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.

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 Selection of Protein Phosphatase 2A Regulatory Subunits Is Mediated by the C Terminus of the Catalytic Subunit

2007 ◽  
Vol 282 (37) ◽  
pp. 26971-26980 ◽  
Author(s):  
Sari Longin ◽  
Karen Zwaenepoel ◽  
Justin V. Louis ◽  
Stephen Dilworth ◽  
Jozef Goris ◽  
...  
Keyword(s):  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Catalytic Subunit ◽  
Regulatory Subunits ◽  
C Terminus ◽  
Phosphatase 2A ◽  
Selection Of

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.

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.

scholarly journals Expression of type 1 and 2A protein phosphatase subunits in the rat corpus luteum across pregnancy

2007 ◽  
Vol 195 (2) ◽  
pp. 293-299
Author(s):  
Manish V Sheth ◽  
Connie J Mark ◽  
Kathleen M Eyster
Keyword(s):  
Corpus Luteum ◽  
Real Time ◽  
Phosphatase Activity ◽  
Protein Phosphatase ◽  
Catalytic Subunit ◽  
Rt Pcr ◽  
Catalytic Subunits ◽  
Regulatory Subunits ◽  
A Subunit

This study was undertaken to test the hypothesis that the reduction in protein phosphatase activity that had been observed at mid-pregnancy in the rat corpus luteum (CL) was due to a decrease in expression of one of the catalytic subunits or an increase in one of the B regulatory subunits of the type 2A protein phosphatase (PP2A). Ovaries were collected from rats on days (d) 1, 3, 7, 14, 20, and 21 of pregnancy, and on day 21 after progesterone treatment on day 20 (n = 6). Real-time RT-PCR was used to analyze the expression of the α and β isoforms of the catalytic subunit, the structural A subunit, and three B regulatory subunits of PP2A, as well as the catalytic subunit of PP1. Expression of the α and β catalytic subunits of PP2A was progesterone responsive. Expression of the PP1 catalytic subunit correlated with the previously reported protein phosphatase activity, but PP2A subunits did not. The data suggest that the decreased protein phosphatase activity at mid-pregnancy was due to a decline in expression of the catalytic subunits of PP1 rather than changes in expression of PP2A subunits.

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 Protein Kinase A Distribution in Meningioma

Cancers ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 1686 ◽  
Author(s):  
Caretta ◽  
Denaro ◽  
D’Avella ◽  
Mucignat-Caretta
Keyword(s):  
Brain Tissue ◽  
Catalytic Subunit ◽  
Therapeutic Interventions ◽  
Normal Brain ◽  
Local Concentration ◽  
Correlation Pattern ◽  
Catalytic Subunits ◽  
Tissue Specimens ◽  
Pka Catalytic Subunit

Deregulation of intracellular signal transduction pathways is a hallmark of cancer cells, clearly differentiating them from healthy cells. Differential intracellular distribution of the cAMP-dependent protein kinases (PKA) was previously detected in cell cultures and in vivo in glioblastoma and medulloblastoma. Our goal is to extend this observation to meningioma, to explore possible differences among tumors of different origins and prospective outcomes. The distribution of regulatory and catalytic subunits of PKA has been examined in tissue specimens obtained during surgery from meningioma patients. PKA RI subunit appeared more evenly distributed throughout the cytoplasm, but it was clearly detectable only in some tumors. RII was present in discrete spots, presumably at high local concentration; these aggregates could also be visualized under equilibrium binding conditions with fluorescent 8-substituted cAMP analogues, at variance with normal brain tissue and other brain tumors. The PKA catalytic subunit showed exactly overlapping pattern to RII and in fixed sections could be visualized by fluorescent cAMP analogues. Gene expression analysis showed that the PKA catalytic subunit revealed a significant correlation pattern with genes involved in meningioma. Hence, meningioma patients show a distinctive distribution pattern of PKA regulatory and catalytic subunits, different from glioblastoma, medulloblastoma, and healthy brain tissue. These observations raise the possibility of exploiting the PKA intracellular pathway as a diagnostic tool and possible therapeutic interventions.

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.

The role of the C-terminus and Kpn loop in the quaternary structure stability of nucleoside diphosphate kinase from Leishmania parasites

2015 ◽  
Vol 192 (3) ◽  
pp. 336-341 ◽  
Author(s):  
Plínio Salmazo Vieira ◽  
Priscila Oliveira de Giuseppe ◽  
Arthur Henrique Cavalcante de Oliveira ◽  
Mario Tyago Murakami
Keyword(s):  
Quaternary Structure ◽  
Nucleoside Diphosphate Kinase ◽  
Structure Stability ◽  
C Terminus ◽  
Leishmania Parasites
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