Dependence of KCC2 K-Cl cotransporter activity on a conserved carboxy terminus tyrosine residue

2000 ◽  
Vol 279 (3) ◽  
pp. C860-C867 ◽  
Author(s):  
Kevin Strange ◽  
Thomas D. Singer ◽  
Rebecca Morrison ◽  
Eric Delpire
Keyword(s):  
Protein Interactions ◽  
Regulatory Protein ◽  
Phosphorylation Site ◽  
Fold Increase ◽  
Tyrosine Residue ◽  
Carboxy Terminus ◽  
Protein Protein Interactions ◽  
Pharmacological Studies ◽  
Osmotic Homeostasis ◽  
Tyrosine Phosphorylation Site

K-Cl cotransporters (KCC) play fundamental roles in ionic and osmotic homeostasis. To date, four mammalian KCC genes have been identified. KCC2 is expressed exclusively in neurons. Injection of Xenopus oocytes with KCC2 cRNA induced a 20-fold increase in Cl−-dependent, furosemide-sensitive K+ uptake. Oocyte swelling increased KCC2 activity 2–3 fold. A canonical tyrosine phosphorylation site is located in the carboxy termini of KCC2 (R1081–Y1087) and KCC4, but not in other KCC isoforms. Pharmacological studies, however, revealed no regulatory role for phosphorylation of KCC2 tyrosine residues. Replacement of Y1087 with aspartate or arginine dramatically reduced K+ uptake under isotonic and hypotonic conditions. Normal or near-normal cotransporter activity was observed when Y1087 was mutated to phenylalanine, alanine, or isoleucine. A tyrosine residue equivalent to Y1087 is conserved in all identified KCCs from nematodes to humans. Mutation of the Y1087 congener in KCC1 to aspartate also dramatically inhibited cotransporter activity. Taken together, these results suggest that replacement of Y1087 and its congeners with charged residues disrupts the conformational state of the carboxy terminus. We postulate that the carboxy terminus plays an essential role in maintaining the functional conformation of KCC cotransporters and/or is involved in essential regulatory protein-protein interactions.

scholarly journals A Novel Phosphorylation Site-Kinase Network-Based Method for the Accurate Prediction of Kinase-Substrate Relationships

2017 ◽  
Vol 2017 ◽  
pp. 1-11 ◽  
Author(s):  
Minghui Wang ◽  
Tao Wang ◽  
Binghua Wang ◽  
Yu Liu ◽  
Ao Li
Keyword(s):  
Protein Interactions ◽  
Phosphorylation Site ◽  
Prediction Performance ◽  
Accurate Prediction ◽  
Phosphorylation Sites ◽  
Protein Protein Interactions ◽  
Biological Mechanisms ◽  
Kinase Substrate ◽  
Prediction Tools ◽  
Local Sequence

Protein phosphorylation is catalyzed by kinases which regulate many aspects that control death, movement, and cell growth. Identification of the phosphorylation site-specific kinase-substrate relationships (ssKSRs) is important for understanding cellular dynamics and provides a fundamental basis for further disease-related research and drug design. Although several computational methods have been developed, most of these methods mainly use local sequence of phosphorylation sites and protein-protein interactions (PPIs) to construct the prediction model. While phosphorylation presents very complicated processes and is usually involved in various biological mechanisms, the aforementioned information is not sufficient for accurate prediction. In this study, we propose a new and powerful computational approach named KSRPred for ssKSRs prediction, by introducing a novel phosphorylation site-kinase network (pSKN) profiles that can efficiently incorporate the relationships between various protein kinases and phosphorylation sites. The experimental results show that the pSKN profiles can efficiently improve the prediction performance in collaboration with local sequence and PPI information. Furthermore, we compare our method with the existing ssKSRs prediction tools and the results demonstrate that KSRPred can significantly improve the prediction performance compared with existing tools.

scholarly journals Positive and Negative Regulation of Poly(A) Nuclease

2004 ◽  
Vol 24 (12) ◽  
pp. 5521-5533 ◽  
Author(s):  
David A. Mangus ◽  
Matthew C. Evans ◽  
Nathan S. Agrin ◽  
Mandy Smith ◽  
Preetam Gongidi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Protein Interactions ◽  
Binding Pocket ◽  
Negative Regulation ◽  
Single Amino Acid ◽  
Carboxy Terminus ◽  
Protein Protein Interactions ◽  
C Terminus

ABSTRACT PAN, a yeast poly(A) nuclease, plays an important nuclear role in the posttranscriptional maturation of mRNA poly(A) tails. The activity of this enzyme is dependent on its Pan2p and Pan3p subunits, as well as the presence of poly(A)-binding protein (Pab1p). We have identified and characterized the associated network of factors controlling the maturation of mRNA poly(A) tails in yeast and defined its relevant protein-protein interactions. Pan3p, a positive regulator of PAN activity, interacts with Pab1p, thus providing substrate specificity for this nuclease. Pab1p also regulates poly(A) tail trimming by interacting with Pbp1p, a factor that appears to negatively regulate PAN. Pan3p and Pbp1p both interact with themselves and with the C terminus of Pab1p. However, the domains required for Pan3p and Pbp1p binding on Pab1p are distinct. Single amino acid changes that disrupt Pan3p interaction with Pab1p have been identified and define a binding pocket in helices 2 and 3 of Pab1p's carboxy terminus. The importance of these amino acids for Pab1p-Pan3p interaction, and poly(A) tail regulation, is underscored by experiments demonstrating that strains harboring substitutions in these residues accumulate mRNAs with long poly(A) tails in vivo.

scholarly journals A proline-rich sequence unique to MEK1 and MEK2 is required for raf binding and regulates MEK function.

1995 ◽  
Vol 15 (10) ◽  
pp. 5214-5225 ◽  
Author(s):  
A D Catling ◽  
H J Schaeffer ◽  
C W Reuter ◽  
G R Reddy ◽  
M J Weber
Keyword(s):  
Protein Interactions ◽  
Critical Role ◽  
Phosphorylation Site ◽  
Specific Protein ◽  
Protein Protein Interactions ◽  
Serum Stimulation ◽  
And Function

Mammalian MEK1 and MEK2 contain a proline-rich (PR) sequence that is absent both from the yeast homologs Ste7 and Byr1 and from a recently cloned activator of the JNK/stress-activated protein kinases, SEK1/MKK4. Since this PR sequence occurs in MEKs that are regulated by Raf family enzymes but is missing from MEKs and SEKs activated independently of Raf, we sought to investigate the role of this sequence in MEK1 and MEK2 regulation and function. Deletion of the PR sequence from MEK1 blocked the ability of MEK1 to associate with members of the Raf family and markedly attenuated activation of the protein in vivo following growth factor stimulation. In addition, this sequence was necessary for efficient activation of MEK1 in vitro by B-Raf but dispensable for activation by a novel MEK1 activator which we have previously detected in fractionated fibroblast extracts. Furthermore, we found that a phosphorylation site within the PR sequence of MEK1 was required for sustained MEK1 activity in response to serum stimulation of quiescent fibroblasts. Consistent with this observation, we observed that MEK2, which lacks a phosphorylation site at the corresponding position, was activated only transiently following serum stimulation. Finally, we found that deletion of the PR sequence from a constitutively activated MEK1 mutant rendered the protein nontransforming in Rat1 fibroblasts. These observations indicate a critical role for the PR sequence in directing specific protein-protein interactions important for the activation, inactivation, and downstream functioning of the MEKs.

scholarly journals Retention Time Prediction Using Neural Networks Increases Identifications in Crosslinking Mass Spectrometry

2021 ◽  
Author(s):  
Sven H. Giese ◽  
Ludwig R. Sinn ◽  
Fritz Wegner ◽  
Juri Rappsilber
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Retention Time ◽  
Fold Increase ◽  
Reversed Phase ◽  
Fanconi Anaemia ◽  
Chromatographic Retention ◽  
Protein Protein Interactions ◽  
Noisy Information ◽  
Strong Anion Exchange

AbstractCrosslinking mass spectrometry (Crosslinking MS) has developed into a robust technique that is increasingly used to investigate the interactomes of organelles and cells. However, the incomplete and noisy information in the spectra limits the numbers of protein-protein interactions (PPIs) that can be confidently identified. Here, we successfully leveraged chromatographic retention time (RT) information to aid the identification of crosslinked peptides from spectra. Our Siamese machine learning model xiRT achieved highly accurate RT predictions of crosslinked peptides in a multi-dimensional separation of crosslinked E. coli lysate. We combined strong cation exchange (SCX), hydrophilic strong anion exchange (hSAX) and reversed-phase (RP) chromatography and reached R2 0.94 in RP and a margin of error of 1 fraction for hSAX in 94%, and SCX in 85% of the predictions. Importantly, supplementing the search engine score with retention time features led to a 1.4-fold increase in PPIs at a 1% false discovery rate. We also demonstrate the value of this approach for the more routine analysis of a crosslinked multiprotein complexes. An increase of 1.7-fold in heteromeric crosslinked residue-pairs was achieved at 1% residue-pair FDR for Fanconi anaemia monoubiquitin ligase complex, solely using reversed-phase RT. Retention times are a powerful complement to mass spectrometric information to increase the sensitivity of Crosslinking MS analyses.

scholarly journals Sequence conservation, domain architectures, and phylogenetic distribution of the HD-GYP type c-di-GMP phosphodiesterases

2021 ◽  
Author(s):  
Michael Y. Galperin ◽  
Shan-Ho Chou
Keyword(s):  
Protein Interactions ◽  
Regulatory Protein ◽  
Second Messenger ◽  
Distribution Patterns ◽  
Distribution Functions ◽  
Amino Acid Residues ◽  
Sequence Motifs ◽  
Protein Protein Interactions ◽  
Conserved Sequence ◽  
Common Domain

The HD-GYP domain, named after two of its conserved sequence motifs, was first described in 1999 as a specialized version of the widespread HD phosphohydrolase domain that had additional highly conserved amino acid residues. Domain associations of HD-GYP indicated its involvement in bacterial signal transduction and distribution patterns of this domain suggested that it could serve as a hydrolase of the bacterial second messenger c-di-GMP, in addition to or instead of the EAL domain. Subsequent studies confirmed the ability of various HD-GYP domains to hydrolyze c-di-GMP to linear pGpG and/or GMP. Certain HD-GYP-containing proteins hydrolyze another second messenger, cGAMP, and some HD-GYP domains participate in regulatory protein-protein interactions. The recently solved structures of HD-GYP domains from four distinct organisms clarified the mechanisms of c-di-GMP binding and metal-assisted hydrolysis. However, the HD-GYP domain is poorly represented in public domain databases, which causes certain confusion about its phylogenic distribution, functions, and domain architectures. Here, we present a refined sequence model for the HD-GYP domain and describe the roles of its most conserved residues in metal and/or substrate binding. We also calculate the numbers of HD-GYPs encoded in various genomes and list the most common domain combinations involving HD-GYP, such as the RpfG (REC-HD-GYP), Bd1817 (DUF3391-HD-GYP), and PmGH (GAF-HD-GYP) protein families. We also provide the descriptions of six HD-GYP-associated domains, including four novel integral membrane sensor domains. This work is expected to stimulate studies of diverse HD-GYP-containing proteins, their N-terminal sensor domains, and the signals to which they respond.

scholarly journals A High-Throughput Solid-Phase Microplate Protein-Binding Assay to Investigate Interactions between Myofilament Proteins

2011 ◽  
Vol 2011 ◽  
pp. 1-8 ◽  
Author(s):  
Brandon J. Biesiadecki ◽  
J.-P. Jin
Keyword(s):  
Protein Binding ◽  
Protein Interactions ◽  
Protein Modification ◽  
Binding Assay ◽  
Solid Phase ◽  
Regulatory Protein ◽  
Protein Isoforms ◽  
Protein Protein Interactions ◽  
Protein Binding Assay ◽  
Phase Protein

To understand the structure-function relationship of muscle-regulatory-protein isoforms, mutations, and posttranslational modifications, it is necessary to probe functional effects at the level of the protein-protein interaction. Traditional methodologies assessing such protein-protein interactions are laborious and require significant amounts of purified protein, while many current methodologies require costly and specialized equipment or modification of the proteins, which may affect their interaction. To address these issues, we developed a novel method of microplate-based solid-phase protein-binding assay over the recent years. This method assesses specific protein-protein interactions at physiological conditions, utilizes relatively small amounts of protein, is free of protein modification, and does not require specialized instrumentation. Here we present detailed methodology for the solid-phase protein-binding assay with examples that we have successfully applied to quantify interactions of myofilament-regulatory proteins. We further provide considerations for optimization of the assay conditions and its broader application in studies of other protein-protein interactions.

scholarly journals Reciprocal Activation by Cyclin-Dependent Kinases 2 and 7 Is Directed by Substrate Specificity Determinants outside the T Loop

2001 ◽  
Vol 21 (1) ◽  
pp. 88-99 ◽  
Author(s):  
Sarah Garrett ◽  
William A. Barton ◽  
Ronald Knights ◽  
Pei Jin ◽  
David O. Morgan ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Protein Interactions ◽  
Phosphorylation Site ◽  
The Body ◽  
Regulatory Subunit ◽  
Minor Role ◽  
Protein Protein Interactions ◽  
Threonine Residue ◽  
A Minor

ABSTRACT Cyclin-dependent kinase 7 (CDK7) is the catalytic subunit of the metazoan CDK-activating kinase (CAK), which activates CDKs, such as CDC2 and CDK2, through phosphorylation of a conserved threonine residue in the T loop. Full activation of CDK7 requires association with a positive regulatory subunit, cyclin H, and phosphorylation of a conserved threonine residue at position 170 in its own T loop. We show that threonine-170 of CDK7 is phosphorylated in vitro by its targets, CDC2 and CDK2, which also phosphorylate serine-164 in the CDK7 T loop, a site that perfectly matches their consensus phosphorylation site. In contrast, neither CDK4 nor CDK7 itself can phosphorylate the CDK7 T loop in vitro. The ability of CDC2 or CDK2 and CDK7 to phosphorylate each other but not themselves implies that each kinase can discriminate among closely related sequences and can recognize a substrate site that diverges from its usual preferred site. To understand the basis for this paradoxical substrate specificity, we constructed a chimeric CDK with the T loop of CDK7 grafted onto the body of CDK2. Surprisingly, the hybrid enzyme, CDK2-7, was efficiently activated in cyclin A-dependent fashion by CDK7 but not at all by CDK2. CDK2-7, moreover, phosphorylated wild-type CDK7 but not CDK2. Our results suggest that the primary amino acid sequence of the T loop plays only a minor role, if any, in determining the specificity of cyclin-dependent CAKs for their CDK substrates and that protein-protein interactions involving sequences outside the T loop can influence substrate specificity both positively and negatively.

scholarly journals Cross-regulatory protein-protein interactions between Hox and Pax transcription factors

2008 ◽  
Vol 105 (36) ◽  
pp. 13439-13444 ◽  
Author(s):  
S. Plaza ◽  
F. Prince ◽  
Y. Adachi ◽  
C. Punzo ◽  
D. L. Cribbs ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Protein Interactions ◽  
Regulatory Protein ◽  
Protein Protein Interactions

scholarly journals Comparative interactomes of HSF1 in stress and disease reveal a role for CTCF in HSF1-mediated gene regulation

2020 ◽  
pp. jbc.RA120.015452
Author(s):  
Eileen T. Burchfiel ◽  
Anniina Vihervaara ◽  
Michael J. Guertin ◽  
Rocio Gomez-Pastor ◽  
Dennis J. Thiele
Keyword(s):  
Heat Shock ◽  
Rna Polymerase Ii ◽  
Protein Interactions ◽  
Target Genes ◽  
Regulatory Protein ◽  
Normal Growth ◽  
Gene Repression ◽  
Interacting Proteins ◽  
Protein Protein Interactions ◽  
Acute And Chronic Stress

Heat Shock Transcription Factor 1 (HSF1) orchestrates cellular stress protection by activating or repressing gene transcription in response to protein misfolding, oncogenic cell proliferation and other environmental stresses. HSF1 is tightly regulated via intramolecular repressive interactions, post-translational modifications, and protein-protein interactions. How these HSF1 regulatory protein interactions are altered in response to acute and chronic stress is largely unknown. To elucidate the profile of HSF1 protein interactions under normal growth, chronic and acutely stressful conditions, quantitative proteomics studies identified interacting proteins in the response to heat shock or in the presence of a poly-glutamine aggregation protein cell-based model of Huntington’s Disease. These studies identified distinct protein interaction partners of HSF1 as well as changes in the magnitude of shared interactions as a function of each stressful condition. Several novel HSF1-interacting proteins were identified that encompass a wide variety of cellular functions, including roles in DNA repair, mRNA processing, regulation of RNA polymerase II and others. One HSF1 partner, CTCF, interacted with HSF1 in a stress-inducible manner and functions in repression of specific HSF1 target genes. Understanding how HSF1 regulates gene repression is a crucial question, given the dysregulation of HSF1 target genes in both cancer and neurodegeration. These studies expand our understanding of HSF1-mediated gene repression and provide key insights into HSF1 regulation via protein-protein interactions.

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