Apoptosis-associated tyrosine kinase scaffolding of protein phosphatase 1 and SPAK reveals a novel pathway for Na-K-2C1 cotransporter regulation

2007 ◽  
Vol 292 (5) ◽  
pp. C1809-C1815 ◽  
Author(s):  
Kenneth B. E. Gagnon ◽  
Roger England ◽  
Lisa Diehl ◽  
Eric Delpire
Keyword(s):  
Xenopus Laevis ◽  
Tyrosine Kinase ◽  
Protein Interaction ◽  
Protein Phosphatase ◽  
Catalytic Domain ◽  
Protein Phosphatase 1 ◽  
Xenopus Laevis Oocytes ◽  
Physical Interaction ◽  
Protein Protein Interaction ◽  
Key Residues

Previous work from our laboratory and others has established that Ste-20-related proline-alanine-rich kinase (SPAK/PASK) is central to the regulation of NKCC1 function. With no lysine (K) kinase (WNK4) has also been implicated in the regulation of NKCC1 activity through upstream activation of SPAK. Because previous studies from our laboratory also demonstrated a protein-protein interaction between SPAK and apoptosis-associated tyrosine kinase (AATYK), we explore here the possibility that AATYK is another component of the regulation of NKCC1. Heterologous expression of AATYK1 in NKCC1-injected Xenopus laevis oocytes markedly inhibited cotransporter activity under isosmotic conditions. Interestingly, mutation of key residues in the catalytic domain of AATYK1 revealed that the kinase activity does not play a role in the suppression of NKCC1 function. However, mutagenesis of the two SPAK-binding motifs in AATYK1 completely abrogated this effect. As protein phosphatase 1 (PP1) also plays a central role in the dephosphorylation and inactivation of NKCC1, we investigated the possibility that AATYK1 interacts with the phosphatase. We identified a PP1 docking motif in AATYK1 and demonstrated interaction using yeast-2-hybrid analysis. Mutation of a key valine residue (V1175) within this motif prevented protein-protein interaction. Furthermore, the physical interaction between PP1 and AATYK was required for inhibition of NKCC1 activity in Xenopus laevis oocytes. Taken together, our data are consistent with AATYK1 indirectly inhibiting the SPAK/WNK4 activation of the cotransporter by scaffolding an inhibitory phosphatase in proximity to a stimulatory kinase.

Functional and molecular characterization of the K-Cl cotransporter of Xenopus laevis oocytes

2001 ◽  
Vol 281 (2) ◽  
pp. C670-C680 ◽  
Author(s):  
Adriana Mercado ◽  
Paola de los Heros ◽  
Norma Vázquez ◽  
Patricia Meade ◽  
David B. Mount ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Protein Phosphatase ◽  
Protein Phosphatase 1 ◽  
Cell Swelling ◽  
Xenopus Laevis Oocytes ◽  
Protein Fragment ◽  
Uptake Mechanism ◽  
Partial Cdna ◽  
Menten Constant

The K-Cl cotransporters (KCCs) have a broad range of physiological roles, in a number of cells and species. We report here that Xenopus laevis oocytes express a K-Cl cotransporter with significant functional and molecular similarity to mammalian KCCs. Under isotonic conditions, defolliculated oocytes exhibit a Cl−-dependent86Rb+ uptake mechanism after activation by the cysteine-reactive compounds N-ethylmaleimide (NEM) and mercuric chloride (HgCl2). The activation of this K-Cl cotransporter by cell swelling is prevented by inhibition of protein phosphatase-1 with calyculin A; NEM activation of the transporter was not blocked by phosphatase inhibition. Kinetic characterization reveals apparent values for the Michaelis-Menten constant of 27.7 ± 3.0 and 15.4 ± 4.7 mM for Rb+ and Cl−, respectively, with an anion selectivity for K+ transport of Cl− = PO[Formula: see text] = Br−> I− > SCN− > gluconate. The oocyte K-Cl cotransporter was sensitive to several inhibitors, including loop diuretics, with apparent half-maximal inhibition values of 200 and 500 μM for furosemide and bumetanide, respectively. A partial cDNA encoding the Xenopus K-Cl cotransporter was cloned from oocyte RNA; the corresponding transcript is widely expressed in Xenopus tissues. The predicted COOH-terminal protein fragment exhibited particular homology to the KCC1/KCC3 subgroup of the mammalian KCCs, and the functional characteristics are the most similar to those of KCC1 (Mercado A, Song L, Vazquez N, Mount DB, and Gamba G. J Biol Chem 275: 30326–30334, 2000).

scholarly journals Protein Phosphatase 1 Regulates Human Cytomegalovirus Protein Translation by Restraining AMPK Signaling

2021 ◽  
Vol 12 ◽  
Author(s):  
Carmen Stecher ◽  
Sanja Marinkov ◽  
Lucia Mayr-Harting ◽  
Ana Katic ◽  
Marie-Theres Kastner ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Protein Phosphatase ◽  
Catalytic Domain ◽  
Human Fibroblasts ◽  
Elongation Factor ◽  
Early Time ◽  
Protein Translation ◽  
Protein Phosphatase 1 ◽  
Ampk Signaling ◽  
Protein Protein Interaction

Human cytomegalovirus (HCMV) carries the human protein phosphatase 1 (PP1) and other human proteins important for protein translation in its tegument layer for a rapid supply upon infection. However, the biological relevance behind PP1 incorporation and its role during infection is unclear. Additionally, PP1 is a difficult molecular target due to its promiscuity and similarities between the catalytic domain of multiple phosphatases. In this study, we circumvented these shortcomings by using 1E7-03, a small molecule protein–protein interaction inhibitor, as a molecular tool of noncatalytic PP1 inhibition. 1E7-03 treatment of human fibroblasts severely impaired HCMV replication and viral protein translation. More specifically, PP1 inhibition led to the deregulation of metabolic signaling pathways starting at very early time points post-infection. This effect was at least partly mediated by the prevention of AMP-activated protein kinase dephosphorylation, leading to elongation factor 2 hyperphosphorylation and reduced translation rates. These findings reveal an important mechanism of PP1 for lytic HCMV infection.

scholarly journals Nonnatural protein-protein interaction-pair design by key residues grafting

2007 ◽  
Vol 104 (13) ◽  
pp. 5330-5335 ◽  
Author(s):  
S. Liu ◽  
S. Liu ◽  
X. Zhu ◽  
H. Liang ◽  
A. Cao ◽  
...  
Keyword(s):  
Protein Interaction ◽  
Protein Protein Interaction ◽  
Key Residues ◽  
Protein Interaction Pair

Modulation of the substrate specificity of the polycation-stimulated protein phosphatase from Xenopus laevis oocytes

1988 ◽  
Vol 173 (1) ◽  
pp. 17-25 ◽  
Author(s):  
Jacques HERMANN ◽  
Xavier CAYLA ◽  
Kathelijn DUMORTIER ◽  
Jozef GORIS ◽  
Rene OZON ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Substrate Specificity ◽  
Protein Phosphatase ◽  
Xenopus Laevis Oocytes

scholarly journals A thorough analysis of the contribution of experimental, derived and sequence-based predicted protein-protein interactions for functional annotation of proteins

2019 ◽  
Author(s):  
Stavros Makrodimitris ◽  
Marcel Reinders ◽  
Roeland van Ham
Keyword(s):  
Protein Interaction ◽  
Protein Interactions ◽  
Biological Process ◽  
Poor Performance ◽  
Protein Interaction Data ◽  
Physical Interaction ◽  
Cellular Functions ◽  
Data Resource ◽  
Protein Protein Interaction ◽  
Ppi Networks

AbstractPhysical interaction between two proteins is strong evidence that the proteins are involved in the same biological process, making Protein-Protein Interaction (PPI) networks a valuable data resource for predicting the cellular functions of proteins. However, PPI networks are largely incomplete for non-model species. Here, we tested to what extened these incomplete networks are still useful for genome-wide function prediction. We used two network-based classifiers to predict Biological Process Gene Ontology terms from protein interaction data in four species: Saccharomyces cerevisiae, Escherichia coli, Arabidopsis thaliana and Solanum lycopersicum (tomato). The classifiers had reasonable performance in the well-studied yeast, but performed poorly in the other species. We showed that this poor performance can be considerably improved by adding edges predicted from various data sources, such as text mining, and that associations from the STRING database are more useful than interactions predicted by a neural network from sequence-based features.

scholarly journals Adipocyte Enhancer-binding Protein-1 Promotes Macrophage Inflammatory Responsiveness by Up-Regulating NF-κB via IκBα Negative Regulation

2007 ◽  
Vol 18 (3) ◽  
pp. 930-942 ◽  
Author(s):  
Amin Majdalawieh ◽  
Lei Zhang ◽  
Hyo-Sung Ro
Keyword(s):  
Protein Interaction ◽  
Target Genes ◽  
Inflammatory Diseases ◽  
Cell Types ◽  
Nuclear Factor Κb ◽  
Physical Interaction ◽  
Eukaryotic Transcription ◽  
Protein Protein Interaction ◽  
Proinflammatory Mediators ◽  
Inhibitory Function

Nuclear factor κB (NF-κB) subunits comprise a family of eukaryotic transcription factors that are critically involved in cell proliferation, inflammation, and apoptosis. Under basal conditions, NF-κB subunits are kept under inhibitory regulation by physical interaction with NF-κB inhibitors (IκB subunits) in the cytosol. Upon stimulation, IκB subunits become phosphorylated, ubiquitinated, and subsequently degraded, allowing NF-κB subunits to translocate to the nucleus and bind as dimers to κB responsive elements of target genes. Previously, we have shown that AEBP1 enhances macrophage inflammatory responsiveness by inducing the expression of various proinflammatory mediators. Herein, we provide evidence suggesting that AEBP1 manifests its proinflammatory function by up-regulating NF-κB activity via hampering IκBα, but not IκBβ, inhibitory function through protein–protein interaction mediated by the discoidin-like domain (DLD) of AEBP1. Such interaction renders IκBα susceptible to enhanced phosphorylation and degradation, subsequently leading to augmented NF-κB activity. Collectively, we propose a novel molecular mechanism whereby NF-κB activity is modulated by means of protein–protein interaction involving AEBP1 and IκBα. Moreover, our study provides a plausible mechanism explaining the differential regulatory functions exhibited by IκBα and IκBβ in various cell types. We speculate that AEBP1 may serve as a potential therapeutic target for the treatment of various chronic inflammatory diseases and cancer.

Discovery and identification of Cdc37-derived peptides targeting the Hsp90–Cdc37 protein–protein interaction

RSC Advances ◽  
2015 ◽  
Vol 5 (116) ◽  
pp. 96138-96145 ◽  
Author(s):  
Lei Wang ◽  
Qi-Chao Bao ◽  
Xiao-Li Xu ◽  
Fen Jiang ◽  
Kai Gu ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulation ◽  
Protein Interaction ◽  
Peptide Inhibitors ◽  
Dynamics Simulation ◽  
Protein Protein Interaction ◽  
Binding Interface ◽  
Combined Strategy ◽  
Key Residues

In order to explore the key residues of the Hsp90–Cdc37 binding interface for further design of peptide inhibitors, a combined strategy of molecular dynamics simulation and MM-PBSA analysis was performed.

scholarly journals A Multimodal Strategy Used by a Large c-di-GMP Network

2018 ◽  
Vol 200 (8) ◽  
Author(s):  
Kurt M. Dahlstrom ◽  
Alan J. Collins ◽  
Georgia Doing ◽  
Jaclyn N. Taroni ◽  
Timothy J. Gauvin ◽  
...  
Keyword(s):  
Protein Interaction ◽  
Biofilm Formation ◽  
Model Building ◽  
Mutational Analysis ◽  
Bacterial Biofilm ◽  
Transcriptional Analysis ◽  
Physical Interaction ◽  
Dependent Manner ◽  
Protein Protein Interaction ◽  
Two Hybrid

ABSTRACTThePseudomonas fluorescensgenome encodes more than 50 proteins predicted to be involved in c-di-GMP signaling. Here, we demonstrated that, tested across 188 nutrients, these enzymes and effectors appeared capable of impacting biofilm formation. Transcriptional analysis of network members across ∼50 nutrient conditions indicates that altered gene expression can explain a subset of but not all biofilm formation responses to the nutrients. Additional organization of the network is likely achieved through physical interaction, as determined via probing ∼2,000 interactions by bacterial two-hybrid assays. Our analysis revealed a multimodal regulatory strategy using combinations of ligand-mediated signals, protein-protein interaction, and/or transcriptional regulation to fine-tune c-di-GMP-mediated responses. These results create a profile of a large c-di-GMP network that is used to make important cellular decisions, opening the door to future model building and the ability to engineer this complex circuitry in other bacteria.IMPORTANCECyclic diguanylate (c-di-GMP) is a key signaling molecule regulating bacterial biofilm formation, and many microbes have up to dozens of proteins that make, break, or bind this dinucleotide. A major open issue in the field is how signaling specificity is conferred in the unpartitioned space of a bacterial cell. Here, we took a systems approach, using mutational analysis, transcriptional studies, and bacterial two-hybrid analysis to interrogate this network. We found that a majority of enzymes are capable of impacting biofilm formation in a context-dependent manner, and we revealed examples of two or more modes of regulation (i.e., transcriptional control with protein-protein interaction) being utilized to generate an observable impact on biofilm formation.

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