Hybrid aspartate transcarbamoylase containing cross-linked subunits

1981 ◽  
Vol 59 (6) ◽  
pp. 461-468 ◽  
Author(s):  
William W.-C. Chan ◽  
Caroline A. Enns
Keyword(s):  
Conformational Changes ◽  
Catalytic Subunit ◽  
Aspartate Transcarbamoylase ◽  
Regulatory Subunit ◽  
Cross Linking ◽  
Substrate Affinity ◽  
Catalytic Subunits ◽  
Regulatory Subunits ◽  
Allosteric Mechanism ◽  
Enzyme Molecules

The role of conformational changes and subunit interactions in the allosteric mechanism of aspartate transcarbamoylase was evaluated by studying hybrid enzyme molecules containing cross-linked subunits. Native enzyme was cross-linked with tartryl diazide in the presence and absence of substrate analogues. The two types of modified enzyme derivatives were each dissociated into catalytic (c3) and regulatory (r2) subunits. Hybrids were constructed with modified catalytic subunits and unmodified regulatory subunits or vice versa. Subunits from different derivatives also formed hybrids.All hybrids containing cross-linked catalytic subunits showed hyperbolic substrate saturation curves while cross-linking in the regulatory subunit alone did not abolish cooperativity. The type of cross-linking in the catalytic subunit had a decisive influence on the substrate affinity of the hybrid as well as its response to the allosteric effectors ATP and CTP. However many effects were also dependent on the presence of regulatory subunits. The results implicate a substantial conformational change in the catalytic subunit upon substrate binding and suggest an important role for the c–r interaction in the allosteric mechanism.

Aspartate transcarbamoylase: loss of homotropic but not heterotropic interactions upon modification of the catalytic subunit with a bifunctional reagent

1979 ◽  
Vol 57 (6) ◽  
pp. 798-805 ◽  
Author(s):  
William W.-C. Chan ◽  
Caroline A. Enns
Keyword(s):  
Conformational Changes ◽  
Substrate Binding ◽  
Catalytic Subunit ◽  
Native Enzyme ◽  
Aspartate Transcarbamoylase ◽  
Substrate Affinity ◽  
Carbamoyl Phosphate ◽  
Catalytic Subunits ◽  
Regulatory Subunits ◽  
Michaelis Constants

The role of conformational changes in the allosteric mechanism of aspartate transcarbamoylase from Escherichia coli was studied by reacting the isolated catalytic subunit with the bifunctional reagent tartryl diazide. Two derivatives differing moderately in substrate affinity were obtained depending on whether the reaction was conducted in the presence or absence of the substrate analogue succinate and carbamoyl phosphate. The modification was not accompanied by aggregation or dissociation. The modified catalytic subunits retained the ability to reassociate with unmodified regulatory subunits and produced hybrids similar in size to the native enzyme. These hybrids were appreciably sensitive to the allosteric effectors ATP and CTP but unlike native enzyme showed no cooperativity in substrate binding. The Michaelis constants of these hybrids for aspartate were intermediate between that of the isolated catalytic subunit and that of the relaxed state. Activation by ATP was caused by a reduction in Km to the value characteristic of the relaxed state whereas CTP inhibited by lowering the Vmax. The properties of the hybrids are strikingly similar to the modified enzyme obtained by Kerbiriou and Hervé from cells grown in the presence of 2-thiouracil. However, the crucial modifications are found in the regulatory subunits of the enzyme studied by these authors whereas they are located in the catalytic subunits of the hybrids reported here. Our results suggest that interactions between the catalytic and regulatory subunits have considerable effects on the state of the substrate binding sites in the native enzyme.

scholarly journals The Role of Protein Phosphatase 2A Catalytic Subunit Cα in Embryogenesis: Evidence from Sequence Analysis and Localization Studies

1999 ◽  
Vol 380 (9) ◽  
pp. 1117-1120 ◽  
Author(s):  
Jürgen Götz ◽  
Wilfried Kues
Keyword(s):  
Protein Phosphatase ◽  
Genomic Organization ◽  
Protein Phosphatase 2A ◽  
Catalytic Subunit ◽  
Regulatory Subunit ◽  
Catalytic Subunits ◽  
Regulatory Subunits ◽  
Phosphatase 2A ◽  
The Brain

AbstractProtein phosphatase 2A (PP2A) constitutes one of the major families of protein serine/threonine phosphatases found in all eukaryotic cells. PP2A holoenzymes are composed of a catalytic subunit complexed with a structural regulatory subunit of 65 kDa. These core subunits associate with regulatory subunits of various sizes to form different heterotrimers which have been purified and evaluated with regard to substrate specificity. In fully differentiated tissues PP2A expression levels are highest in the brain, however, relatively little is known about expression in the developing embryo.In order to determine the composition of PP2A catalytic subunits in the mouse, cDNAs were cloned and the genomic organization of PP2A Cα was determined.By a gene targeting approach in the mouse, we have previously shown that the absence of the major catalytic subunit of PP2A, Cα, resulted in embryonic lethality around embryonic day E6.5. No mesoderm was formed which implied that PP2A plays a crucial role in gastrulation.Here, we extended our studies and analyzed wildtype embryos for Cα expression at subsequent stages of development. After gastrulation is completed, we find high expression of Cα restricted to the neural folds, which suggests that PP2A plays an additional pivotal role in neurulation.

scholarly journals Langevin Dynamics Simulation of AKAP-PKA Complex: Re-Envisioning the Local Concentration Mechanism for Directing PKA Phosphorylation

2017 ◽  
Author(s):  
Marc Rigatti ◽  
Paul J. Michalski ◽  
Kimberly L. Dodge-Kafka ◽  
Ion I. Moraru
Keyword(s):  
Molecular Mechanism ◽  
Characteristic Time ◽  
Langevin Dynamics ◽  
Catalytic Subunit ◽  
Dynamics Simulation ◽  
Regulatory Subunit ◽  
Signaling System ◽  
Average Characteristic ◽  
Catalytic Subunits ◽  
Regulatory Subunits

AbstractThe second messenger cAMP and its effector cAMP-dependent protein kinase A (PKA) constitute a ubiquitous cell signaling system. In its inactive state PKA is composed of two regulatory subunits that dimerize, and two catalytic subunits that are inhibited by the regulatory subunits. Activation of the catalytic subunits occurs upon binding of two molecules of cAMP to each regulatory subunit. Although many receptor types existing within the same cell may use this signaling system, compartmentation of signaling is thought to occur due to A-Kinase Anchoring Proteins (AKAPs), which act to co-localize PKA with specific substrates. However, the molecular mechanism allowing AKAPs to direct PKA phosphorylation to a particular substrate remained elusive, as prior evidence suggested that the catalytic subunit, which is highly diffusible, is released after cAMP binding to the regulatory subunit. Recent evidence from Smith et al. suggests that in the cell, the catalytic subunit may in fact not be released from the AKAP complex [1, 2]. They further demonstrated that alterations in the structure of the PKA regulatory subunit tether affect substrate phosphorylation. We use a novel computational software based on Langevin dynamics, SpringSaLaD, to simulate the AKAP-PKA complex in order to determine a molecular mechanism for the changes in phosphorylation seen with alteration in tether length and flexibility, and to demonstrate whether or not AKAPs can effectively direct PKA phosphorylation to a particular substrate upon release of the catalytic subunit from the complex. We find that short and flexible tethers contribute to a decrease in the average characteristic time of binding, allowing the catalytic subunit to spend more time in a bound state with the substrate, which yields faster characteristic times of phosphorylation. We further demonstrate that release of the catalytic subunit from the AKAP complex abrogates the effect of tethering, with characteristic times of phosphorylation similar to non-AKAP bound PKA. The data demonstrates that AKAPs likely do not release the catalytic subunit in directing PKA phosphorylation to AKAP bound substrates. In combination with the changes in characteristic time of phosphorylation which are driven by tether structure, this work indicates that the purpose of AKAPs may be to increase the efficiency of phosphorylation of particular AKAP substrates.

scholarly journals PRKAR2A deficiency protects mice from experimental colitis by increasing IFN-stimulated gene expression and modulating the intestinal microbiota

2021 ◽  
Author(s):  
Lumin Wei ◽  
Rongjing Zhang ◽  
Jinzhao Zhang ◽  
Juanjuan Li ◽  
Deping Kong ◽  
...  
Keyword(s):  
Experimental Colitis ◽  
Regulatory Subunit ◽  
Protective Effects ◽  
Intestinal Epithelial ◽  
Catalytic Subunits ◽  
Regulatory Subunits ◽  
Pka Regulatory Subunit ◽  
Cross Fostering ◽  
Specific Deletion

AbstractProtein kinase A (PKA) plays an important role in regulating inflammation via its catalytic subunits. Recently, PKA regulatory subunits have been reported to directly modulate some signaling pathways and alleviate inflammation. However, the role of PKA regulatory subunits in colonic inflammation remains unclear. Therefore, we conducted this study to investigate the role of the PKA regulatory subunit PRKAR2A in colitis. We observed that PRKAR2A deficiency protected mice from dextran sulfate sodium (DSS)-induced experimental colitis. Our experiments revealed that the intestinal epithelial cell-specific deletion of Prkar2a contributed to this protection. Mechanistically, the loss of PRKAR2A in Prkar2a−/− mice resulted in an increased IFN-stimulated gene (ISG) expression and altered gut microbiota. Inhibition of ISGs partially reversed the protective effects against DSS-induced colitis in Prkar2a−/− mice. Antibiotic treatment and cross-fostering experiments demonstrated that the protection against DSS-induced colitis in Prkar2a−/− mice was largely dependent on the gut microflora. Altogether, our work demonstrates a previously unidentified function of PRKAR2A in promoting DSS-induced colitis.

scholarly journals Positive and Negative Regulation of Phosphoinositide 3-Kinase-Dependent Signaling Pathways by Three Different Gene Products of the p85α Regulatory Subunit

2000 ◽  
Vol 20 (21) ◽  
pp. 8035-8046 ◽  
Author(s):  
Kohjiro Ueki ◽  
Petra Algenstaedt ◽  
Franck Mauvais-Jarvis ◽  
C. Ronald Kahn
Keyword(s):  
Glucose Transport ◽  
Kinase Activity ◽  
Glycogen Synthesis ◽  
Catalytic Subunit ◽  
Regulatory Subunit ◽  
Regulatory Subunits ◽  
Irs Proteins ◽  
Terminal Structure ◽  
Stimulation Of ◽  
In Cells

ABSTRACT Phosphoinositide (PI) 3-kinase is a key mediator of insulin-dependent metabolic actions, including stimulation of glucose transport and glycogen synthesis. The gene for the p85α regulatory subunit yields three splicing variants, p85α, AS53/p55α, and p50α. All three have (i) a C-terminal structure consisting of two Src homology 2 domains flanking the p110 catalytic subunit-binding domain and (ii) a unique N-terminal region of 304, 34, and 6 amino acids, respectively. To determine if these regulatory subunits differ in their effects on enzyme activity and signal transduction from insulin receptor substrate (IRS) proteins under physiological conditions, we expressed each regulatory subunit in fully differentiated L6 myotubes using adenovirus-mediated gene transfer with or without coexpression of the p110α catalytic subunit. PI 3-kinase activity associated with p50α was greater than that associated with p85α or AS53. Increasing the level of p85α or AS53, but not p50α, inhibited both phosphotyrosine-associated and p110-associated PI 3-kinase activities. Expression of a p85α mutant lacking the p110-binding site (Δp85) also inhibited phosphotyrosine-associated PI 3-kinase activity but not p110-associated activity. Insulin stimulation of two kinases downstream from PI-3 kinase, Akt and p70 S6 kinase (p70S6K), was decreased in cells expressing p85α or AS53 but not in cells expressing p50α. Similar inhibition of PI 3-kinase, Akt, and p70S6K was observed, even when p110α was coexpressed with p85α or AS53. Expression of p110α alone dramatically increased glucose transport but decreased glycogen synthase activity. This effect was reduced when p110α was coexpressed with any of the three regulatory subunits. Thus, the three different isoforms of regulatory subunit can relay the signal from IRS proteins to the p110 catalytic subunit with different efficiencies. They also negatively modulate the PI 3-kinase catalytic activity but to different extents, dependent on the unique N-terminal structure of each isoform. These data also suggest the existence of a mechanism by which regulatory subunits modulate the PI 3-kinase-mediated signals, independent of the kinase activity, possibly through subcellular localization of the catalytic subunit or interaction with additional signaling molecules.

scholarly journals Methylated C-terminal leucine residue of PP2A catalytic subunit is important for binding of regulatory Bα subunit

1999 ◽  
Vol 339 (2) ◽  
pp. 241-246 ◽  
Author(s):  
Jeffrey C. BRYANT ◽  
Ryan S. WESTPHAL ◽  
Brian E. WADZINSKI
Keyword(s):  
Catalytic Subunit ◽  
Regulatory Subunit ◽  
Post Translational Modification ◽  
Regulatory Subunits ◽  
Leucine Residue ◽  
Cell Extracts ◽  
C Subunit ◽  
Phosphatase 2A ◽  
A Subunit

Methylation of the C-terminal leucine residue (Leu309) of protein serine/threonine phosphatase 2A catalytic subunit (PP2AC) is known to regulate catalytic activity in vitro, but the functional consequence(s) of this post-translational modification in the context of the cell remain unclear. Alkali-induced demethylation of PP2AC in purified PP2A heterotrimer (ABαC), but not in purified PP2A heterodimer (AC), indicated that a larger fraction of PP2AC is carboxymethylated in ABαC than in AC. To explore the role of Leu309 in PP2A holoenzyme assembly, epitope-tagged PP2A catalytic subunit (HA-PP2A) and a mutant of HA-PP2A containing an alanine residue in place of Leu309 (HA-PP2A-L309A) were transiently expressed in COS cells. Both recombinant proteins exhibited serine/threonine phosphatase activity when immunoisolated from COS cell extracts. HA-PP2A, but not HA-PP2A-L309A, was carboxymethylated in vitro. A chromatographic analysis of cell extracts indicated that most endogenous PP2AC and HA-PP2A were co-eluted with the A and Bα regulatory subunits of PP2A, whereas most HA-PP2A-L309A seemed to elute with the A subunit as a smaller complex or, alternatively, as free catalytic (C) subunit. The A subunit co-immunoisolated with both tagged proteins; however, substantially less Bα subunit co-immunoisolated with HA-PP2A-L309A than with HA-PP2A. These results demonstrate that the reversibly methylated C-terminal leucine residue of PP2AC is important for Bα regulatory subunit binding. Furthermore, the results provide evidence for an interrelationship between PP2AC carboxymethylation and PP2A holoenzyme assembly.

scholarly journals Choice of PP1 catalytic subunit affects neither requirement for G-actin nor insensitivity to Sephin1 of PPP1R15A-regulated eIF2αP dephosphorylation

2017 ◽  
Author(s):  
Ana Crespillo-Casado ◽  
Zander Claes ◽  
Meng S. Choy ◽  
Wolfgang Peti ◽  
Mathieu Bollen ◽  
...  
Keyword(s):  
Protein Misfolding ◽  
Catalytic Subunit ◽  
Translation Initiation Factor ◽  
Terminal Repeat ◽  
Initiation Factor ◽  
Regulatory Subunit ◽  
Rabbit Muscle ◽  
Α Subunit ◽  
Regulatory Subunits ◽  
Initiation Factor 2

ABSTRACTThe integrated stress response (ISR) is regulated by kinases that phosphorylate the α subunit of translation initiation factor 2 and phosphatases that dephosphorylate it. Genetic and biochemical observations indicate that the eIF2αP-directed holophosphatase - a therapeutic target in diseases of protein misfolding - is comprised of a regulatory, PPP1R15, and a catalytic, Protein Phosphatase 1 (PP1) subunit. In mammals, there are two isoforms of the regulatory subunit, PPP1R15A and PPP1R15B, with overlapping roles in promoting the essential function of eIF2αP dephosphorylation. However, conflicting reports have appeared regarding the requirement for an additional co-factor, G-actin, in enabling substrate-specific de-phosphorylation by PPP1R15-containing PP1 holoenzymes. An additional concern relates to the sensitivity of the PPP1R15A-containing PP1 holoenzyme to the [(ochlorobenzylidene)amino]guanidines (Sephin1 or Guanabenz), small molecule proteostasis modulators. It has been suggested that the source and method of purification of the PP1 catalytic subunit and the presence or absence of an N-terminal repeat-containing region in the PPP1R15A regulatory subunit might influence both the requirement for G-actin by the eIF2αP-directed holophosphatase and its sensitivity to inhibitors. Here we report that in the absence of G-actin, PPP1R15A regulatory subunits were unable to accelerate eIF2αP dephosphorylation beyond that affected by a catalytic subunit alone, whether PPP1R15A regulatory subunit had or lacked the N-terminal repeat-containing region and whether paired with native PP1 purified from rabbit muscle, or recombinant PP1 expressed in and purified from bacteria. Furthermore, none of the PPP1R15A-containing PP1c holophosphatases were inhibited by Sephin1 or Guanabenz.

scholarly journals Chemical cross-linking of cyclic AMP-dependent protein kinase and its dissimilar subunits

1983 ◽  
Vol 209 (3) ◽  
pp. 581-586 ◽  
Author(s):  
J P Charlton ◽  
C H Huang ◽  
L C Huang
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Ternary Complex ◽  
Kinetic Studies ◽  
Catalytic Subunit ◽  
Regulatory Subunit ◽  
Cross Linking ◽  
Dependent Protein Kinase ◽  
Native Form ◽  
Dependent Protein

Previous kinetic studies have demonstrated that the activation of cyclic AMP-dependent protein kinase by cyclic AMP involves the formation of a ternary complex of cyclic AMP, the regulatory subunit (R) and the catalytic subunit (C). It is suggested that only this ternary complex breaks down to liberate the enzymically active catalytic subunit. We have performed cross-linking experiments with the holoenzyme and its dissimilar subunits in the presence of MgATP and various concentrations of cyclic AMP. Results from these cross-linking studies indicate that regulatory subunits exist as dimers in the native form. Moreover, dissociation of the holoenzyme or the reconstituted enzyme is promoted by cyclic AMP, and the effect of MgATP is to stabilize the enzyme in the tetrameric form. The success in cross-linking the regulatory and the catalytic subunits of protein kinase with the lysine-specific bifunctional cross-linking reagent dimethyl suberimidate may be attributed to the presence of a large number of lysine residues in the enzyme.

scholarly journals Quantitative kinase and phosphatase profiling reveal that CDK1 phosphorylates PP2Ac to promote mitotic entry

2020 ◽  
Vol 13 (648) ◽  
pp. eaba7823 ◽  
Author(s):  
Isha Nasa ◽  
Lauren E. Cressey ◽  
Thomas Kruse ◽  
Emil P. T. Hertz ◽  
Jiang Gui ◽  
...  
Keyword(s):  
Cell Cycle Progression ◽  
Regulatory Subunit ◽  
Reciprocal Regulation ◽  
Chemical Proteomics ◽  
Catalytic Subunits ◽  
Regulatory Subunits ◽  
Mitotic Entry ◽  
Proteomics Approach ◽  
And Control

The reciprocal regulation of phosphoprotein phosphatases (PPPs) by protein kinases is essential to cell cycle progression and control, particularly during mitosis for which the role of kinases has been extensively studied. PPPs perform much of the serine/threonine dephosphorylation in eukaryotic cells and achieve substrate selectivity and specificity through the interaction of distinct regulatory subunits with conserved catalytic subunits in holoenzyme complexes. Using a mass spectrometry–based chemical proteomics approach to enrich, identify, and quantify endogenous PPP holoenzyme complexes combined with kinase profiling, we investigated the phosphorylation-dependent regulation of PPP holoenzymes in mitotic cells. We found that cyclin-dependent kinase 1 (CDK1) phosphorylated a threonine residue on the catalytic subunit of the phosphatase PP2A, which disrupted its holoenzyme formation with the regulatory subunit B55. The consequent decrease in the dephosphorylation of PP2A-B55 substrates promoted mitotic entry. This direct phosphorylation by CDK1 was in addition to a previously reported indirect mechanism, thus adding a layer to the interaction between CDK1 and PP2A in regulating mitotic entry.

scholarly journals Plasmin alters the activity and quaternary structure of human plasma carboxypeptidase N

2005 ◽  
Vol 388 (1) ◽  
pp. 81-91 ◽  
Author(s):  
Mercy O. QUAGRAINE ◽  
Fulong TAN ◽  
Hironori TAMEI ◽  
Ervin G. ERDÖS ◽  
Randal A. SKIDGEL
Keyword(s):  
Quaternary Structure ◽  
Gel Filtration ◽  
Catalytic Subunit ◽  
Surface Proteins ◽  
Covalent Bonds ◽  
Catalytic Subunits ◽  
Regulatory Subunits ◽  
C Terminus ◽  
Carboxypeptidase N ◽  
Structure And Activity

Human CPN (carboxypeptidase N) is a tetrameric plasma enzyme containing two glycosylated 83 kDa non-catalytic/regulatory subunits that carry and protect two active catalytic subunits. Because CPN can regulate the level of plasminogen binding to cell surface proteins, we investigated how plasmin cleaves CPN and the consequences. The products of hydrolysis were analysed by activity assays, Western blotting, gel filtration and sequencing. When incubated with intact CPN tetramer, plasmin rapidly cleaved the 83 kDa subunit at the Arg457–Ser458 bond near the C-terminus to produce fragments of 72 and 13 kDa, thereby releasing an active 142 kDa heterodimer, and also cleaved the active subunit, decreasing its size from 55 kDa to 48 kDa. Further evidence for the heterodimeric form of CPN was obtained by re-complexing the non-catalytic 72 kDa fragment with recombinant catalytic subunit or by immunoprecipitation of the catalytic subunit after plasmin treatment of CPN using an antibody specific for the 83 kDa subunit. Upon longer incubation, plasmin cleaved the catalytic subunit at Arg218–Arg219 to generate fragments of 27 kDa and 21 kDa, held together by non-covalent bonds, that were more active than the native enzyme. These data show that plasmin can alter CPN structure and activity, and that the C-terminal 13 kDa fragment of the CPN 83 kDa subunit is a docking peptide that is necessary to maintain the stable active tetrameric form of human CPN in plasma.

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