Glutaredoxin AtGRXC2 catalyses inhibitory glutathionylation of Arabidopsis BRI1-associated receptor-like kinase 1 (BAK1) in vitro

2015 ◽  
Vol 467 (3) ◽  
pp. 399-413 ◽  
Author(s):  
Kyle W. Bender ◽  
Xuejun Wang ◽  
George B. Cheng ◽  
Hyoung Seok Kim ◽  
Raymond E. Zielinski ◽  
...  
Keyword(s):  
Protein Kinases ◽  
Redox Regulation ◽  
Kinase Activity ◽  
Cytoplasmic Domain ◽  
Post Translational Modification ◽  
Cysteine Oxidation ◽  
Receptor Like Kinase ◽  
Reversible Protein Phosphorylation ◽  
Two Hybrid

Reversible protein phosphorylation, catalysed by protein kinases, is the most widely studied post-translational modification (PTM), whereas the analysis of other modifications such as S-thiolation is in its relative infancy. In a yeast-two-hybrid (Y2H) screen, we identified a number of novel putative brassinosteroid insensitive 1 (BR1)-associated receptor-like kinase 1 (BAK1) interacting proteins including several proteins related to redox regulation. Glutaredoxin (GRX) C2 (AtGRXC2) was among candidate proteins identified in the Y2H screen and its interaction with recombinant Flag–BAK1 cytoplasmic domain was confirmed using an in vitro pull-down approach. We show that BAK1 peptide kinase activity is sensitive to the oxidizing agents H2O2 and diamide in vitro, suggesting that cysteine oxidation might contribute to control of BAK1 activity. Furthermore, BAK1 was glutathionylated and this reaction could occur via a thiolate-dependent reaction with GSSG or a H2O2-dependent reaction with GSH and inhibited kinase activity. Surprisingly, both reactions were catalysed by AtGRXC2 at lower concentrations of GSSG or GSH than reacted non-enzymatically. Using MALDI–TOF MS, we identified Cys353, Cys374 and Cys408 as potential sites of glutathionylation on the BAK1 cytoplasmic domain and directed mutagenesis suggests that Cys353 and Cys408 are major sites of GRXC2-mediated glutathionylation. Collectively, these results highlight the potential for redox control of BAK1 and demonstrate the ability of AtGRXC2 to catalyse protein glutathionylation, a function not previously described for any plant GRX. The present work presents a foundation for future studies of glutathionylation of plant receptor-like protein kinases (RLKs) as well as for the analysis of activities of plant GRXs.

Abstract 546: S-palmitoylation Mediates Caveolae Localization and Limits Cysteine Oxidation of Gc-1 in Cardiomyocytes

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Christos Tzimas ◽  
Ilaria Russo ◽  
Wen Dun ◽  
Sevde Yilmaz ◽  
Sudarsan Rajan ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Redox Regulation ◽  
Soluble Guanylyl Cyclase ◽  
Fold Increase ◽  
Transcript Level ◽  
Neonatal Rat ◽  
Membrane Microdomains ◽  
Post Translational Modification ◽  
Cysteine Oxidation ◽  
Rat Cardiomyocytes

Objectives: The nitric oxide (NO) receptor soluble guanylyl cyclase (GC-1) can localize to cell membrane microdomains, including caveolae. In failing hearts, GC-1 relocalizes away from caveolae and becomes oxidized. How this occurs is unknown. We hypothesized that S-palmitoylation, a reversible, enzyme-catalyzed, post-translational modification that enhances membrane affinity of protein substrates, mediates caveolae-localization and modulates redox regulation of GC-1. Methods: Adult male C57BL/6 mice were subjected to transverse aortic constriction (TAC) versus thoracotomy (Sham). Modified acyl-biotin exchange (ABE) assays were performed to assess S-palmitoylation of GC-1 in failing TAC vs non-failing Sham hearts. Using bioinformatics, we identified 5 conserved cysteines in the GC-1α subunit as potential S-palmitoylation sites. We created cysteine-serine mutants of GC-1α at each and all 5 sites (C3S, C15S, C176S, C497S, C595S, CS*). Mutants and WT GC-1α were expressed in neonatal rat cardiomyocytes (NRCM) and their palmitoylation status compared by ABE. We assessed localization and oxidation of GC-1α (CS* vs WT) by confocal microscopy and redox Western, respectively. Results: In TAC hearts, S-palmitoylation of GC-1α was half of that in Sham ( p =0.02). Likewise, palmitoyl-transferase ZDHHC16 was downregulated in TAC vs Sham by 31% ( p =0.01) at the transcript level and by 56% ( p =0.03) at the protein level. In NRCMs, S-palmitoylation of CS* was markedly diminished (6.9%, p <0.001), while that of C15S, C176S, and C497S were modestly diminished (23.8%, p=0.03; 23.2%, p=0.01; 22.8%, p=0.01), compared to WT (54.7%). Moreover, S-palmitoylation of WT but not CS* was decreased by palmitoylation inhibitor 2-BP (17.8%, p<0.0001). Confocal microscopy showed more diffuse cytosolic distribution of CS* compared to WT. Redox Westerns revealed increased oxidation of CS* vs WT upon H 2 O 2 treatment (1.7-fold increase, p=0.008). Conclusions: Our data support that GC-1α is S-palmitoylated at C15, C176, and C497. S-palmitoylation of GC-1α at these 3 cysteines appears to protect GC-1α against oxidation and to mediate its membrane localization.

Phosphorylation of the dimeric cytoplasmic domain of the phytosulfokine receptor, PSKR1

2016 ◽  
Vol 473 (19) ◽  
pp. 3081-3098 ◽  
Author(s):  
Victor Muleya ◽  
Claudius Marondedze ◽  
Janet I. Wheeler ◽  
Ludivine Thomas ◽  
Yee-Fong Mok ◽  
...  
Keyword(s):  
Guanylate Cyclase ◽  
Kinase Activity ◽  
Analytical Ultracentrifugation ◽  
Domain Architecture ◽  
Defense Responses ◽  
Cytoplasmic Domain ◽  
Size Exclusion ◽  
Cyclase Activity ◽  
Guanylate Cyclase Activity

Phytosulfokines (PSKs) are plant peptide hormones that co-regulate plant growth, differentiation and defense responses. PSKs signal through a plasma membrane localized leucine-rich repeat receptor-like kinase (phytosulfokine receptor 1, PSKR1) that also contains a functional cytosolic guanylate cyclase with its cyclase catalytic center embedded within the kinase domain. To functionally characterize this novel type of overlapping dual catalytic function, we investigated the phosphorylation of PSKR1 in vitro. Tandem mass spectrometry of the cytoplasmic domain of PSKR1 (PSKR1cd) revealed at least 11 phosphorylation sites (8 serines, 2 threonines and 1 tyrosine) within the PSKR1cd. Phosphomimetic mutations of three serine residues (Ser686, Ser696 and Ser698) in tandem at the juxta-membrane position resulted in enhanced kinase activity in the on-mutant that was suppressed in the off-mutant, but both mutations reduced guanylate cyclase activity. Both the on and off phosphomimetic mutations of the phosphotyrosine (Tyr888) residue in the activation loop suppressed kinase activity, while neither mutation affected guanylate cyclase activity. Size exclusion and analytical ultracentrifugation analysis of the PSKR1cd suggest that it is reversibly dimeric in solution, which was further confirmed by biflourescence complementation. Taken together, these data suggest that in this novel type of receptor domain architecture, specific phosphorylation and dimerization are possibly essential mechanisms for ligand-mediated catalysis and signaling.

scholarly journals Std1p (Msn3p) Positively Regulates the Snf1 Kinase in Saccharomyces cerevisiae

Genetics ◽  
2003 ◽  
Vol 163 (2) ◽  
pp. 507-514 ◽  
Author(s):  
Sergei Kuchin ◽  
Valmik K Vyas ◽  
Ellen Kanter ◽  
Seung-Pyo Hong ◽  
Marian Carlson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Kinase Activity ◽  
Catalytic Domain ◽  
Snf1 Kinase ◽  
Glucose Signaling ◽  
Catalytic Domains ◽  
Upstream Kinase ◽  
Two Hybrid

Abstract The Snf1 protein kinase of the glucose signaling pathway in Saccharomyces cerevisiae is regulated by an autoinhibitory interaction between the regulatory and catalytic domains of Snf1p. Transitions between the autoinhibited and active states are controlled by an upstream kinase and the Reg1p-Glc7p protein phosphatase 1. Previous studies suggested that Snf1 kinase activity is also modulated by Std1p (Msn3p), which interacts physically with Snf1p and also interacts with glucose sensors. Here we address the relationship between Std1p and the Snf1 kinase. Two-hybrid assays showed that Std1p interacts with the catalytic domain of Snf1p, and analysis of mutant kinases suggested that this interaction is incompatible with the autoinhibitory interaction of the regulatory and catalytic domains. Overexpression of Std1p increased the two-hybrid interaction of Snf1p with its activating subunit Snf4p, which is diagnostic of an open, uninhibited conformation of the kinase complex. Overexpression of Std1p elevated Snf1 kinase activity in both in vitro and in vivo assays. These findings suggest that Std1p stimulates the Snf1 kinase by an interaction with the catalytic domain that antagonizes autoinhibition and promotes an active conformation of the kinase.

325 ACTIVITY OF MATURATION-PROMOTING FACTOR (MPF) AND MITOGEN-ACTIVATED PROTEIN KINASES DURING IN VITRO MATURATION OF BUFFALO OOCYTES (BUBALUS BUBALIS)

2006 ◽  
Vol 18 (2) ◽  
pp. 270
Author(s):  
B. Gasparrini ◽  
G. Leoni ◽  
L. Boccia ◽  
M. Galiotto ◽  
S. Ledda ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Kinases ◽  
In Vitro Maturation ◽  
Map Kinases ◽  
Kinase Activity ◽  
Buffer System ◽  
Mitogen Activated Protein Kinases ◽  
Mitogen Activated Protein ◽  
Maturation Promoting Factor

The maturation promoting factor (MPF) and mitogen-activated protein kinases (MAPK) are the key regulators of both meiotic and mitotic cell cycles. The absence of data on the activity of the major cell cycle kinases in buffalo oocytes during meiotic progression provided the bases for this study. More specifically we assayed the MPF and MAP kinase activity of buffalo oocytes during meiosis. Abattoir-derived cumulus-oocyte complexes (COCs) with a compact, non-atretic cumulus and a homogeneous cytoplasm were utilized for the study. The COCs (n = 293, over four replicates) were matured in vitro in TCM-199 supplemented with 10% fetal calf serum (FCS), 0.2 mM sodium pyruvate, 0.5 �g/mL FSH, 5 �g/mL LH, 1 �g/mL 17�-estradiol, 50 �M of cysteamine, and 50 �g/mL kanamycin (B199). In vitro maturation (IVM) was carried out at 38.5�C under a controlled gas atmosphere of 5% CO2 in humidified air. At scale times during the culture (0, 3, 6, 9, 12, 15, 18, 21, 24 h) groups of oocytes were stained with Hoechst 33342 to assess chromatin configuration and stored according to the maturation stage (GV, GVDB, MI, and MII) at -80�C pending protein analysis. SDS-polyacrylamide gel electrophoresis wase performed using Laemmli discontinuous buffer system (Laemmli 1970 Nature 227, 680) with a 12% running gel. Groups of oocytes were analyzed for MPF activity (n = 65) by histone H1 kinase activity (Naito and Toyoda 1991 J. Reprod. Fertil. 93, 467-473) and for MAPK activity (n = 48) by myelin basic protein assays (Chesnel et al. 1995 Biol. Reprod. 52, 895-902). The activity of both MPF and MAP kinases was quantified by measuring the density of the bands on the autoradiographic film with a densitometer. Differences in the levels of the kinases among groups were analyzed by ANOVA. It was assumed that the value of MPF and MAPK was 100% in metaphase II (MII) stage oocytes. The lowest levels of MPF and MAPK activities were found in the oocytes at the GV (0-6 h post-IVM: 40% and 17.2%, respectively) and at the GVBD (6-9 h post-IVM: 41.2% and 18%) stages. The activities increased at metaphase I (MI) stage (9-15 h post-IVM) and at MII (21-24 post-IVM). Interestingly, although similar levels of MAP kinases were found at MI and MII stages (95.1% vs. 100%), MPF levels were significantly lower (P < 0.01) at the MI stage compared to those detected at MII (82.8% vs. 100%). The fluctuations of the MPF levels in buffalo appear different compared to those observed in other species; in particular, no differences were recorded between the GV and the GVBD stages whereas a significant increase of the MPF levels was found at MII compared to the MI stage. It seems that MPF and MAPK could differently guide meiotic resumption and progression to the MII arrest in this species. To our knowledge, this is the first report on biochemical analysis of the cell cycle regulation in buffalo oocytes.

scholarly journals PamgeneAnalyzeR: open and reproducible pipeline for kinase profiling

2020 ◽  
Vol 36 (20) ◽  
pp. 5117-5119 ◽  
Author(s):  
Amel Bekkar ◽  
Anita Nasrallah ◽  
Nicolas Guex ◽  
Lluis Fajas ◽  
Ioannis Xenarios ◽  
...  
Keyword(s):  
Protein Kinases ◽  
High Throughput ◽  
Kinase Activity ◽  
Experimental Approach ◽  
R Package ◽  
Activity Measurement ◽  
Post Translational Modification ◽  
Peptide Microarrays ◽  
Disease States ◽  
Downstream Analysis

Abstract   Protein phosphorylation––catalyzed by protein kinases–is the most common post-translational modification. It increases the functional diversity of the proteome and influences various aspects of normal physiology and can be altered in disease states. High throughput profiling of kinases is becoming an essential experimental approach to investigate their activity and this can be achieved using technologies such as PamChip® arrays provided by PamGene for kinase activity measurement. Here, we present ‘pamgeneAnalyzeR’, an R package developed as an alternative to the manual steps necessary to extract the data from PamChip® peptide microarrays images in a reproducible and robust manner. The extracted data can be directly used for downstream analysis. Availability and implementation PamgeneAnalyzeR is implemented in R and can be obtained from https://github.com/amelbek/pamgeneAnalyzeR.

scholarly journals The PRK2 kinase is a potential effector target of both Rho and Rac GTPases and regulates actin cytoskeletal organization.

1997 ◽  
Vol 17 (4) ◽  
pp. 2247-2256 ◽  
Author(s):  
S Vincent ◽  
J Settleman
Keyword(s):  
Protein Kinases ◽  
Kinase Activity ◽  
Rho Kinase ◽  
Close Relative ◽  
Rho Proteins ◽  
Cellular Processes ◽  
Rac Gtpases ◽  
Downstream Effector ◽  
Rho Family Gtpases

The Ras-related Rho family GTPases mediate signal transduction pathways that regulate a variety of cellular processes. Like Ras, the Rho proteins (which include Rho, Rac, and CDC42) interact directly with protein kinases, which are likely to serve as downstream effector targets of the activated GTPase. Activated RhoA has recently been reported to interact directly with several protein kinases, p120 PKN, p150 ROK alpha and -beta, p160 ROCK, and p164 Rho kinase. Here, we describe the purification of a novel Rho-associated kinase, p140, which appears to be the major Rho-associated kinase activity in most tissues. Peptide microsequencing revealed that p140 is probably identical to the previously reported PRK2 kinase, a close relative of PKN. However, unlike the previously described Rho-binding kinases, which are Rho specific, p140 associates with Rac as well as Rho. Moreover, the interaction of p140 with Rho in vitro is nucleotide independent, whereas the interaction with Rac is completely GTP dependent. The association of p140 with either GTPase promotes kinase activity substantially, and expression of a kinase-deficient form of p140 in microinjected fibroblasts disrupts actin stress fibers. These results indicate that p140 may be a shared kinase target of both Rho and Rac GTPases that mediates their effects on rearrangements of the actin cytoskeleton.

scholarly journals Altered Plant and Nodule Development and Protein S-Nitrosylation in Lotus japonicus Mutants Deficient in S-Nitrosoglutathione Reductases

2019 ◽  
Vol 61 (1) ◽  
pp. 105-117 ◽  
Author(s):  
Manuel A Matamoros ◽  
Maria C Cutrona ◽  
Stefanie Wienkoop ◽  
Juan C Begara-Morales ◽  
Niels Sandal ◽  
...  
Keyword(s):  
Protein Function ◽  
Stress Protein ◽  
Lotus Japonicus ◽  
Protein S ◽  
Shotgun Proteomics ◽  
Nodule Development ◽  
Post Translational Modification ◽  
Cysteine Oxidation ◽  
No Donor

Abstract Nitric oxide (NO) is a crucial signaling molecule that conveys its bioactivity mainly through protein S-nitrosylation. This is a reversible post-translational modification (PTM) that may affect protein function. S-nitrosoglutathione (GSNO) is a cellular NO reservoir and NO donor in protein S-nitrosylation. The enzyme S-nitrosoglutathione reductase (GSNOR) degrades GSNO, thereby regulating indirectly signaling cascades associated with this PTM. Here, the two GSNORs of the legume Lotus japonicus, LjGSNOR1 and LjGSNOR2, have been functionally characterized. The LjGSNOR1 gene is very active in leaves and roots, whereas LjGSNOR2 is highly expressed in nodules. The enzyme activities are regulated in vitro by redox-based PTMs. Reducing conditions and hydrogen sulfide-mediated cysteine persulfidation induced both activities, whereas cysteine oxidation or glutathionylation inhibited them. Ljgsnor1 knockout mutants contained higher levels of S-nitrosothiols. Affinity chromatography and subsequent shotgun proteomics allowed us to identify 19 proteins that are differentially S-nitrosylated in the mutant and the wild-type. These include proteins involved in biotic stress, protein degradation, antioxidant protection and photosynthesis. We propose that, in the mutant plants, deregulated protein S-nitrosylation contributes to developmental alterations, such as growth inhibition, impaired nodulation and delayed flowering and fruiting. Our results highlight the importance of GSNOR function in legume biology.

225. Characterisation of a diverse secretome generated by the mouse preimplantation embryo

2008 ◽  
Vol 20 (9) ◽  
pp. 25
Author(s):  
C. O.'Neill ◽  
A. Beardsley
Keyword(s):  
Mass Spectrometry ◽  
Redox Regulation ◽  
Preimplantation Embryo ◽  
Early Embryo ◽  
Arginine Deiminase ◽  
Post Translational Modification ◽  
Preimplantation Embryo Development ◽  
Mouse Preimplantation Embryo ◽  
Esi Mass Spectrometry

This study investigated the suitability of surface-enhanced laser desorption and ionisation time-of-flight (SELDI-TOF) and electro-spray ionisation (ESI) mass spectrometry for the analysis of the proteins released by the mouse preimplantation embryo in vitro. SELDI-TOF analysis with CM10 or IMAC30 (but not Q10) chips detected a protein peak at m/z 8570 released by both C57BL6 and hybrid embryos. No other peaks unique to the embryo were identified with this method. ESI mass spectrometry of tryptic digests of embryo conditioned media identified a total of 20 proteins released during development from the zygote to blastocysts stage. Four proteins were expressed in at least 7 out of 8 cultures tested, one of these (lactate dehydrogenase B) was in all cultures. A further five proteins were in at least half of the cultures and 11 more putative proteins were detected in at least one culture. The pattern of protein secretion was not obviously different for C57BL6 or hyrid embryos. The expression of two of these proteins is essential for preimplantation embryo development (NLR family, pyrin domain containing 5 protein and peptidyl arginine deiminase, type VI). A further four proteins detected have roles in redox regulation of cells, and three others are capable of inducing post-translational modification of proteins. This study shows the feasibility of ESI mass spectrometry and the limitations of SELD-TOF mass spectrometry for identifying the proteins scereted by the preimplantation embryo in vitro. This analysis identifies a range of targets that now require detailed functional analysis to assess whether their release by the embryo is an important property of the early embryo, or an artefact of in vitro culture.

scholarly journals p53 Forms Redox-Dependent Protein–Protein Interactions through Cysteine 277

Antioxidants ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 1578
Author(s):  
Tao Shi ◽  
Paulien E. Polderman ◽  
Marc Pagès-Gallego ◽  
Robert M. van Es ◽  
Harmjan R. Vos ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Fine Tuning ◽  
Protein Protein Interactions ◽  
Post Translational Modification ◽  
Cysteine Oxidation ◽  
Interaction Partners ◽  
Dependent Protein ◽  
And Function

Reversible cysteine oxidation plays an essential role in redox signaling by reversibly altering protein structure and function. Cysteine oxidation may lead to intra- and intermolecular disulfide formation, and the latter can drastically stabilize protein–protein interactions in a more oxidizing milieu. The activity of the tumor suppressor p53 is regulated at multiple levels, including various post-translational modification (PTM) and protein–protein interactions. In the past few decades, p53 has been shown to be a redox-sensitive protein, and undergoes reversible cysteine oxidation both in vitro and in vivo. It is not clear, however, whether p53 also forms intermolecular disulfides with interacting proteins and whether these redox-dependent interactions contribute to the regulation of p53. In the present study, by combining (co-)immunoprecipitation, quantitative mass spectrometry and Western blot we found that p53 forms disulfide-dependent interactions with several proteins under oxidizing conditions. Cysteine 277 is required for most of the disulfide-dependent interactions of p53, including those with 14-3-3q and 53BP1. These interaction partners may play a role in fine-tuning p53 activity under oxidizing conditions.

scholarly journals PamgeneAnalyzeR open and reproducible pipeline for kinase profiling

2019 ◽  
Author(s):  
Amel Bekkar ◽  
Anita Nasrallah ◽  
Nicolas Guex ◽  
Lluis Fajas ◽  
Ioannis Xenarios ◽  
...  
Keyword(s):  
Protein Kinases ◽  
High Throughput ◽  
Kinase Activity ◽  
Experimental Approach ◽  
R Package ◽  
Activity Measurement ◽  
Post Translational Modification ◽  
Peptide Microarrays ◽  
Disease States ◽  
Downstream Analysis

AbstractProtein phosphorylation - catalyzed by protein kinases - is the most common post-translational modification. It increases the functional diversity of the proteome and influences various aspects of normal physiology and can be altered in disease states. High throughput profiling of kinases is becoming an essential experimental approach to investigate their activity and this can be achieved using technologies such as PamChip® arrays provided by PamGene for kinase activity measurement. Here we present “pamgeneAnalyzeR”, an R package developed as an alternative to the manual steps necessary to extract the data from PamChip® peptide microarrays images in a reproducible and robust manner. The extracted data can be directly used for downstream analysis.Availability and implementationPamgeneAnalyzeR is implemented in R and can be obtained from https://github.com/amelbek/pamgeneAnalyzeR.

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