease. We found that there was massive up-regulation of genes encoding the fleshfly peptide sapecin, as well as the protein transferrin. We also detected down-regulation of, or no change in, the expression of genes that encode prophenoloxidase 1 and 2, storage-binding protein, cathepsin L, sarcocystatin, and 26/29 kDa protease.

• MeSH
• Diptera genetics   immunology   microbiology   MeSH
• Escherichia coli immunology   MeSH
• Financing, Organized MeSH
• Larva genetics   immunology   microbiology   MeSH
• Reverse Transcriptase Polymerase Chain Reaction MeSH
• Pseudomonas aeruginosa immunology   MeSH
• Gene Expression Regulation MeSH
• Staphylococcus immunology   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH

The orphan G-protein-coupled receptor (GPCR) GPR158 is expressed in the brain, where it is involved in the osteocalcin effect on cognitive processes, and at the periphery, where it may contribute to glaucoma and cancers. GPR158 forms a complex with RGS7-β5, leading to the regulation of neighboring GPCR-induced Go protein activity. GPR158 also interacts with αo, although no canonical Go coupling has been reported. GPR158 displays three VCPWE motifs in its C-terminal domain that are putatively involved in G-protein regulation. Here, we addressed the scaffolding function of GPR158 and its VCPWE motifs on Go. We observed that GPR158 interacted with and stabilized the amount of RGS7-β5 through a 50-residue region downstream of its transmembrane domain and upstream of the VCPWE motifs. We show that two VCPWE motifs are involved in αo binding. Using a Gαo-βγ bioluminescence resonance energy transfer (BRET) sensor, we found that GPR158 decreases the BRET signal as observed upon G-protein activation; however, no constitutive activity of GPR158 could be detected through the measurement of various G-protein-mediated downstream responses. We propose that the effect of GPR158 on Go is unlikely due to a canonical activation of Go, but rather to the trapping of Gαo by the VCPWE motifs, possibly leading to its dissociation from βγ Such action of GPR158 is expected to prolong the βγ activity, as also observed with some activators of G-protein signaling. Taken together, our data revealed a complex functional scaffolding or signaling role for GPR158 controlling Go through an original mechanism.

• MeSH
• Amino Acid Motifs MeSH
• HEK293 Cells MeSH
• Humans MeSH
• Mutagenesis, Site-Directed MeSH
• RGS Proteins metabolism   MeSH
• GTP-Binding Protein alpha Subunits, Gi-Go metabolism   MeSH
• Receptors, G-Protein-Coupled chemistry   genetics   metabolism   MeSH
• Gene Expression Regulation MeSH
• Bioluminescence Resonance Energy Transfer Techniques MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

BACKGROUND: Frequent truncation mutations of the histone lysine N-methyltransferase KMT2C have been detected by whole exome sequencing studies in various cancers, including malignancies of the prostate. However, the biological consequences of these alterations in prostate cancer have not yet been elucidated. METHODS: To investigate the functional effects of these mutations, we deleted the C-terminal catalytic core motif of Kmt2c specifically in mouse prostate epithelium. We analysed the effect of Kmt2c SET domain deletion in a Pten-deficient PCa mouse model in vivo and of truncation mutations of KMT2C in a large number of prostate cancer patients. RESULTS: We show here for the first time that impaired KMT2C methyltransferase activity drives proliferation and PIN formation and, when combined with loss of the tumour suppressor PTEN, triggers loss of senescence, metastatic dissemination and dramatically reduces life expectancy. In Kmt2c-mutated tumours we show enrichment of proliferative MYC gene signatures and loss of expression of the cell cycle repressor p16INK4A. In addition, we observe a striking reduction in disease-free survival of patients with KMT2C-mutated prostate cancer. CONCLUSIONS: We identified truncating events of KMT2C as drivers of proliferation and PIN formation. Loss of PTEN and KMT2C in prostate cancer results in loss of senescence, metastatic dissemination and reduced life expectancy. Our data demonstrate the prognostic significance of KMT2C mutation status in prostate cancer patients. Inhibition of the MYC signalling axis may be a viable treatment option for patients with KMT2C truncations and therefore poor prognosis.

• MeSH
• DNA-Binding Proteins physiology   MeSH
• Cyclin-Dependent Kinase Inhibitor p16 genetics   metabolism   MeSH
• Humans MeSH
• Methyltransferases * genetics   MeSH
• Mutation MeSH
• Mice MeSH
• Prostatic Neoplasms * metabolism   MeSH
• Exome Sequencing MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Redirection of intercellular auxin fluxes via relocalization of the PIN-FORMED 3 (PIN3) and PIN7 auxin efflux carriers has been suggested to be necessary for the root gravitropic response. Cytokinins have also been proposed to play a role in controlling root gravitropism, but conclusive evidence is lacking. We present a detailed study of the dynamics of root bending early after gravistimulation, which revealed a delayed gravitropic response in transgenic lines with depleted endogenous cytokinins (Pro35S:AtCKX) and cytokinin signaling mutants. Pro35S:AtCKX lines, as well as a cytokinin receptor mutant ahk3, showed aberrations in the auxin response distribution in columella cells consistent with defects in the auxin transport machinery. Using in vivo real-time imaging of PIN3-GFP and PIN7-GFP in AtCKX3 overexpression and ahk3 backgrounds, we observed wild-type-like relocalization of PIN proteins in the columella early after gravistimulation, with gravity-induced relocalization of PIN7 faster than that of PIN3. Nonetheless, the cellular distribution of PIN3 and PIN7 and expression of PIN7 and the auxin influx carrier AUX1 was affected in AtCKX overexpression lines. Based on the retained cytokinin sensitivity in pin3 pin4 pin7 mutant, we propose the AUX1-mediated auxin transport rather than columella-located PIN proteins as a target of endogenous cytokinins in the control of root gravitropism.

• MeSH
• Arabidopsis drug effects   physiology   MeSH
• Models, Biological MeSH
• Biological Transport drug effects   MeSH
• Cytokinins pharmacology   MeSH
• Gravitation MeSH
• Gravitropism drug effects   MeSH
• Plant Roots drug effects   physiology   MeSH
• Indoleacetic Acids metabolism   MeSH
• Meristem drug effects   physiology   MeSH
• Arabidopsis Proteins metabolism   MeSH
• Signal Transduction drug effects   MeSH
• Green Fluorescent Proteins metabolism   MeSH
• Publication type
• Journal Article MeSH