Studies of cytokinin receptor–phosphotransmitter interaction provide evidences for the initiation of cytokinin signalling in the endoplasmic reticulum

2018 ◽  
Vol 45 (2) ◽  
pp. 192 ◽  
Author(s):  
Sergey N. Lomin ◽  
Yulia A. Myakushina ◽  
Dmitry V. Arkhipov ◽  
Olga G. Leonova ◽  
Vladimir I. Popenko ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Endoplasmic Reticulum ◽  
Active Form ◽  
Kinase Assay ◽  
Phosphoryl Group ◽  
Subcellular Localisation ◽  
In Planta ◽  
Cytokinin Receptors ◽  
Receptor Dimers

Cytokinin receptors were shown recently to be localised mainly to the endoplasmic reticulum (ER); however, the activity of ER-located receptors was not proven. We have therefore tested the functionality of ER-located Arabidopsis receptors. The first step of cytokinin signal transduction is the transfer of a phosphoryl group from the activated receptor to a phosphotransfer protein. To determine the subcellular localisation of receptor–phosphotransmitter interaction in planta, BiFC experiments were performed. Receptors ARABIDOPSIS HISTIDINE KINASE 2 (AHK2), AHK3 and AHK4 (CRE1) and phosphotransmitters ARABIDOPSIS HISTIDINE-CONTAINING PHOSPHOTRANSMITTER 1 (AHP1), AHP2 and AHP3 fused to split-eYFP were transiently expressed in Nicotiana benthamiana leaves. Receptor–phosphotransmitter pairs were shown to interact in every possible combination in a pattern reflecting the ER. Receptor dimers, an active form of the receptors, were also detected in the ER. According to BiFC and protease protection data, the catalytic part of AHK3 was located in the cytoplasm whereas the hormone binding module faced the ER lumen. This topology is consistent with receptor signalling from the ER membrane. Finally, the functionality of receptors in different membrane fractions was tested using an in vitro kinase assay visualising the phosphorylation of phosphotransfer proteins. The detected cytokinin-dependent phosphotransfer activity was confined mainly to the ER-enriched fraction. Collectively, our data demonstrate that ER-located cytokinin receptors are active in cytokinin signal transduction. Hence, intracellular cytokinins appear to play an essential role in cytokinin signalling. An updated model for the spatial organisation of cytokinin transport form activation, intracellular trafficking and signalling from the ER is proposed.

scholarly journals An Intramolecular Association between Two Domains of the Protein Kinase Fused Is Necessary for Hedgehog Signaling

2004 ◽  
Vol 24 (23) ◽  
pp. 10397-10405 ◽  
Author(s):  
Manuel Ascano ◽  
David J. Robbins
Keyword(s):  
Signal Transduction ◽  
Protein Kinase ◽  
Hedgehog Signaling ◽  
Membrane Vesicles ◽  
Kinase Domain ◽  
Kinase Assay ◽  
Dependent Manner ◽  
Intramolecular Association

ABSTRACT The protein kinase Fused (Fu) is an integral member of the Hedgehog (Hh) signaling pathway. Although genetic studies demonstrate that Fu is required for the regulation of the Hh pathway, the mechanistic role that it plays remains largely unknown. Given our difficulty in developing an in vitro kinase assay for Fu, we reasoned that the catalytic activity of Fu might be highly regulated. Several mechanisms are known to regulate protein kinases, including self-association in either an intra- or an intermolecular fashion. Here, we provide evidence that Hh regulates Fu through intramolecular association between its kinase domain (ΔFu) and its carboxyl-terminal domain (Fu-tail). We show that ΔFu and Fu-tail can interact in trans, with or without the kinesin-related protein Costal 2 (Cos2). However, since the majority of Fu is found associated with Cos2 in vivo, we hypothesized that Fu-tail, which binds Cos2 directly, would be able to tether ΔFu to Cos2. We demonstrate that ΔFu colocalizes with Cos2 in the presence of Fu-tail and that this colocalization occurs on a subset of membrane vesicles previously characterized to be important for Hh signal transduction. Additionally, expression of Fu-tail in fu mutant flies that normally express only the kinase domain rescues the fu wing phenotype. Therefore, reestablishing the association between these two domains of Fu in trans is sufficient to restore Hh signal transduction in vivo. In such a manner we validate our hypothesis, demonstrating that Fu self-associates and is functional in an Hh-dependent manner. Our results here enhance our understanding of one of the least characterized, yet critical, components of Hh signal transduction.

scholarly journals FUS3 phosphorylates multiple components of the mating signal transduction cascade: evidence for STE12 and FAR1.

1993 ◽  
Vol 4 (5) ◽  
pp. 495-510 ◽  
Author(s):  
E A Elion ◽  
B Satterberg ◽  
J E Kranz
Keyword(s):  
Signal Transduction ◽  
Map Kinase ◽  
Maximal Activity ◽  
Kinase Assay ◽  
G1 Arrest ◽  
Multicopy Suppressor ◽  
Signal Transduction Cascade ◽  
Kinase Homologue

The mitogen-activated protein (MAP) kinase homologue FUS3 mediates both transcription and G1 arrest in a pheromone-induced signal transduction cascade in Saccharomyces cerevisiae. We report an in vitro kinase assay for FUS3 and its use in identifying candidate substrates. The assay requires catalytically active FUS3 and pheromone induction. STE7, a MAP kinase kinase homologue, is needed for maximal activity. At least seven proteins that specifically associate with FUS3 are phosphorylated in the assay. Many of these substrates are physiologically relevant and are affected by in vivo levels of numerous signal transduction components. One substrate is likely to be the transcription factor STE12. A second is likely to be FAR1, a protein required for G1 arrest. FAR1 was isolated as a multicopy suppressor of a nonarresting fus3 mutant and interacts with FUS3 in a two hybrid system. Consistent with this FAR1 is a good substrate in vitro and generates a FUS3-associated substrate of expected size. These data support a model in which FUS3 mediates transcription and G1 arrest by direct activation of STE12 and FAR1 and phosphorylates many other proteins involved in the response to pheromone.

Biochemical identification of the OsMKK6–OsMPK3 signalling pathway for chilling stress tolerance in rice1

2012 ◽  
Vol 443 (1) ◽  
pp. 95-102 ◽  
Author(s):  
Guosheng Xie ◽  
Hideki Kato ◽  
Ryozo Imai
Keyword(s):  
Protein Kinase ◽  
Low Temperature ◽  
Transgenic Plants ◽  
Stress Tolerance ◽  
Chilling Stress ◽  
Active Form ◽  
Signalling Pathway ◽  
Kinase Assay ◽  
Mapk Kinase

MAPK (mitogen-activated protein kinase) pathways have been implicated in stress signalling in plants. In the present study, we performed yeast two-hybrid screening to identify partner MAPKs for OsMKK (Oryza sativa MAPK kinase) 6, a rice MAPK kinase, and revealed specific interactions of OsMKK6 with OsMPK3 and OsMPK6. OsMPK3 and OsMPK6 each co-immunoprecipitated OsMKK6, and both were directly phosphorylated by OsMKK6 in vitro. An MBP (myelin basic protein) kinase assay of the immunoprecipitation complex indicated that OsMPK3 and OsMPK6 were activated in response to a moderately low temperature (12°C), but not a severely low temperature (4°C) in rice seedlings. A constitutively active form of OsMKK6, OsMKK6DD, showed elevated phosphorylation activity against OsMPK3 and OsMPK6 in vitro. OsMPK3, but not OsMPK6, was constitutively activated in transgenic plants overexpressing OsMKK6DD, indicating that OsMPK3 is an in vivo target of OsMKK6. Enhanced chilling tolerance was observed in the transgenic plants overexpressing OsMKK6DD. Taken together, our data suggest that OsMKK6 and OsMPK3 constitute a moderately low-temperature signalling pathway and regulate cold stress tolerance in rice.

scholarly journals High-affinity binding of glycogen-synthase phosphatase to glycogen particles in the liver. Role of glycogen in the inhibition of synthase phosphatase by phosphorylase a

1987 ◽  
Vol 246 (2) ◽  
pp. 367-374 ◽  
Author(s):  
L Mvumbi ◽  
W Stalmans
Keyword(s):  
Endoplasmic Reticulum ◽  
High Speed ◽  
Glycogen Synthase ◽  
Smooth Endoplasmic Reticulum ◽  
Active Form ◽  
Glycogen Concentration ◽  
High Affinity ◽  
Glycogen Particles

1. Post-mitochondrial supernatants were prepared from the livers of 24 h-fasted rats. Upon centrifugation at high speed, the major part of the glycogen-synthase phosphatase activity sedimented with the microsomal fraction. However, two approaches showed that the enzyme was associated with residual glycogen rather than with vesicles of the endoplasmic reticulum. Indeed, the activity was entirely solubilized when the remaining glycogen was degraded either by glucagon treatment in vivo or by alpha-amylolysis in vitro. No evidence could be found for an association of glycogen-synthase phosphatase with the smooth endoplasmic reticulum, as isolated with the use of discontinuous sucrose gradients. 2. After solubilization by glucagon treatment in vivo, synthase phosphatase could be transferred to glycogen particles with very high affinity. Half-maximal binding occurred at a glycogen concentration of about 0.25 mg/ml, whereas glycogen synthase and phosphorylase required 1.5-2 mg/ml. 3. In gel-filtered extracts prepared from glycogen-depleted livers, the activation of glycogen synthase was not inhibited at all by phosphorylase alpha. The inhibition was restored when the liver homogenates were prepared in a glycogen-containing buffer. The effect was half-maximal at a glycogen concentration of about 0.25 mg/ml, and virtually complete at 1 mg/ml. These findings explain long-standing observations that in fasted animals the liver contains appreciable amounts of both synthase and phosphorylase in the active form.

scholarly journals Two-Dimensional Interfacial Exchange Diffusion Has the Potential to Augment Spatiotemporal Precision of Ca2+ Signaling

2022 ◽  
Vol 23 (2) ◽  
pp. 850
Author(s):  
Cornelis van Breemen ◽  
Nicola Fameli ◽  
Klaus Groschner
Keyword(s):  
Signal Transduction ◽  
Endoplasmic Reticulum ◽  
Recent Literature ◽  
Stochastic Simulations ◽  
Two Dimensional ◽  
Phospholipid Membrane ◽  
Exchange Diffusion ◽  
Lipid Interactions ◽  
Aqueous Volume

Nano-junctions between the endoplasmic reticulum and cytoplasmic surfaces of the plasma membrane and other organelles shape the spatiotemporal features of biological Ca2+ signals. Herein, we propose that 2D Ca2+ exchange diffusion on the negatively charged phospholipid surface lining nano-junctions participates in guiding Ca2+ from its source (channel or carrier) to its target (transport protein or enzyme). Evidence provided by in vitro Ca2+ flux experiments using an artificial phospholipid membrane is presented in support of the above proposed concept, and results from stochastic simulations of Ca2+ trajectories within nano-junctions are discussed in order to substantiate its possible requirements. Finally, we analyze recent literature on Ca2+ lipid interactions, which suggests that 2D interfacial Ca2+ diffusion may represent an important mechanism of signal transduction in biological systems characterized by high phospholipid surface to aqueous volume ratios.

scholarly journals Cytokinin activity ofN6-benzyladenine derivatives assayed by interaction with the receptorsin planta, in vitro, andin silico

2018 ◽  
Author(s):  
Ekaterina M. Savelieva ◽  
Vladimir E. Oslovsky ◽  
Dmitry S. Karlov ◽  
Nikolay N. Kurochkin ◽  
Irina A. Getman ◽  
...  
Keyword(s):  
Specific Binding ◽  
Biological Effects ◽  
Test System ◽  
Membrane Receptors ◽  
Receptor Interaction ◽  
Cytokinin Activity ◽  
Interaction Patterns ◽  
In Planta ◽  
Cytokinin Receptors

AbstractBiological effects of hormones in both plants and animals are based on high-affrnity interaction with cognate receptors resulting in their activation. The signal of cytokinins, classical plant hormones, is perceived inArabidopsisby three homologous membrane receptors: AHK2, AHK3, and CRE1/AHK4. To study the cytokinin–receptor interaction, we used 25 derivatives of potent cytokininN6-benzyladenine (BA) with substituents in the purine heterocycle and/or in the side chain. The study was focused primarily on individual cytokinin receptors fromArabidopsis. The mainin plantaassay system was based onArabidopsisdouble mutants retaining only one isoform of cytokinin receptors and harboring cytokinin-sensitive reporter gene. Classical cytokinin biotest withAmaranthusseedlings was used as an additional biotest. In parallel, the binding of ligands to individual cytokinin receptors was assessed in thein vitrotest system. Quantitative comparison of results of different assays confirmed the partial similarity of ligand-binding properties of receptor isoforms. Substituents at positions 8 and 9 of adenine moiety, elongated linker up to 4 methylene units, replacement ofN6by sulfur or oxygen, resulted in suppression of cytokinin activity of the derivative towards all receptors. Introduction of a halogen into position 2 of adenine moiety, on the contrary, often increased the ligand activity, especially toward AHK3. Features both common and distinctive of cytokinin receptors inArabidopsisandAmaranthuswere revealed, highlighting species specificity of the cytokinin perception apparatus. Correlations between extent of compound binding to a receptorin vitroand its ability to activate the same receptorin plantawere evaluated for each AHK protein. Interaction patterns between individual receptors and ligands were rationalized by structure analysis and molecular docking in sensory modules of AHK receptors. The best correlation between docking scores and specific binding was observed for AHK3. In addition, receptor-specific ligands have been discovered with unique properties to predominantly activate or block distinct cytokinin receptors. These ligands are promising for practical application and as molecular tools in the study of the cytokinin perception by plant cells.Graphical abstractIndividual cytokinin receptors fromArabidopsiswere assayedin planta,in vitroandin silicowith 25 different 6-benzyladenine derivatives, new receptor-specific cytokinins were revealed.

scholarly journals Sensitivity of an Epstein-Barr Virus-Positive Tumor Line, Daudi, to Alpha Interferon Correlates with Expression of a GC-Rich Viral Transcript

1999 ◽  
Vol 19 (11) ◽  
pp. 7305-7313 ◽  
Author(s):  
Yanning Gao ◽  
Shao-an Xue ◽  
Beverly E. Griffin
Keyword(s):  
Epstein Barr Virus ◽  
Active Form ◽  
Type I Interferons ◽  
Alpha Interferon ◽  
Kinase Assay ◽  
Type I ◽  
Viral Transcript ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT The exquisite sensitivity of the Burkitt’s lymphoma (BL)-derived cell line Daudi to type I interferons has not previously been explained. Here we show that expression of an Epstein-Barr virus (EBV) transcript, designated D-HIT (Y. Gao et al., J. Virol. 71:84–94, 1997), correlates with the sensitivity of different Daudi cell isolates (or that of other EBV-carrying cells, where known) to alpha interferon (IFN-α). D-HIT, transcribed from a GC-rich repetitive region (IR4) of the viral genome, is highly structured, responding to RNase digestion in a manner akin to double-stranded RNA. Comparing EBV-carrying BL cell lines with differing responses to IFN-α, we found the protein levels of the dsRNA-activated kinase, PKR, to be similar, whereas the levels of the autophosphorylated active form of PKR varied in a manner that correlated with endogenous levels of D-HIT expression. In a classical in vitro kinase assay, addition of either poly(I)-poly(C) or an in vitro-transcribed D-HIT homolog stimulated the autophosphorylation activity of PKR from IFN-α-treated cells in both EBV-positive and EBV-negative B lymphocytes. By transfection experiments, these RNAs were shown to reduce cell proliferation and to sensitize otherwise relatively insensitive Raji cells to IFN-α. The data lead to a model wherein the D-HIT viral RNA also serves as a possible transcriptional activator of IFN-α or cellular genes regulated by this cytokine.

In Vitro Induction of Crystals of MT Protein by Vinblastine-Colcemid in Mouse Spermatids

1974 ◽  
Vol 32 ◽  
pp. 132-133
Author(s):  
John J. Wolosewick ◽  
John H. D. Bryan
Keyword(s):  
Endoplasmic Reticulum ◽  
Smooth Endoplasmic Reticulum ◽  
Nuclear Ring ◽  
Rough Er ◽  
Distal Direction ◽  
In Vitro Induction

Early in spermiogenesis the manchette is rapidly assembled in a distal direction from the nuclear-ring-densities. The association of vesicles of smooth endoplasmic reticulum (SER) and the manchette microtubules (MTS) has been reported. In the mouse, osmophilic densities at the distal ends of the manchette are the organizing centers (MTOCS), and are associated with the SER. Rapid MT assembly and the lack of rough ER suggests that there is an existing pool of MT protein. Colcemid potentiates the reaction of vinblastine with tubulin and was used in this investigation to detect this protein.

Sign in / Sign up

Export Citation Format

Share Document